ABSORBING SURFACE AREA OF SOUTHERN PINE ROOT SYSTEMS:
PHOSPHORUS AND POTASSIUM UPTAKE BY ROOT SYSTEMS OF TWELVE-

YEAR-OLD SLASH PINE TREES

By

JOSE A. ESCAMILLA-BENCOMO

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1997

ACKNOWLEDGMENTS

-Hey you , what are you going to do when hope dies?

-Hope does not die. Hope has relatives and patrons

to come to its aid ; there are plenty of very devout,
optimistic people in the world, who will protect hope.

Jose Joaquin Fernandez de Lizardi
El PERIQUILLO SARMIENTO

Through the course of the last four years of my life,
many people have provided assistance and support which, to a
large degree, have made a reality of my Ph.D. program and
this dissertation.

First, I thank my main adviser, Dr. Nicholas Comer ford
("Nick"), for providing constructive criticism, intellectual
support and friendship. I also appreciate Dr. Konda R. Reddy,
Dr. Suresh Rao, Dr. David Sylvia and Dr. Kimberlyn Williams
for serving on my committee.

Second, I am grateful for the financial support provided
for this project by USDA Grant No. 722122712 and by the
industrial members of the Program of Pine Productivity
Research (P3R) at the University of Florida. I also
acknowledge Jef ferson-Smurf it Corporation for providing the
land for field studies. Also for providing funds during my
graduate education I acknowledge the following organizations:
National Council of Science and Technology in Mexico
(CONACYT) , Centro de Investigacion Cientifica de Yucatan

11

(CICY), and the Florida Mexico Institute of the Latin
American and Caribbean Center.

My special thanks to Mary McLeod for logistic support,
help in all phases of lab work, and her friendship. Thanks
also to Randall McCrady for his essential help and
camaraderie during greenhouse and field work. I also
recognize the vital and tedious root separation work done by
Karen Wenner-Pedersen and Sharon Wright.

, Friends are as important as academic advice. Pauline
Grierson and John Tredinnick provided a home away from home,
during their time in Gainesville. Special thanks to Pauline
for eliciting key scientific discussions and lengthy
manuscript editing. Thanks to Andres Nazario for his
counseling and support, and to Arlete Freitas, Leslie
Cooperband, Christine Bliss, Karen Wenner-Pedersen, Dave
Farmer, Steve Trabue, Ian Bjorsvik and Scott Brinton for
their friendship. They all were players in those fun events
that kept me going throughout these years of graduate school.

Lastly, but not less important, I thank my friends and
family in Mexico, too numerous to list. However, none of this
has been possible without my parents, Armando y Beatriz, and
their unconditional love, encouragement and understanding
during this very long process.

in

TABLE OF CONTENTS

ACKNOWLEDGMENTS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

CHAPTERS

1 GENERAL INTRODUCTION

. ii
vii
. ix
. xi

1

2 NUTRIENT ABSORBING-SURFACES OF TREES AND THE NUTRIENT
UPTAKE PROCESS

Introduction

Definition of Absorbing-Surfaces of Trees

Nutrient Uptake for the Different Root Surfaces of
Trees

Pathways of Nutrient Uptake !!!!!!!!!!!!!

Nutrient Uptake by Root Trees

Evidence of Nutrient Uptake in Brown Roots . ’. *. *. ! *. *. ]
Michaelis-Menten Kinetics as a Description of Active
Nutrient Uptake

Appropriateness of the Equation for Root Uptake . .
Active Root Uptake Mechanism and Root Anatomy ....
Importance of Root Uptake Kinetics under Field
Conditions

^ and C^ under field conditions

Temporal and spatial changes in root uptake

kinetics under field conditions

Concluding Remarks

. 6
. 7

11

11

14

18

20

20

23

26

26

30

34

3 A METHOD FOR MEASURING NUTRIENT DEPLETION BY ROOTS OF
MATURE TREES IN THE FIELD

Introduction

Materials and Methods

Site Description ’ [

Root Regeneration System . .* .* .* .* .* .* .* .* ’ ’ * ’
Nutrient Uptake Root Chamber Design and

Construction

In-situ Nutrient Uptake ’system !*.’.!!!!*.!

35

40

40

41

43

46

IV

Testing of the Nutrient Uptake System

Laboratory studies

In-situ field studies

Chemical Analyses

Data Analyses

Laboratory studies

Field studies

Results

Laboratory Studies (No roots Present in Chambers)

Field Studies

Root regeneration system

Nutrient depletion with air

Nutrient depletion with N2 gas

Oxygen saturation with air and N2 gas

Discussion

4 PHOSPHORUS AND POTASSIUM DEPLETION BY ROOTS OF FIELD-
GROWN SLASH PINE: AEROBIC AND HYPOXIC CONDITIONS

Introduction

Materials and Methods

Site Description

Root Regeneration System

Field Nutrient Uptake System Installation

Depletion Studies

Pre-depletion treatment

Experiment 1 : Nutrient depletion study with air . . . .
Experiment 2: Nutrient depletion study with N

gas \

Experiment 3: Nutrient depletion study with air
after N^ gas

Experiments 4, 5 and 6: Nutrient depletion study
with air/ N gas/ air

Chemical Analyses

Data Analyses

Results " ’

Oxygen Concentration and Temperature in the
Chambers

Potassium Depletion with Alternating Aerobic and

Hypoxic Conditions

Potassium Depletion Under Hypoxic Conditions

Phosphorus Depletion with Alternating Aerobic and
Hypoxic conditions

. Phosphorus Depletion Under Hypoxic Conditions .*.* !!!!***
Discussion

Implications of P and K uptake by roots of pines in

reduced soil conditions in the field

Conclusions

51

51

53

55

56

56

56

57

57

58

58

58

58

61

63

68

68

75

75

75

76

77

77

78

78

79

79

80

80

81

81

83

84

84

84

88

93

95

v

5 PHOSPHORUS AND POTASSIUM DEPLETION BY WOODY-ROOTS OF

TWELVE-YEAR-OLD SLASH PINE TREES 97

Introduction

Materials and Methods

Root System Description

Nutrient Uptake system installation

Depletion Studies

Pre-depletion treatment

Study #1.P and K depletion by brown roots:

Isolation of white roots by waxing

Study #2 (1995) and study #3 (1996). P and K
depletion by brown roots: Isolation of white

roots by cutting

Study #4 . P and K depletion by whole root

systems subjected to NaOCl solution

Laboratory Incubation Studies

Inoculation with root scrapings

Inoculation with nutrient solution

Ion Mass in the Root Free Space

Root Measurements

Anatomical Observations of Root Sections

Chemical Analyses

Data Analyses

Oxygen saturation, and nutrient solution

temperature

Phosphorus and K uptake and mass flow influx.*!

Phosphorus and K influx

Phosphorus and K uptake, NaOCl solution study*.*

Laboratory incubation studies

Results

Anatomical Observations and Root System
Descriptions

02 Concentration and Temperature in the Chambers .

Microbial Interferences

Potassium and Phosphorus Influx

Discussion

. 97
101
101
102
103

103

104

. 106

. 106
. 107
. 107
. 107
. 109
. 109
. 110
. Ill
. Ill

. Ill
. 112
. 112
. 114
. 116
. 116

. 116
. 121
. 121
. 124
. 127

6 OVERALL RESULTS ,
LIST OF REFERENCES

136

140

BIOGRAPHICAL SKETCH

155

vi

LIST OF TABLES

Table

page

2- 1. Categories of fine-roots in a perennial, dicot,

woody- root system 8

3- 1. List of parts and materials for constructing a

nutrient uptake chamber 48

3- 2. List of parts and materials for constructing a

Mariotte flask system 52

4- 1. Temperature and Dissolved Oxygen concentration in
nutrient solution, during consecutive experiments of

P and K depletion by pine roots 82

5- 1. Experimental design of Covariance analysis used for

P and K Influx data obtained from depletion experiments
of studies 1,2 and 3 115

5-2. Description of root systems used in study one, where
roots were subjected to waxing (1995 depletion
experiments .)

5-3. Description of root systems used in study two, where
roots were subjected to cutting (1995 depletion
experiments .) 218

5-4. Description of root systems used in study three,
where roots were subjected to cutting. 1996 depletion
experiments

5-5. Temperature and Dissolved Oxygen concentrations in
the nutrient solution during consecutive experiments
of P and K depletion by pine roots. 1995 studies 122

5-6. Temperature and Dissolved Oxygen concentrations in
the nutrient solution during consecutive experiments
of P and K depletion by pine roots. 1996 studies 123

5-7. Comparison of constant rate of depletion (r) and
C for P and K depleted pine roots treated with 1%
NaOCl. Study four

vix

128

5-8. Covariance analysis for ? and K influx (Ln( Influx))
by pine root systems of studies 1,2 and 3

5-9. Mean P and K influx for woody roots and whole

roots of 12-year-old slash pine. These untransformed
values (jumol cm'1 h'1) are a summary of the three
separate studies performed in 1995 and 1996. Cut
refers to the studies when white roots were removed
by cutting while waxed refers to the study where white
roots were covered with wax

129

viii

LIST OF FIGURES

Figure page

2- 1. A series of Michaelis-Menten curves 28

3- 1. The root regeneration system 42

3-2. Nutrient uptake root chamber 44

3-3. In-sit u nutrient uptake system 47

3-4. Mariotte Flask system 50

3-5. Phosphorus and Potassium depletion by seventeen root
systems of Pinus elliottii var. elliottii. The roots
were gassed with air during the experiment 59

3-6. Phosphorus and Potassium depletion by seventeen root
systems of Pinus elliottii var. elliottii. The roots
were gassed with N2 gas during the experiment 60

3- 7. Nutrient solution temperature and Oxygen saturation

in root chambers with lateral root systems of Pinus
elliottii var. elliottii

4- 1 . Potassium depletion by seventeen root systems of

Pinus elliottii var. elliottii. under aerobic and
hypoxic conditions

4-2. Potassium depletion by root systems of Pinus

elliottii var. elliottii. under hypoxic conditions 86

4-3. Potassium depletion by seventeen root systems of
Pinus elliottii var. elliottii. under series of aerobic
conditions

4-4. Phosphorus depletion by root systems of Pinus
elliottii var. elliottii . under aerobic and hypoxic
conditions on

IX

Figure

page

4-5. Phosphorus depletion by seventeen root systems of
Pinus elliottii var. elliottii . under series of
aerobic conditions 90

4-6. Phosphorus depletion by root systems of Pinus

elliottii var. elliottii. under hypoxic conditions 91

4- 7. Potassium uptake potential of slash pine roots when
subjected to cycles of aerobic and hypoxic conditions .... 96

5- 1. Calculation of potassium depletion due to mass flow
by eight root systems of Pinus elliottii var.

elliottii 126

5-2. Calculation of phosphorus depletion due to mass flow
by eleven root systems of Pinus elliottii var.
elliottii

5-3. Calculation of phosphorus depletion due to mass flow
by eleven root systems of Pinus elliottii var.
elliottii. The root chambers were gassed with N2 gas 131

x

Abstr^c-t of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ABSORBING SURFACE AREA OF SOUTHERN PINE ROOT
PHOSPHORUS AND POTASSIUM UPTAKE BY ROOT SYSTEMS

YEAR-OLD SLASH PINE TREES

SYSTEMS:

OF TWELVE -

By

Jose A. Escamilla-Bencomo
December, 1997

Chairman: Nicholas B. Comerford

Major Department: Soil and Water Science Department

Our understanding of ion uptake by pine trees is
principally based on studies of white roots of seedlings.
However, root systems of trees consist of white, brown (woody
and nonwoody) and mycorrhizal roots including external
hyphae. It is unclear whether brown roots are functional in
nutrient absorption. However, brown and woody roots may
contribute over 80% of the fine-root system of slash pine

{Pinus Engelm. var. elliotii) . The aim of this

research was to study P and K uptake by root systems of
twelve-year-old slash pine growing under field conditions.

The first objective of my research was to develop a
field method for measuring nutrient uptake by roots of trees.
The design included (i) a root chamber, (ii) a system for

xi

controlling the 02 level of the root chamber, and (iii) a
Mariotte flask system.

The second objective was to (i) compare P and K
depletion by lateral root systems of slash pine when the
roots are subjected to a short-term, hypoxic treatment, and
( ii ) to document the ability of roots to absorb P and K
following the removal of the hypoxic treatment. N gas was

used to achieve hypoxic conditions during the experiments
(<50 p M 0^ ) . Slash pine is grown extensively on soils where

surface horizons are subject to short-term hypoxic conditions
due to seasonal, fluctuating watertables. I demonstrated that
that pine roots grown in aerobic soil conditions were capable
of p uptake during short-duration hypoxic conditions. In
contrast, K depletion was totally inhibited. Once the hypoxic
condition was corrected, uptake of both P and K resumed.

The final objective of my research was to compare P and
K influx rates between whole and woody roots of slash pine,
while evaluating the mechanism of nutrient uptake by woody
roots through the use of N2 gas as a metabolic inhibitor of

ion absorption. I concluded that woody roots absorb P and K
and that rates of ion uptake were similar to whole roots. I
also documented an active K uptake mechanism by woody roots.

The results indicated that estimates of the surface area
of woody roots and sensitivity of K uptake under aerobic and
hypoxic conditions, should be included in nutrient uptake
models of slash pine ecosystems.

xii

CHAPTER 1

GENERAL INTRODUCTION

Water and mineral nutrient limitations occur frequently
in southern pine forest (Neary et al., 1990; Peters, 1990).
These limitations are compounded by the fact that intensive
forest harvesting and site preparation can change the
nutrient capital and availability of nutrients for the next
rotation of trees (Morris and Miller, 1994). Therefore,
nutrient management in forest plantations increasingly is
focusing on matching nutrient supply, from soil or
fertilizers with nutrient demand by crop (Smethurst and
Nambiar, 1990). Since nutrient availability is a limitation
to productivity, then an understanding of nutrient uptake,
allocation and use by pine trees is fundamental to predicting
the consequences of harvesting, as well as to ameliorating
negative harvesting effects.

Predictions of nutrient uptake and demand by the tree at
the root surface can be contrasted with the ability of the
soil to supply nutrients. Tree nutrient uptake can be
described as a set of processes occurring simultaneously in
three phases (Comerford et al., 1994a). The first phase is
the effect of processes that release nutrients from their
solid form to the soil solution. The second phase is the
nutrient movement through the soil solution to the absorbing
surface of the tree (via mass flow and/or diffusion). The

1

2

last phase is the uptake process at the absorbing surface of
the tree. If the rate of absorption is less than supply by
mass flow nutrients will accumulate at the root surface.
Whereas a zone of depletion will occur if mass flow is less
than uptake (Barber, 1984; Nye and Tinker, 1977). In the case
of depletion, uptake is an active process because the plant
must transport these nutrients into cells against a
concentration gradient (Clarkson and Grignon, 1991). The
absorption of nutrients by roots depends on aerobic
metabolism to produce high energy potentials to drive active
membrane transport processes (Mengel, 1974). Sudden oxygen
deprivation reduces or elimiminates active nutrient uptake.
Hence uptake is governed by root kinetics, and is often
described mathematically by Michaellis-Menten kinetics
(Claassen and Barber, 1976).

Total nutrient accumulation of plants can be predicted
reasonably well from nutrient uptake model calculations.

These calculations are based on parameters such as root
kinetics of the plant species, and nutrient concentrations in
the soil solution (Barber and Cushman, 1981; Nye and Tinker,
1977). Such predictions from model calculations are less
successful for plants grown at low nutrient supply (Jungk and
Claasen, 1986) and in tree species under field conditions
(Smethurst and Comerford, 1993). Calculations of average
uptake rates over the entire root system of a plant are
inappropriate in tree species under field conditions because
(i) tree roots are irregularly distributed in the soil

3

(Comerford et al., 1994b), (ii) tree root systems are
comprised of root zones with different morphology and
physiology (Clarkson et al., 1978) and (iii) soil supply of
nutrients is highly heterogeneous both spatially and
temporally (Lechowiez and Bell, 1991; Koch and Matzner,

1993) .

Root kinetic parameters have been studied for most ions
either with excised roots, or with whole root systems of
intact plants (reviewed by Epstein, 1972; Nye and Tinker,
1977). However, these investigations have been largely
restricted to annual species or herbaceous perennials. Pine
root systems include from white roots to mycorrhizal roots to
brown roots with secondary growth (woody roots).

Consequently, differences in root morphology could influence
nutrient uptake to varying extents. There is no question that
white roots and mycorrhizal roots of forest tree species are
active in nutrient uptake. However a main question is whether
woody roots function in uptake, and if so, is it active
uptake (VanRees and Comerford, 1990). The question is
important because of the tremendous woody fine root surface
area in pine trees (Comerford et al., 1994a). Information of
this kind is very limited for tree species (Atkinson and
Wilson, 1979; Bhat, 1981), and only few root kinetics
parameters have been determined for commercial forest tree
species, and in all cases for tree seedlings (Van Rees et
al., 1990; Kelly and Barber, 1991).

4

Pine roots of the southern United States encounter
shallow water tables that severely reduce soil-02 supply to
large parts of the root system (Comerford et al., 1996).
Forested Spodosols of the Southeastern lower Coastal Plain
typically have perched, fluctuating water tables. Sudden
oxygen deprivation reduces or eliminates active nutrient
uptake of white roots (Fisher and Stone, 1990b; Topa and
McLeod, 1986b). A main question is whether under field
conditions if a short-term reduced 02 supply will affect

permanently pine roots ability for ion uptake from the soil
solution.

Clarkson et al. (1978), stated that the nutrient uptake
effectiveness of a root system depends on (i) the relative
amounts of different absorbing surfaces present, (ii) the
nutrient absorption rates of each type of surface, (iii)
differential response of the absorbing surfaces to
environmental variables. A major limitation to test the above
hypothesis with roots of forest trees is the lack of
methodology for field studies of nutrient uptake.

This dissertation describes a nutrient uptake system
that I had to implement to document if (i) woody fine root
function in nutrient absorption, and (ii) differential

response of fine pine roots to changing aeration in the soil
environment .

The overall objective of my research was to investigate
absorption of phosphorus (P) and potassium (K) by the root
surface area of twelve-year-old slash pine trees under field

5

conditions. Chapter 2 is a literature review of the
definition of the nutrient-absorbing surface of trees with a
discussion of the active uptake process in the context of
root kinetics . Chapter 3 is the description and testing of a
field method that I developed to measure nutrient depletion
by roots of trees under field conditions. Chapter 4 addresses
the effect of low oxygen on the uptake of P and K by roots of
pine trees under field conditions. Chapter 5 is a study of P
and K depletion by woody roots of pine trees under field
conditions. Each of the following 4 chapters is an
independent manuscript for journal publication. I chose to
study K and P uptake because (i) p availability limits growth
of pine (Pritchett and Comerford, 1982; Waring, 1981); ii) p
and K uptake by slash pine seedlings were successfully
predicted under field conditions (Van Rees at al., 1990a;
Smethurst and Comerford, 1993), (iii) P and K uptake by roots
have contrasting responses to low oxygen supply, and (iv) K
and P have contrasting nutrient cycles.

CHAPTER 2

NUTRIENT-ABSORBING SURFACES OF TREES AND THE NUTRIENT UPTAKE

PROCESS

Introduction

This review addresses the question of nutrient uptake
and focuses on the root-hypha absorbing system of the plant.
It is based mainly on a review article published by Comerford
et al. (1994a), of which I was a coauthor. This review
includes sections from that article, which are relevant to
the topics covered in my dissertation.

Clarkson et al. (1978), stated that the nutrient uptake
effectiveness of a root system depends on (i) the relative
amounts of different absorbing surfaces present, (ii) the
nutrient absorption rates of each type of surface, (iii)
feren-tial response of the absorbing surfaces to
environmental variables .

I address these issues in relation to pine trees by
considering first the definition of the nutrient-absorbing
surface of trees. Once the root categories are defined, this
review will address what are the pathways of nutrient uptake
for different root surfaces and whether all categories have a
nutrient uptake function. Then I discuss the evidence for
nutrient uptake by fine brown and fine woody roots, and the
potential importance of this uptake in pine forests based on

6

7

the account that they are a large proportion of the total
fine root surface area. In turn I discuss the active uptake
process in the context of Michaelis-Menten kinetics, the
biofeedback between the plant and the uptake process, and the
response of the root uptake process to changing soil
environments, specifically under low-oxygen or hypoxic
condition. Last, I framed this information in the the overall
objectives of the research described in the following four
chapters .

Definition of Absorbing-Surfaces of Trees

A perennial, dicot, woody root system is interesting
because secondary xylem growth occurs, and this "woody"
portion of the root system may be the major contributor to
the fine root population. Fine roots in pine trees are
defined as roots < 2 mm in diameter (Van Rees and Comer ford,
1986). As an illustration, mature stands of Pinus elliottii
Engelm. var. elliottii (slash pine) had fine roots accounting
for about 90% of the total root length (Van Rees and
Comerford, 1986), while mature Pinus taeda (loblolly pine),
had 67% of its root system surface area in roots with
diameters less than 1.3 mm (Kramer and Bullock, 1966).

A perennial dicot, woody, fine root system can be
described by considering the types of roots that constitute
it. The following fine root classification (Table 2-1) is
based on the definitions of Sutton and Tinus

(1983), the

8

Table 2 1. Categories of fine-roots in a perennial, dicot,
woody-root system (Comerford et al., 1994a).

ROOT CATEGORY

DESCRIPTION

LATERAL ROOTS
White Roots

New primary lateral roots white in
color

Brown Roots
Primary

Lateral roots, still having primary
growth but the epidermis /cortex has
turned brown from either suberin/
lignin deposition or cortical cell
decay

Woody

Roots showing evidence of secondary
growth by having secondary xylem

SHORT ROOTS

A root of diminutive length (often
less than 2 mm) arising from a
lateral root. It may or may not be
bifurcated and is not considered
capable of further growth

Mycorrhizal

A short root infected with
symbiotic fungi. May have a fungal
mantle surrounding it if
ectomycorrhizal

Non-mycorrhizal

A short root not infected with
symbiotic fungi

ROOT HAIRS

A small tubular outgrowth from an
epidermal cell.

EXTRAMATRICAL HYPHAE

Hyphae and rhizomorphs emanating
from the mycorrhiza and distributed
in the soil.

9

descriptions of Chung and Kramer (1975) and Kramer and
Bullock (1966). Most studies of fine-root systems have
generally not recognized nor included all these categories in
their analyses .

However, most root studies in forest species have
recognized that brown roots are the dominant fine-root
category while white roots represent less than 1% of the
surface area (Kramer and Bullock, 1966; Van Rees and
Comerford, 1990). The majority, or even all, of the tree
fi-ne-roo't system can be brown depending on seasonal and
environmental conditions (Hendrick and Pregitzer, 1992).
Loblolly pine, growing in the Piedmont of North Carolina
(Kramer and Bullock, 1966), had the following surface area
distribution (i) white roots 0.3%; (ii) mycorrhizas (not
including extramatrical hyphae) 2.7%; (iii) brown roots <1.3
mm in diameter 64.1%; and brown roots >1.3 in diameter 32.7%.
In this study, as in most root biomass studies, it is unclear
if the brown root category is woody or just brown in color
with no secondary growth (primary).

Fine roots of most species common in forests (e.g.,
spruce, fir, pine, beech, oak, birch, poplar) are colonised
by ectomycorrhizal fungi (Harley and Harley, 1987). Other
tree species (e.g., elm, ash, maple, fruit trees) are
colonised by endomycorrhizal (arbuscular mycorrizal) fungi
(George and Marschner, 1996). In a number of tree species,
both ectomycorrhizal and arbuscular mycorrhizal, can occur
simultaneously (Lodge, 1989). Some roots of all trees, in

10

particular the fast-growing long roots, usually remain non-
mycorrhizal. The surface area of extramatrical mycorrhizas
found in pine forest might very well dwarf the surface area
of the other root categories. Under controlled conditions
external mycelium of mycorrhyzal pine seedlins accounted for
75% of the total surface area (Rousseau et al., 1994)
however, this contribution is likely to be lower under more
natural conditions due to presence of competitors and
predators . With current technology it is not possible to
measure mycorrhizal hyphae length or surface area under field
conditions. Therefore there are no reliable estimates of
extramatrical mycorrhizal hyphal length or surface area in
natural environments.

Root hairs are part of the epidermis of the root. They
are involved in water and ionic exchange between the plant
and the growth medium. Therefore, when root hairs are present
the root surface can be considerably enlarged depending on
root hair length and density of hairs on the epidermis. Root
hair length varies between 80 and 1500 pm, with diameters of
5-20 pm, depending on species and cultivars (Dittmer, 1949;
Caradus, 1979). Although the location of the root hair zone
is generally known in some plant species (1 to 4 cm long and
behind the zone of active root elongation (Jaunin and Hofer,
1986), root hairs are not always present. Little work has
been done to investigate the abundance of root hairs on pine
roots. However Kozlowski and Scholtes (1948) reported root
hairs on roots of 7 -week-old loblolly pine seedling with hair

11

densities of 217 hair cm"2 Roots developing an
ectomycorrhizal structure will not have root hairs (Mexal et
al . , 1979), but root hairs are common on some endomycorrhizal
roots (Lyford, 1975).

Most studies on nutrient absorption by root hairs have
been done in agronomic crops where they have been shown to be
important for increasing root surface area. (Itoh and Barber,
1983). Still, their contribution to the root system surface
area of trees is largely unkown.

Nutrient Uptake for the Different Root-Surfaces of Trees

Once the root categories are defined, the next question
is whether all categories have a nutrient uptake function. I
will address this question by first describing the pathways
for nutrient uptake and then reviewing the nutrient uptake by
different root surfaces of trees.

Pathways of Nutrient Uptake

In any root there exists two pathways for nutrient
uptake. These are the apoplastic and symplastic pathways. As
defined by Dumbroff and Pierson (1971) the apoplastic pathway
is a route through the apoplast where the ion does not have
to pass a selectively permeable membrane but moves through
intercellular space, while the symplastic pathway is where
the ion must pass a semipermeable membrane to enter the
cytoplasm and moves radially in the cytoplasm by passing from
cell to cell via plasmodesmata. Transport of water and ions

12

through the apoplast of the root outside the endodermis is
only stopped by Casparian bands on the inner cortical layer
in the endodermis .

A white root includes the area from the root tip to some
point in the zone of primary tissues. Therefore, a white root
can have no endodermis, can have a developing endodermis, and
can have a fully developed endodermis; depending on position
along the root. In the youngest white roots, the casparian
bands are absent but the xylem vessels are immature, so only
very limited apoplastic entry to the xylem is possible. As
the root matures the casparian band becomes continuous and
forms a barrier to apoplastic nutrient flow.

In some cases a Casparian band can develop in the
hypodermis, sometimes referred to as the exodermis (Peterson
et al., 1981; Perumalla and Peterson, 1986). This has been
shown in some plants to be an effective barrier to apoplastic
flow (Peterson et al., 1981). Yet, it is not clear if this
strip is fully equivalent to that of the endodermis as
proposed by Shone and Clarkson (1986). The presence and
degree of development of the suberin deposition is related to
stress, with high water stress causing it to form closer to
the tip of the root.

In brown roots, a fully developed endodermis can be
compromised by short and lateral root development leaving a
partial pathway for apoplastic water and nutrient flow where
the roots exit the pericycle through the endodermis. If
secondary xylem growth occurs, it can cause corruption of the

13

endodermis with the cortex progressively sloughing away
(Troughton, 1957). With time, cork tissue with a cork cambium
develops, becoming another possible barrier to apoplastic
flow.

In short roots with ectomycorrhiza, the fungal sheath
becomes the surface area for absorption of both water and
nutrients . Ectomycorrhizae may have a well developed sheath
which forms a tightly packed layer between the soil and root
extending around the root apex, so that the zone of
ift^ximum water and mineral uptake of the root is separated
from the soil by fungal tissue. Therefore all substances
absorbed from the soil must pass through the fungal sheath or
mantle before coming in contact with the outer cells of the
root (Harley and Smith, 1983). Like the root tissue, the
fungal sheath is divided into symplastic and apoplastic
compartments (Ashford et al., 1988; 1989). Cellufluor has
been used to test the apoplasmic permeability of the fungal
sheath in Pisonia mycorrhizae. Ashford et al. (1988)
documented that the apoplastic tracers penetrated as far as
the root epidermal cells in tip regions in the immediate
vicinity of the mycorrhizal root cap. Behind this area, where
both the root and mycorrhiza are likely to be active, the
sheath was impermeable to the tracer. Blockage of the
apoplast was believed to be at the interface between fungus
and root. Therefore it was concluded that the symplastic
pathway within the fungus was very important at this stage
with the hartig net in a position to deliver water and

14

nutrients into the intercellular cortex. Any obstructions to
apoplastic flow that exist at that point of entry as
described above then apply. In short roots with
endomycorrhiza the fungal mantle is not present.

Root hairs generally arise from the epidermis. A root
hair may increase the absorbing surface area and hence
increase the ion and water absorption per unit length of
root, but nutrients must still pass a symplastic route. When
root hairs develop, a cuticle has been known to develop over
them and the epidermis (Curl and Truelove, 1986). The
hydrophobic waxy components of the cuticle may increase
resistance to water and nutrient flow, but experimental data
are lacking making definitive statements difficult. The
relative rapid deterioration of the cytoplasm of root hairs
of maize roots suggests that root hairs may not function for
very long (Fusseder, 1987).

Nutrient Uptake by Root TrPPs

Most of the research on root nutrient uptake by trees
comes almost exclusively from experiments with tree seedlings
under laboratory or greenhouse conditions. There is no
question that white 'roots, short roots and mycorrhizal hyphae
are active in nutrient uptake of trees (George and Marschner,
1996). However much less is known about nutrient absorption
by intact older fine roots of perennial plants in field
conditions . The main question in nutrient uptake by trees is
whether brown roots function in uptake, and if so, is it by

15

active uptake? The importance of this question for trees is
obvious when one considers the tremendous root length and
surface area in this category. For illustration Comerford
and Neary (1991) used nutrient uptake models to show that in
a semi-mature slash pine stand, the only way one can predict
phosphorus (P) root uptake based on observed P soil solution
concentrations, was to either adjust mineralization rates or
not allow a very large part of root system to function in P
uptake. This would suggests that a significant portion of the
fine root (brown-woody) system is not absorbing P. It might
be that our idea of an "effective" root length density is
incorrect. Yet, one would normally include these potentially
non-absorbing roots in estimates of root length density when
using nutrient uptake models. While the lack of a mechanistic
definition of an "effective" root length density has not been
a problem in agriculture crops, its importance is more
critical in tree root systems. Not only are tree roots
irregularly distributed in the soil (Comerford et al.,

1994b), but tree root systems also comprise roots with very
different morphology and physiology (Table 2-1). Also, the
plant available nutrients are distributed with higher
heterogeneity in forest soils (Lechowiez and Bell, 1991).

In forest trees, root biomass represents less than 20%
of the total biomass (Keyes and Grier, 1981). This relatively
lower root biomass is mainly caused by the high turnover
rates of tree fine roots. Fine roots of forest trees have a
short-life span (George and Marschner, 1996), and a large

16

amount of nutrients is used each year for the growth of new
roots (van Praag et al., 1988; Eissenstat and Van Rees,

1994). The yearly nutrient uptake in adequately growing
forest depends on the tree species, but to take up those
nutrients, trees need efficient root systems adapted to the
usually nutrient-poor forest soils (Koch and Matzner 1993).

If absorption of nutrients by roots of trees is
confined to new root growth, is it the browning of roots
(accompanied by suberization) that curtails the nutrient
uptake activity of roots of trees? Long lived root elements
would seem to be advantageous for a plant given energy and
nutrient availability considerations. Still the question of
root longevity may be viewed in a different perspective.
Maintenance of long lived, fine root elements, may in fact be
a poor evolutionary strategy. It may be less energetically
expensive to form a new root element which systematically
explores new microsites for relative inmobile elements such
as phosphorus, than to maintain a long lived absorbing root
element in sufficient density to meet plant nutrient demand.
Studies have demonstrated that either alternative, can be
favorable to the plant.

With annual plants it is well known that uptake rates of
roots change with age (distance along the root axis) (Bowen,
1969; Barber, 1984; Marschner, 1991) and that the important
factor in zonality of ion uptake is the structural
differences m walls of epidermal cells and in peripheral
cells of the cortex, endodermis and stele. For both annual

17

and perennial plants the term "root browning" has been
loosely applied to several developmental processes including
those of root dormancy, cortical breakdown and periderm
formation (Sutton and Tinus, 1983). Field observations of
growing root systems generally corroborate the common
assumption that only the new growing root elements are free
of the browning (darkening) in color, considered to be a
development of suberin layers in older roots (Head, 1967).
Even though suberin is impermeable to water, the deposition
of suberin layers is not always continuous, particularly if
they are deposited in the walls of the exodermis. Even in the
case of periderm formation, water could still enter through

lenticels, wounds or other breaks or discontinuities in the
suberin layer.

The most detailed investigation related to the
histochemical nature of root browning was done in jack pine
roots and eucalyptus by McKenzie and Peterson (1995a and
1995b). Root browning was not linked to suberization but was
caused by deposition of condensed tannins in the walls of all
cells external to the stele. The term suberization should not
be associated with browning, since the white zones of jack
pme and eucalyptus roots were not colored internally,
despite the presence of suberin in the endodermis of jack
pme and m both the endodermis and exodermis of eucalyptus.
Therefore, a white root is not necessarily an unsuberized
root, nor is a suberized tissue layer necessarily brown. This

study documented that an apoplastic tracer was unable to
penetrate the periderm in secondary roots .

18

Evidence of Nutrient Uptake in Brown Roots

An important question in nutrient uptake is whether
brown roots function in uptake, and if so, is it by active
uptake? Uptake by brown (woody?) roots has been reported, but
the data base is not extensive. Citrus and Pinus taeda have
both been shown to have brown roots that absorb P04 (Crider
1933; Chung and Kramer, 1975). The work by Chung and Kramer
even suggested that an effective ion barrier existed allowing
selective ion absorption. Kramer (1946) suggested that the
selective barrier was either the cork cambium, the vascular
cambium or both. Similar uptake rates for Prunus were
subsequently reported (Atkinson and Wilson, 1979). The same
study by Crider (1933) also indicated active uptake of N03 by
woody (brown?) roots in Citrus, again with an effective ion
selective barrier implicating active uptake.

The rate of K uptake by brown (woody?) roots of Prunus
and Malus (Atkinson and Wilson, 1980) and slash pine (Van
Rees and Comerford,- 1990) was shown to be lower or similar to
the uptake rate of white roots. In the work on Pinus
elliottn (Van Rees and Comerford, 1990), K accumulated at a
rate faster than that attributable to mass flow, suggesting
an active uptake mechanism

19

So there appears to be some evidence that, as with white
roots and short roots , brown roots are capable of nutrient
uptake. However, it is not clear if uptake is dominated by
mass flow or active uptake mechanisms. If uptake in brown
roots is via apoplastic pathways, these pathways could
include lenticels, wounds at the base of branch roots
(Addoms, 1946) and breaks in the endodermis (Dumbroff and
Pierson, 1971; Peterson et al., 1981). However, where active
uptake is indicated it is not clear what the barrier to
apoplastic flow is.

All of the research on root nutrient uptake by brown
roots comes almost exclusively from experiments with either
sections of roots (Chung and Kramer 1975) or intact roots
from tree seedlings under laboratory or greenhouse
conditions. Also in most studies, is it unclear what category
of brown roots was being investigated.

Model experiments by Bhat (1981, 1983) showed that the
rate of P uptake by whole root systems of 3.5 years-old
apples trees was 2.5 lower than root systems of apple tree
seedlings . In both cases the trees were grown under
greenhouse conditions. Unfortunately such detailed uptake
studies with root trees in field conditions are rare. Up to
now, only very few attempts have been made to develop
techniques to study the nutrient uptake of root systems in

forest sites (Glavac and Ebben 1986; Marschner et al., 1991;
Niederholzer et al., 1994).

20

Michaelis-Menten Kinetics as a Description of Active Nutrient

Uptake

Appropriateness of the Equation for Root Uptake

Nutrient concentrations in the cytoplasm of soil grown
roots are usually much higher than in the soil solution at
the rhizoplane. Metabolic energy is required for nutrient
uptake to occur against this chemical gradient. The transport
of nutrients across cell membranes is enzymatically mediated
and nutrient specific (Nye and Tinker, 1977), but chemical
details about transport to the enzyme carrier, the nature of
the enzyme, movement via carrier, and release inside the cell
remain largely matters of speculation.

Because active nutrient uptake at low concentrations
(e.g. < 1 mM) is enzymatic, the principles of enzyme kinetics
have been applied to the mathematical description of nutrient
uptake within the appropriate ranges of concentrations. L.
Michael is and M.L. Menten in 1913 developed a general theory
of enzyme kinetics, which was later extended by G.E. Briggs
and J.B.S. Haldane (Lehninger, 1970). They considered the
situation where an enzyme combined with a substrate, then
dissociated to release the product. Both steps were
associated with forward and reverse rate constants. Once
simplifying assumptions were made, this situation resolved
mathematically to an asymptotic function between the velocity
of the reaction and the concentration of the substrate, i.e.

v= Vmx*S/(Ka +S)

(1)

21

Where V is the velocity of the reaction, S is the
concentration of the substrate, Vmx is the maximum velocity
attainable, and K is the S at half V „ and is also related to
the rate constants of the reactions and thereby the affinity
of the enzyme for the substrate. The constants Vmx and Ka are
normally empirically fit but can be calculated if forward and
reverse rate constants are known. The function is known as
the Michaelis-Menten equation and describes the saturation
phenomenon observed in enzyme studies at low concentrations.

Claassen and Barber (1976) and Nielsen and Barber
(1978), applied this function to nutrient uptake by roots,
modified it by including a minimum concentration below which
there was no further uptake ( CKln ) , and substituted the terms
I (inflow to a root) for V, and C (concentration) for S,
i.e.

1 = *.x* (C-C^J / (Km +( C-C (2)

Hence, this function is a description of nutrient uptake
using Michaelis-Menten kinetics (Figure 2-1).

Although this function has fit observations of inflow
versus concentration, i.e. 1(C), for many nutrients and root
types, there are several reasons why observations might
deviate from this theoretical form. Nutrient uptake may be
more complex than the simple enzyme system on which
Michaelis-Menten kinetics are based. For instance, more than
one enzyme or type of enzymes may be involved, diffusion to
and from the carrier enzyme might be a limitation, energy
supply to the enzyme may be a limitation, the rate constants

may be nonlinear with substrate concentration (Lehninger,
1970), and more than one uptake mechanism might be at work
(Nye and Tinker, 1977).

22

This latter remark refers to the phenomenon of
multiphasic uptake (Epstein, 1972; Hodges, 1973; Nissen,

1980) whereby Michaelis-Menten kinetics seem to be an
adequate description of uptake at normal soil solution
concentrations, e.g. < 1 mM, but at higher concentrations the
process is not active and hence is unaffected if induction of
the carrier enzyme is blocked (Omata et al., 1989; Oaks,
1992). However, the high concentration at which this second
mechanism dominates is much higher than the concentration of
most nutrients under natural and most managed conditions.

With the exception of N03 under limited soil management
conditions, multiphase uptake is likely to be of no concern
for understanding growth under low fertility.

A further complication is that efflux of nutrients from
roots may not be related to the activity of the uptake system
because it is thought to be largely a diffusive process
dependent upon internal and external nutrient concentrations
and the integrity of the membrane.

Although Michaelis-Menten kinetics remain a useful
description of nutrient uptake by roots at most soil solution
concentrations, it is best thought of as an empirical
description of nutrient uptake relative to solution
concentration, 1(C), than a theoretical approach. While based
in theory, its application will only approach a theoretical

23

level when the process is better understood, when active and
passive uptake mechanisms are defined for each root category,
and when feedback mechanisms regulating uptake are
mechanistically incorporated.

Active Root Uptake Mechanism and Root Anatomy

Most evidence suggests that an active mechanism is
responsible for the bulk of ion uptake by roots, but many
physiologists believe that passive mechanisms also provide at
least a small portion of the ions that are absorbed by roots
(Brouwer, 1965). In this review I am focusing on P and K
uptake by roots of trees; therefore I will only discuss the
uptake mechanism of the above mentioned nutrients .

Phosphorus uptake by roots is active as measured by
electrochemical gradients (Ulrich-Eberius et al., 1981) and
appears to be operated by the pH gradient across the
plasmalemma as indicated by Dunlop (1989) and Bowling (1981).
Phosphate flux in the roots is affected by transpiration
which ensures the flow of solutes in plant tissues. Active P
uptake by roots of trees has been demonstrated for loblloly
pine (Topa and McLeod, 1986b).

Potassium uptake by root tissue is active (Higinbotham
et al., i967). This was proved by the use of inhibitors of
energetic metabolism. In sunflower and wheat roots, K uptake
was inhibited by 2,4-DNP (Pettersson, 1981). Antibiotics of
the ionophore" type showed a stimulatory effect on K uptake.
Gramicidine-D and nigericine stimulated K influx into the

24

roots up to from 4- to 8-fold (Hodges et al., 1971). The
ability of roots to regulate influx decreases with increasing
external ion K concentrations suggesting a feedback mechanism
(Petterson and Jensen, 1979). Active K uptake by roots of
trees has been demonstrated for plum (Rosen and Carlson,

1984) and for slash pine species (Fisher and Stone, 1990b;

Van Rees and Comer ford, 1990).

Ion transfer across the plasmalemma and ion influx or
efflux from the apoplast into the symplast is directed by
regulation of active processes of metabolic reactions. There
is much experimental evidence for the prevalence of
symplastic over the apoplastic transport of ions. Anderson
(1975) assumed that over 90% of the ions passed into the
stele moved through the symplast-apoplast mechanism (not
plasmodesmata) . For instance P-phosphate ions are transported
mainly through the symplast (Clarkson and Sanderson, 1974).
However starving roots first take up phosphate by diffusion
into the apoplast. After the saturation of the apoplast and
when the steady-state of the external solution-tissue system
is exceeded, the transport proceeds actively (symplastic)

( Ulrich-Eberius et al., 1981). in comparison, K ions are

transported mainly through the symplast (Lauchli et al.,

1973) .

Only few studies have examined the potential role of
brown (woody?) and white roots roots in water and nutrient
uptake. Moon et al . , (1986) found that the uptake of ions and
water in roots of mangrove occurred primarily through the

25

symplast of the younger (nonwoody?), intact roots (apoplastic
flow was estimated at 1%). Furthermore, Moon et al., (1986)
found that disturbing roots by removing them from the soil
increased estimates of apoplastic water flux by 57X. However
time was not allowed for roots to heal from the injury
sustained nor were injuries sealed with wax, thus allowing an
abnormally high number of direct entry points for apoplastic
water flow. Van Rees and Comerford (1990) reported that fine
woody roots of slash pine seedlings were nearly as effective
in water uptake as were entire root systems (nonwoody and
woody). However, Van Rees and Comerford (1990) found that
water absorption by fine woody roots with cut ends (the cut
ends had several day to callous) was similar to that of
woody-roots coated with paraffin wax. The dependence of flow
upon development of suberized layers in the endodermal cell
is not crucial for apoplastic movement. There are known to be
regions of discontinuity in the Casparium band in roots
through which unhindered transport in apoplast might occur.
Such regions occur at the root where the endodermal cells are
not yet mature (Robards and Jackson, 1976) and sites of
secondary root initiation. During their development, laterals
root cause a gap in the structure of the endodermis. They
arise from meristematic initials in the pericycle; as they
divide, a dome of cells is produced and the endodermis divide
to form the epidermal layer of the emerging lateral (McCully,
1975). At the junction of the lateral and the endodermis
there is a ring of cells which lack Casparian bands (Dumbroff

26

and Peirson, 1971)) which seems to permit apoplast continuity
between the cortex and the stele. Apoplastic tracers were
seen to enter the stele at this point (Peterson et al., 1981)
and water uptake in the zone of lateral emergency of barley
roots was higher than elsewhere and very responsive to
changes in transpiration rate (Sanderson, 1983). Therefore,
probable mass flow pathways are provided at one stage during
branch development. Yet, the physiological significance of
this apoplastic pathway and a potential passive uptake of
nutr^-ents has not been studied in field conditions. All of
the research on the mechanisms of nutrient uptake by roots
comes from experiments with tree seedlings under laboratory
or greenhouse conditions .

Importance of Root Uptake Kinetics Under Field Conditions

— Kg— and Under Field Conditions

The importance of the Michaelis-Menten parameters under
field conditions will depend mainly on the concentration of
the nutrient in solution at the absorbing surface (Cla). The
profile of nutrient concentration in solution relative to
radial distance from the root can take three basic profiles.
When plant uptake is equal to the supply by mass flow, is
equal to the bulk soil solution concentration. If demand at
the root surface is greater than supply by mass flow in the
soil, a depletion zone will develop and Cla will be less than
the bulk soil solution. The higher the plant demand and the

27

slower the rates of mass flow and diffusion supply, the lower
will be Cla. However, if mass flow supply is more than the
demand at the root surface, a surplus will build at the root
surface and Cla will be greater than the bulk solution
concentration .

The sensitivity of nutrient uptake to the value of the
Michaelis-Menten parameters depends on Cla. When mass flow is
a dominant source of supply and is approaching or exceeding
plant demand, then Cu is high, approaching the solution
concentration at which 1^ occurs. Under these conditions,
uptake will be dependent on the value of lB However, if mass
flow does not contribute much to soil supply and the supply
by diffusion is significantly less than demand by the plant,
Cla becomes very low. If Cla gets low enough, it gets to the
point on the curves where they begin to converge and now
plant uptake is not sensitive to i^. There is a similar but
reverse explanation to a plant's uptake sensitivity to CM As
cu approaches the value of C^, plant nutrient uptake will be
very sensitive to the value of (Figure 2-1, case A)
However, when Cla is much higher than (C^ is very low
relative to Cla), changes in will have little effect on
plant uptake. (Figure 2-1, case B). For illustration Fescue
and Canary grass roots with low I_ values relative to corn
(Figure 2-1), can deplete P from the soil solution under
lower soil p concentrations when compared with corn roots.

28

Imax and C min evaluation (Comerford et al. , 1994a)

Concentration at absorbing surface ( i/Mol cm 3 )

Figure 2-1. A series of Michaelis-Menten curves. Case A
represents the condition where the solution concentration is
so low that sensitivity to 1^ is unimportant. Case B
represents the condition where the influx would be sensitive
to the value of 1^.

29

Conversely at high soil P concentration corn roots will
deplete P faster from the soil solution because higher 1^
values when compared with Fescue and Canary grass roots. This
is extremely important since soil nutrient concentrations
will be closer to C^ values in forest soils. Although it is
possible that these parameters could affect a plant’s
competitive ability, little is known about their genetic or
environmental controls. Nevertheless, there is evidence that
Km is under genetic and environmental control, and that plants
continually presented with nutrients at concentrations
approximating Km will grow at high relative growth rates in
the absence of other limitations (Bloom et al., 1993).

Mycorrhizal hyphae may have lower C^ values than the
root with which they are associated, thereby enhancing P
uptake. However, work by Li et al. (1991) indicated that this
was not the case for Glomus mosseae colonizing Trifolium
repens because both surfaces depleted P to similar
concentrations. However knowledge of nutrient uptake by
ectomycorhizal fungi comes almost from experiments in the
laboratory where small tree seedlings are inoculated with
isolates of some species of mycorrhizal fungi. Some of the
fungi used in these experiments are not common in forests and
often substrates other than forest soils are used (George and
Marschner, 1996). Unfortunately, an excavation of mycorrhizal
fine roots from soils disrupts the hyphae growing into the
soil dissecting the mycorrhizal roots from its major uptake
organs. Therefore, uptake studies with intact mycorrhizal

30

fine roots are only possible in greenhouse conditions
(Anderson and Riegwickz, 1991).

The Iioax parameter has received more attention than K or

m

Cmin' probably because 1^ is more easily measured and it is

quite responsive to imbalances between nutrient uptake rates

and demand by the plant. For instance, Jungk et al. (1990)

found that the 1^ of soybean roots increased 376% due to a

decrease in pretreatment P concentrations from 30 to 0.03 mM,

while Km values decreased by only 60%. Jackson and Caldwell

(1992) have shown for field grown Agropyron desertorum roots

that the P uptake capacity (i.e., im) can be increased by at

least 73% in response to increases in P supply.

Temporal and Spatial Chances in Root Uptake Kinetics Under
Field Conditions

Not only do the uptake kinetics of a root change as a
root ages (i.e. with distance behind the root tip), but the
uptake kinetics of root systems may vary temporally over
short periods, e.g., 2 minutes (Ayling, 1993), or spatially
over small distances, e.g., 2 centimeters (Bowen, 1969).

The potential significance of spatial and temporal
variability of root, uptake kinetics for nutrient competition
between plants has been illustrated experimentally and
theoretically by Jackson and Caldwell (1992) and Caldwell et
al., (1992). They measured P uptake by excised roots of A.
desertorum grown in microsites to which water or water with
nutrients had been added. Plants were 0.5 m apart and control
and enriched microsites were located on opposite sides of

31

individual plants. Four days after treatment, uptake rates of
P by excised roots from enriched microsites were up to 73%
higher than those by roots from control areas when immersed
ini, 10 or 20 mM P, but there was no comparable effect on N
uptake in the range 50-1000 mM N. Model simulations indicated
that this mechanism for enhanced P uptake from fertile
microsites was more important than root proliferation.

Dighton and Harrison (1990) reported that, despite year
to year variation in the P uptake capacity of fine roots of
seven Picea sitchensis stands aged 14-26 years, increased
growth rates after the addition of p fertilizer were
accompanied by a decrease in the P uptake capacity by fine
roots in fertilized plots. The change in P uptake capacity
after fertilization was considered a better indication of P
deficiency in these stands than concentrations of p in
foliage.

Short-term feedback mechanisms may operate by regulating
the energy supply to ion carriers in cell membranes or by
altering the configuration of the membrane so that transport
of the ion to, with and from the carrier is retarded or
enhanced. Long-term feedback on uptake kinetics probably
involve regulation of the number of carriers. For example,
enzymes like nitrate transporters require a lag phase of
exposure to nitrate before uptake can proceed.

Under field conditions absorbing surface areas of trees
can also influence nutrient soil-solution concentration,
modulating the supply of nutrients to the surface, and

32

arfecting uptake rate. Processes important for forest soils
or the southeastern united states include organic exudates
and redox potentials. For example several types of organic
acids (such as citrate and oxalate) are excreted by roots and
mycorrhizal hyphae in response to P deficiency, improving P
availability in the soil-solution. Examples of this process
m forest soils are given for Pinus by Fox et al. (1990) and
Fox and Comerford (1990).

Also, roots of some forest species growing in anaerobic
conditions have been shown to oxidize their rhizosphere.
Taproots of pine trees occur in soil zones which are
perenially or temporarilly water saturated producing anoxic
conditions. Roots of slash pine ( Pinus elliottii) (Fisher and
Stone, 1990a), lodgpole pine ( Pinus contorts Douglas ex
Louden) (Coutss, 1982) and pond pine (Pinus serotina Michx.)
growing in oxygen depleted soils induced localized
hypertrophy of lenticels on taproots and lateral roots and
produced continuous gas filled lacunae in the roots. Internal
02 transport of these roots provides the ability to absorb
nutrient on poorly drained soil. Such a mechanism allows
active K uptake under what would appear to be anaerobic
conditions (Fisher and Stone, 1990b). This process is
important for forest plantations of the southeastern United
States where seasonal water tables severly reduce 02 supply to
large parts of the root system. Forested Spodosols of the
southeastern lower coastal plain typically have perched,
fluctuating water tables. At any single location the

33

saturated zone can range from near the soil surface to depths
> 150 cm during a single year, and often during a single
growing season (Phillips et al., 1989). Pine roots growing in
those soils encounter shallow water tables which promote
reduced soil conditions (Comerford et al., 1996). Reduced
soil conditions affect root respiration and, as soil oxygen
tension decreases, the uptake of ions falls, particularly
under hypoxic conditions (Hopkins et al., 1950; Hopkins,

1956). However maintenance of ion uptake by submerged roots
of trees depends on the ion studied, and time of root
acclimation to hypoxic conditions.

The 02 dependent nature of ion influx in unacclimated or
hypoxic intolerant root tissues suggest that fermentative
respiration does not produce sufficient levels of ATP to
drive the plasmalemma H+-ATPase (Drew, 1988). Investigations
with wheat suggest that xylem loading of K is more sensitive
to 02 deficiency than influx at the plasmalemma (Thompson et
al., 1989). Recent investigations by Topa and Cheeseman
(1993) concluded that 32P transport to shoots of pond pine
seedlings may be more sensitive to 02 deficient soil
conditions than uptake at the epidermal or cortical
plasmalemma of pine roots, possibly because stelar tissue may
experience 02 deficiency before and more frequently than outer
cortical cells. From the point of view of mineral nutrition
of trees under field conditions, it is not the ion transport
mechanism that is important, but the way in which the
transport mechanism is controlled by the tree. Clarkson et

34

al. (1978) stated that the nutrient uptake effectiveness of a
root system depends on the differential response of the
absorbing surfaces to environmental variables.

Concluding Remarks

From the concepts presented above I conclude that uptake
and transport of ions takes place in a complex structure of
various root tissues (eg., apoplastic and symplastic
pathway); at different stages of growth and development (e.g.
cell division, elongation and cambial growth); and along the
length of roots. Thus anatomical, as well as physiological
characteristics of both young and old roots of perennial
plants deserve to be studied under field conditions. The
overall objective of the research described in the following
four chapters was to investigate the absorption of phosphorus
(P) and potassium (K) by the root surface area of twelve-
year-old slash pine trees under field conditions. Chapter 3
is the description and testing of a field method that I
developed to measure nutrient depletion by roots of trees
under field conditions. Chapter 4 addresses the effect of low
oxygen in the uptake of K and P by roots of pine trees under
field conditions. Chapter 5 is a study of P and K depletion
by fine woody roots of pine trees under field conditions.

CHAPTER 3

A METHOD FOR MEASURING NUTRIENT
DEPLETION BY ROOTS OF MATURE TREES IN THE FIELD

Introduction

Current understanding of water and ion uptake by roots
generally comes from studies of transport phenomena using
new, white roots of young plants (Bowen and Rovira, 1967;

Bhat and Nye, 1973; Ginsburg and Ginzburg, 1970). Much less
is known about nutrient absorption in older roots of
perennial plants, yet their investigation is fundamental for
a complete description of the "effective" nutrient absorbing
surface area of a root system. "Effective root surface" is
defined, in water and nutrient uptake models, as the fraction
of the total surface area contributing to water and ion
uptake (Baldwin et al., 1972). Root surface area can be the
single most important factor determining nutrient uptake by
plants (Barber, 1984). However, a root's ability to absorb
nutrients can change with age and stage of growth development
(Fusseder, 1987; Nayakekorala and Taylor, 1990). Types of
root surface areas have been categorized as (i) white roots
(with and without root hairs), (ii) mycorrhiza, (iii) other
nonwoody roots (nonwoody roots undergoing root browning which
may be due to metaculization, suberization, or cortical cell
decay, and iv) woody-roots (those containing secondary xylem)

35

36

(Chung and Kramer, 1975; Sutton and Tinus, 1983; Comerford et
al* ' 1994a). Nutrient uptake by roots has, in most cases,
been studied in solution culture. It has been described by
relating the rate of ion uptake by the root to the external
ion solution concentration. Net ion influx has been described
by equations derived from the Michaelis-Menten equation
(Claassen and Barber, 1974). Parameters of these equations
may vary according to the type of nutrient, plant species
(Clarkson, 1985; Foehse et al., 1988), genotype (Nielsen and
Barber, 1978), and age of plants (Jungk and Barber, 1975).
Therefore, it is reasonable to believe that uptake
characteristics also change with different types of root
surface area. Determining the kinetics of ion uptake by roots
is of fundamental importance to the study of plant mineral
nutrition, yet field studies of root uptake tend to be
complicated due to access to plant roots below the soil,
plant age and plant size. This is especially true when
considering mature trees.

Uptake kinetic studies have been accomplished either by
using excised roots (Noggle and Fried, 1960) or intact plants
(Loneragan and Asher, 1967). Uptake studies using excised
roots may not be relevant because (i) roots are not attached
to the sink (the shoot), ( ii ) a steady state condition might
not be established in the short uptake periods used (e.g. 20
minutes) and, (iii) changes in root metabolic processes may
occur due to injury stress (Bloom and Caldwell, 1988).

37

Longer-term depletion studies by intact roots have been
conducted under (i) steady-state conditions, or (ii)
transient conditions. Steady-state conditions occur when the
influx of the ion into the system occurs at a rate equal to
root uptake of the ion, so the quantity of the monitored ion
remains steady over time, even though ion uptake is on-going.
The amount of the ion that must enter or leave the system per
unit time to maintain a steady-state becomes a measurement of
the rate of the reaction. Nutrient flow systems that monitor
steady-state ion absorption by intact plants were described
by Bloom (1989), Bloom and Chapin (1981), and Glass et al.
(1987). The systems are sophisticated, and have been used
under greenhouse conditions. Cost and technical requirements
are a constraining for implementing a steady-state system
that provides field measurements of ion uptake for roots of
mature trees. Also, the steady-state ion absorption method
has received some criticism in that the procedure may not
adequately represent soil solution conditions in the
rhizosphere of plants growing under field conditions.

Transient conditions to measure root uptake by intact
plants are based on the time required to deplete the ion
concentration m a solution; hence they are called depletion
methods. A depletion method can be accomplished by either a)
exposing root systems to a graded series of initial ion
concentrations (Nye and Tinker, 1977); or, b) measuring rates
of ion depletion from an initial ion concentration (Claassen
and Barber, 1974). A major requirement for the first case is

38

to have enough root systems and replicates for the chosen
graded series of initial ion concentrations. Uniformity of
root systems could be a major constraint in measuring in situ
ion uptake on roots of mature trees using this approach. The
second case is less laborious than the first one, requiring
only a system to maintain a constant solution volume during
the whole experiment. In this fashion, the ion uptake rate
can be calculated from changes in the ion concentration in
the solution. The few ion uptake studies using forest tree
roots have employed the depletion method (Beck, 1979; Van
Rees and Comerford, 1990).

In order to measure ion uptake by intact roots of mature
trees in the field, two major requirements must be met (i) to
grow the root system in a way that allows access and
minimizes disturbance; and (ii) to enclose the root system in
a chamber where environmental conditions can be controlled.
Meeting these two conditions in the field is challenging. Not
surprisingly, I found only two useful examples of nutrient
depletion studies in the field and a handful of related
studies. The first example was published by Marschner et al.
(1991). Non-mycorrhizal, intact long roots of Norway spruce
( Picea abies (L.) Karst.) were carefully excavated, lifted
from the soil, and placed into containers where a solution
with nutrient concentrations similar to those in soil
solution was supplied. The experiment was designed to study
the preferential uptake of nitrogen ions (NH4+vs. N03J from
the nutrient solution. The second example was recently

39

published by Goutouly and Habib (1996). Whole root systems of
3 to 5 year-old, non-bearing peach (Prunus persica (L.)
Batsch. ) trees were excavated. Each root system was split in
half and put into nutrient solution containers. The whole
root system was maintained at 1 or 5 mM N03_ . Hourly nitrate
uptake rates of each root subsystem were measured under
natural climatic conditions. Besides these two, a few related
studies include descriptions of techniques for measuring ion
uptake by intact roots of mature trees under field
conditions. Glavac and Ebben (1986) described a root chamber
to test the reaction of roots to different nutrients and
toxic chemicals. The system utilized a square container made
of 1 mm2 polyvinylchloride (PVC)-net, filled with washed sand,
a ceramic plate, and a remote solution reservoir. Capillary
action provided by the ceramic plate in equilibrium with the
^ the chamber resulted in a constant supply of
nutrients and chemicals to the root system. This technique
was designed to investigate root morphological changes
following four weeks of exposure to different chemical
environments. As described, however, the system was
inappropriate for nutrient depletion studies. Niederholzer et
al. (1994) described a method for measuring root development
of field-grown, mature peach trees. They developed a novel
system to measure root regeneration and elongation from the
cut ends of roots placed into root-impenetrable cloth bags.
Sisson (1983) described in detail a system for determining

40

-espiration rates of intact Yucca elata Engelm. roots. The
system is a good example of meeting aeration requirements.

There does not appear to be a system appropriate for the
study of nutrient depletion by roots of mature trees in the
-ield. The objectives of this study were (i) to present the
design and construction of a methodology for nutrient uptake
by lateral root systems of mature trees in the field, and
(ii) to test the procedure under laboratory and field
conditions .

Materials and Methods

Site Description

Twelve-year-old slash pine trees (Pinus elliotii Engelm.
var. elliotii ) averaging 16.7 m (std=3.7) tall and 19.4 cm
(std-2.7) dbh (diameter-base-height) were selected from a
research plot in north Florida that received complete weed
control. The study area is a long-term field experiment,
initiated in 1983 by the Intensive Management Practices
Assessment Center (IMPAC) of the USDA Forest Service, to
assess potential biological productivity of pine and the
processes controlling it (Colbert et al., 1990). A complete
description of the site is given in Swindel et al. (1988) and
Neary et al. (1990). Weeds were controlled with a combination
of herbicide and mechanical means. The study site is about 10
km north of Gainesville, FL (29° 80' N, 82° 20' W) . The climate
is warm, temperate-subtropical with a mean annual

41

precipitation of 1332 mm, most of which occurs in summer,
with the least falling in autumn and spring. Mean annual
temperature is 21 °C. The soil is a poorly-drained Pomona fine
sand (sandy, siliceous, hyperthermic Ultic Alaquod).

Root Regeneration System

The root regeneration system was a variation of the
method used by Lyford and Wilson (1966). Four lateral roots
1-1.5 cm in diameter were carefully excavated at the base of
a tree so that the fine root system was preserved as much as
possible. Each lateral root was pruned to a length that
varied from 20 to 40 cm. If a ramification of the lateral
root was present, the cut was made after the branching. A
five-cm inside diameter (i.d.) PVC cap was then threaded over
each root through a 2.2 cm hole drilled in the middle of the
cap, until the cap rested near the base of the root where it
was attached to the tree. Each root was then placed in a 50 x
25 x 6 cm black plastic tray and the trays were filled with
sieved soil from the A horizon of the study plot (Figure 3-
1). The PVC cap was placed outside the tray at the point of
entry for the root. Black plastic mesh was placed over the
tray and covered with pine litter from the plot. The root
system was irrigated with nutrient solution every other day
to keep the soil moist. The nutrient solution consisted of
110 pM nitrogen (90 pM as ammonium-N and 20 pM as nitrate-N),
20 pM phosphate-P, 77 pM potassium, 65 pM calcium, 10 pM
magnesium, 10 pM sulfate-S, 0.24 pM borate-B, 0.20 pM iron,

42

Figure 3-1. The root regeneration system. Each root system was
covered with moistened wiper paper sheets. A PVC cap (A) was
threaded over each root system, before entering the root tray
(B). (C) Tree trunk

43

0.02 pM manganese, 0.02 p M zinc, 0.005 pM molybdate-Mo, and
0.005 pM copper. These nutrient concentrations are typical of
soil solutions of a Florida Spodosol (Van Rees and Comerford,
1990). Excess drained through holes drilled in the bottom of
each tray. Watering was suspended when rain was plentiful.

The PVC cap was used as one end of the "nutrient uptake root
chamber". The design of the chamber is described in detail in
the following section. Because this cap part of the chamber
is installed proximal to new root growth, it was convenient
to install it before root growth occurred, thus preventing
damage to the root system at the time of the nutrient
depletion study. This method was applied to twenty trees (80
trays) between July 24 to July 28, 1994. Fourteen of these
trees were used for this study.

Nutrient Uptake Root Chamber Design and Construction

Root uptake chambers were constructed from PVC (ASTM-D-
2665) with a 5 cm i.d. and walls 4 mm thick (Figure 3-2).

Each column consisted of 3 parts: (a) the main column, (b) L-
type PVC connector with both ends made to fit 5 cm i.d. PVC
pipes, and (c) a 10. cm long PVC pipe to fit into part (b).

Root chambers varied in length from 30-40 cm to accommodate
various root system sizes. Figure 3-2 is a schematic
representation of the nutrient uptake root chamber. Part (a)
has a 3 -cm diameter hole, sealed with a No 6.5 solid neoprene
rubber stopper (1). The hole was used to completely drain the
nutrient solution from the chamber. Parts (b) and (c) were

44

Figure 3-2. Nutrient uptake root chamber, (a, c) PVC DiDes*
<b) PVC elbow; (d, 2) PVC cap; (e) cross sechon of the Sit
chamber; (1, 3, 4) rubber stoppers; (5) plastic L
connectors; (6, 9) plastic syringe; (7) one way stopcock
valve; (8) disposable syringe filter.

00 N.

45

used to raise the level of the nutrient solution so the root
system could be completely immersed within the root chamber.
Part (c) was attached to a 5 cm i.d. PVC cap (2) with a 2.2
cm hole drilled in the middle (3). The hole was used to
insert a tube that gassed the solution with either compressed
air, or N2 gas. Parts (a), (b), and (c) were attached with
silicone lubricant (Dow Corning). Modeling clay (Rose Arts
Industries, Inc., Livingston NJ) was used to externally seal
the chamber. By using silicone lubricant and modeling clay I
was able to assemble and disassemble the chamber in the
field. The root chamber could then be connected to the root
system by fitting it to the 5 cm i.d. PVC cap (d) which was
previously threaded over each root system (as described in
the root regeneration system section). Sealing the root-PVC
hole intersection was achieved by using a No. 4 solid
neoprene rubber stopper (4). A hole, slightly larger than the
lateral root diameter (3-5 cm) was drilled in the center of
the rubber stopper, then radialy cut, so that the stopper
could be inserted around the lateral root. The rubber stopper
was used to seal the PVC cap hole. Epoxy putty, a two-part
semisolid resin that cures in one hour (Cole Parmer H-08785-
10) was used to seal externally the root-stopper junction.

Diagram (e). Figure 3-2, is a cross-sectional
representation of the assembled root chamber. It consisted of
two polypropylene elbow connectors (i.d. 4 mm) attached to
mam PVC column ( 5 ) . They were inserted in the middle of the
chamber opposite each other. One of the elbow

connectors was

46

attached to a 10 mL plastic burette (6). The plastic burette
was constructed by inserting the barrel of a 10 mL
polypropylene syringe into one end of the elbow connector.
This burette was used to maintain a constant volume in the
root chamber utilizing a Mariotte flask system. The other
elbow connector was attached to a one-way stopcock valve,
with a male Luer lock ( 7 ) . The male lock end was attached to
a 2 5 -mm cellulose acetate disposable syringe filter (8)
connected to a 10-mL disposable polypropylene syringe (9).
This system was used to retrieve or add a known amount of
solution to the chamber. When needed, connections were sealed
with a hot glue gun. A complete list of parts and materials
for constructing a nutrient uptake chamber is in Table 3-1.

In-situ Nutrient Uptake Rym-m

A view of the in situ nutrient uptake system is shown in
Figure 3-3. Prior to lab and field experiments the whole
system was flushed with NaOCl (5%) and diluted sulfuric acid
and then rinsed well with distilled water. The three
components of the system were (a) the gas system, (b) the
root uptake chamber and, (c) the Mariotte flask system. The
system was used as follows. The root chamber was gassed with
compressed gas tanks (air, or N2 gas) (1) and the gas was
delivered from the tanks to a controlling board (2). The
controlling board had a main flowmeter (3) that, through the
use of a needle valve (4), controlled the gas pressure
delivered to each tree. Trees were 1-2 m apart, with four

47

Figure 3-3. In situ nutrient uptake system, (a) aeration
system; (b) root chamber; (c) Mariotte flask system; (1) gas
tank; (2) controlling board; (3) main needle valve; (4)
needle valves for each tree; (5) needle valves for each root.

48

Table 3-1. List of parts and materials for constructing a

nutrient uptake chamber.

Material

DescriDtion

Qtv.

Refer

to

Fiaure

Suqqested suDDlier
( Cataloq

number/ descriDtion \

PVC cap

2

3-2.2

Hardware stores

(5 cm i.d. )

3-2. d

(ASTM-D 2665)

PVC pipe
(5 cm i.d.)

2

3-2. a
3-2 .c

is

L-PVC connector

1

3-2. b

IS

(5 cm i.d.)

Rubber stopper

3

3-2.1

Fisher /Cole-Parmer

No. 6.5, 4 )

3-2.3

3-2.4

(Solid Neoprene)

Polypropylene elbow
connectors (i.d. 4 mm)

2

3-2.5

Cole-Parmer

(H-06285-10)

One-way male stopcock

1

3-2.7

Cole-Parmer
(H-06464-71 )

Disposable cellulose
acetate syringe filter

1

3-2.8

Cole-Parmer
(H-02915-62 1

(25 mm)

Disposable,

2

3-2.6

Fi shef

polypropylene syringe

3-2.9

( 14-823-2A*

Luer-tip (10 mL)

Epoxy putty

sealant

Cole-Parmer
( H-08785-10

Plastaline

sealant

Handicraft stores
(modeling clay)

Hot-glue for guns

sealant

Handicraft stores

Silicone lubricant
(Dow corning grease)

grease

Fisher
( 14-635-5D)

49

root chambers each. The gas delivered to each root chamber
was controlled by another needle valve (5). Aeration to each
root chamber was checked by measuring dissolved oxygen in
root chambers with a Yellow Springs Instrument (YSI) portable
oxygen meter and a Clark type membrane-covered probe
( accuracy 0.1 mg L'1 02 ) .

Details of the root uptake chamber and the Mariotte
flask system are presented in Figure 3-4. The root uptake
chamber was placed in a shallow pit excavated around the
lateral roots. A plywood (1) stand for the chamber was
inserted into the ground to stabilize each chamber. The whole
chamber was covered with a black plastic mesh to avoid direct
sunlight.

The Mariotte flask system was built using a 250-mL
straight-sided glass digestion tube (2), a rubber stopper
(3), nalgene-VI grade tubing (i.d. 0.31 mm) (4), a 1-mL
polystyrene disposable pipette ( 5 ) , and two one-way stopcock
valves, with a male Luer lock (6). The glass digestion tube
flask system was held by a burette support stand ( 7 ) . When
needed, connections were sealed with a hot glue gun and
Teflon tape. As the solution level dropped in the chamber,
the solution in the glass tube moved into the chamber due to
atmospheric pressure. The glass digestion tube (2) was marked
with milimetric paper and calibrated, so that the volume of
water used to replace the root chamber solution level was
known. The glass tube can be refilled by connecting it to a

50

51

reservoir. A complete list of parts and materials for
constructing the Mariotte flask system is in Table 3-2.

Testing of the Nutrient Uptake System
Laboratory studies

All laboratory tests of the nutrient uptake chamber
system were performed without roots in the chambers. Four
nutrient uptake chambers were assembled in the laboratory, and
sealed as described above. A Mariotte flask system was
assembled for each chamber, and gassing was provided using
compressed air (Medical Grade). Each chamber was filled with
1 L of nutrient solution containing 6.2 /jM P (ca. 0.20 mg L'1),
25.6 pM K (ca. 1.00 mg L'1), 110 pM nitrogen (90 /jM as
ammonium-N and 20 pM as nitrate-N), 65 jiM calcium, 10 fj M
magnesium, 10 pM sulfate-S, 0.24 pM borate-B, 0.20 piM iron,

0.02 pM manganese, 0.02 pM zinc, 0.005 jj M molybdate-Mo, and
0.005 pM copper. The nutrient solution was adjusted to pH 4.5.

Solution samples (8 mL) were collected from each chamber
every 8 hours for 48 hours using the syringe described in
Figure 3-2, Label 9. Mariotte flasks were filled with
distilled water (pH 4.5), and were connected to each chamber
to maintain a constant solution volume. The volume of water
replenished by the Mariotte flasks between sampling intervals
was recorded. Dissolved oxygen saturation, and temperature
were measured for each chamber at each sampling interval with

52

Table 3-2. List of parts and materials for constructing a

Mariotte flask system.

Material

•

>

-p

ol

Refer

Suqcrested suDDlier

DescriDtion

to

Fiaure

( Cataloa

number /descriDtion )

Glass digestion tube
( 250 mL)

1

3-4.2

Fisher

(TC1000-0155 )

Rubber stopper
(No. 4)

1

3-4.3

Fisher

(Solid Neoprene)

PVC tubing

(Nalgene i.d. 0.31 mm)

3-4.4

Fisher

( 180-clear VI grade)

Polystyrene pipette
(1 mL)

1

3-4.5

Fisher

(Disposable)

One-way male
stopcock

2

3-4.6

Cole-Parmer
(H-06464-71 )

Burette stand

1

3-4.7

Fisher

Teflon tape

sealant

Fisher

( 14-831-300A)

Hot-glue for guns

sealant

Handicraft stores

53

a Yellow Springs Instrument (YSI) portable oxygen meter, and
a Clark type membrane-covered probe (accuracy 0.1 mg L'1 02).

In-situ field studies

Root regeneration system. Nine months after the
installation of the root trays, thirty-six intact lateral
roots from 9 trees were gently uncovered and washed with

water from soil particles. The root regeneration
system was evaluated based on the presence or absence of new
root growth. Eleven months later, twenty lateral roots from
another 5 trees were also uncovered and evaluated as
described previously.

Nutrient uptake system installation. Chambers were
fitted to seventeen lateral roots on 5 trees as described
above. Sealing the root-stopper junction was the most
critical part. The junction was checked for leaks before
connecting the cap to the root uptake chamber. During this
procedure the root system was wrapped in several layers of
soggy sheets of delicate task paper wiper (38 x 43 cm 2-ply
) , and covered with a black plastic mesh to avoid direct
sunlight. The chamber was connected to the root as described
above. Lastly, the Mariotte flask system was placed in the
shallow pit as shown in Figure 3-4. The setting was
accomplished by a two-person team. In order to minimize the
risk of breaking the lateral root, I left enough length of
lateral root between the tree trunk and the root-stopper-cap
junction to allow easy flexible movement of the root. This

54

length facilitated the movement needed when assembling the
chamber system. After installation, the portion of root
external to the chamber was covered with soil.

Pre-depletion treatment. The chamber was filled with the
nutrient solution described in the laboratory study section,
but lacking P and K. Roots in the chambers were continuously
gassed with compressed air for 24 hours. During that time the
system was monitored for leaks. Modeling clay was used to
seal leaks. The aeration and Mariotte flask systems were also
monitored for proper performance. Each chamber was covered
with a black plastic mesh to avoid direct sunlight. This
prevented the softening of the modeling clay by sunlight and
kept the nutrient solution temperature uniform. The chamber
was drained before the nutrient depletion study and then
flushed with distilled water.

Nutrient depletion study with air. The root chambers
containing roots were filled with known amounts of nutrient
solution (800 to 1200 mL). Initial P and K concentrations for
the study were 6.25 pM P (ca. 0.20 mg IT1 ) and 25.66 pM K
(ca. 1.00 mg L'1), plus other nutrients as described in the
previous section. The nutrient solution was adjusted to pH
4.5. Solution samples (8 mL) were collected every 6 hours for
a period of 72 hours. An equal volume of distilled water (8
mL (pH 4.5) was replaced. Water uptake was measured by
calculating the volume of water replenished by the Mariotte
flasks between sampling intervals. The system was
continuously aerated with pressurized air. Dissolved oxygen

55

saturation and temperature were measured for each root
chamber at each sampling interval.

Nutrient depletion study with N: gas . After completion
of the previous experiment, all root chambers were drained
and flushed with distilled water. They were then subjected to
the pre-depletion treatment for 24 hours. Afterwards, the
root chambers were filled with known amounts of nutrient
solution (c.a. 800-1200 mL). Nutrient concentrations for the
study were the same as for the air study. The nutrient
solution was purged with N2 gas for 2 hours before the
depletion study experiment. Solution samples (8 mL) were
collected from each chamber every 8 hours for 48 hours. An
equal volume of distilled water (8 mL pH 4.5) was added back
after taking each sample. Root water uptake was measured by
calculating the volume of water replenished by the Mariotte
flasks between sampling intervals. The system was
continuously gassed with N2 gas (ultra-high purity grade) to
decrease the oxygen solution concentration (Steward et al.,
1936). Dissolved oxygen saturation, and temperature were
measured for each root chamber at each sampling interval.

Chemical analyses

Nutrient solution samples were kept on ice until the
depletion study was completed. They were analyzed within 24
hours of the completion of each depletion study.

Concentration of p was determined by the method of Murphy and

56

Riley (1962). Potassium concentration was determined by
atomic absorption spectroscopy.

Data Analyses
Laboratory studies

Statistical comparisons of P and K concentrations,
volume of water replenished by the Mariotte flask system and
oxygen saturation were made using a repeated measures
analysis (SAS Institute, 1985). The chambers (without roots)
were the measurement units and the repeated measures were the
time intervals .

Field studies

Periodic P and K uptake were calculated by expressing
the P and K concentration at each sampling interval as a
fraction of the initial nutrient concentration. Corrections
were made for the dilution caused by sample withdrawal.
Statistical comparisons for P and K concentrations , oxygen
saturation, and nutrient .solution temperature were made using
a repeated measures .analysis (SAS Institute, 1985).

Treatments were the aeration environments caused by air or N2
gas. The blocks were trees while the replicates were the
roots from each tree. The measurement unit was the chamber
(with root) and the repeated measures were the time
intervals .

57

Results

Laboratory Studies (No Roots Present In Chambers!

Phosphorus and K solution concentrations in the chamber
were not significantly different at sampling intervals, nor
among chambers. Average solution concentrations of the
nutrient solution were 6.09 pM (c.a. 0.195 mg L'1) for P
( SE=1 .16 x 10-3), and 26.28 pM (c.a. 1.024 mg L'1 ) for K
( SE=7 . 49 x 10‘2) .

The Mariotte flask system accurately replenished the
water removed by sampling. The average volume of water
replenished by the Mariotte flask system was 8.4 mL. (SE=0.15
mL). The volume of solution withdrawn from each chamber was
56 mL (7 samples x 8 mL) . The average volume of solution
replenished by the Mariotte flask system was 58.8 mL (7 x 8.4
mL) . This represents a 5% error per volume sampled. The

dilution of the solution concentration in the chamber was
5.8%.

Oxygen saturation in the chambers was not significantly
different at sampling intervals, nor among chambers. Nutrient
solution temperature was 25 °C and the average oxygen
saturation was 98.3%.

58

Field Studies

Root regeneration system

A total of 56 lateral roots from 14 trees were uncovered
after 9 and 20 months from the initial installation of root
trays. From this total, thirty-two (57%) lateral roots had
new white roots. Sixteen (29%) more lateral roots had new
fine roots that were brown in color. Two lateral root systems
did not have fine roots but the lateral was still alive (4%).
Six xateral roots (10%) were dead, apparently broken during
the initial excavation. Therefore, 86% of the fifty-six
lateral roots that were evaluated had new growth.

Nutrient depletion with air

Phosphorus and K solution concentrations (expressed as a
fraction of the initial) were significantly different at
sampling intervals (p < 0.001), but were not significantly
among root chambers. The percentage of nutrient
depleted was different for P than for K. It was as high as

92% for P while for K only 25% of the initial K concentration
was depleted. (Figure 3-5).

Nutrient depletion with gas

Potassium depletion was effectively inhibited by N2 gas .
The chamber K concentrations for the sampling intervals were
not significantly different (Figure 3-6). In contrast, the

59

1.0

<0.

O'

t 0.8-1

*J\%

o 0.7H
| 0.6-1
< 0.5-
| 0.4-
O 0.3-
O 0-2-
O 0.1-

0.0

1

AIR

\

\

\

\

Potassium

Phosphorus

■*

"I I I I — I — I — I — I — I — l — l — r~

6 12 18 24 30 36 42 48 54 60 66 72 78

Time (Hours)

Figure 3-5. Phosphorus and Potassium depletion by seventeen
root systems of Pinus elliottii var. elliottii. Each symbol
represent an average with standard error of seventeen root
chambers Values are expressed as fraction of initial
phosphorus ( # ) and potassium ( ■ ) concentration in the

nutrient uptake chamber. The roots were gassed with air during
the pxnpnmonf ’

60

8 1.4- n2gas

o

H

<

on

1.2-

LU
O
z
O
o

-J

<

= 0.6-1 l

1.0-

0.8*

• 4+

V

l

LL

o

0.4-

o

o

<

on

Potassium

Phosphorus

IZ 0.2-

0.0-

4

"i i i i i — i — i — i — i — i — r~

6 12 18 24 30 36 42 48 54 60 66 72
Time (Hours)

Figure 3-6. Phosphorus and Potassium depletion by seventeen
root systems of Pinus elliottii var. elliottii. Each symbol
represent an average with standard error of seventeen root
chambers. Values are expressed as fraction of initial
phosphorus ( # ) and potassium ( ■ ) concentration in the

nutrient uptake chamber. The roots were gassed with N, gas
during the experiment.

61

initial P concentrations in the roots chambers gassed with N„
were depleted as much as 92% (Figure 3-6), and were depleted

in a similar trend as when roots were gassed with air (Figure
3-5) .

Oxygen saturation with air and N. gas

The oxygen saturation and the nutrient solution
temperature in the root chambers during the depletion
experiment with air and N2 gas are presented in Figure 3-7.

For either air or N2 gas treatment the oxygen saturation
values were not significantly different at sampling
intervals, nor among root chambers (Figure 3-7b and 3-7c).
However the N2 gas treatment decreased the oxygen saturation
significantly as compared with the air gas treatment (p <
0.001). The average oxygen saturation was effectively reduced
from 96.3% (SE 1.69 % ) to 4.2 % (SE= 2.7 %) when the
gassing was changed from air to N2 gas. The average nutrient
solution temperature when roots were gassed with air was 25.9
C. ( SE= 5.67 °C), and was not different from nutrient solution
temperature when roots were gassed with N, gas
( average=2 3 . 9°C , SE= 5. 7°C). Diurnal air temperature
fluctuations influenced the nutrient solution temperature.

They were as low as 18 °C at the 6:00 a.m. samplings and as
high as 35 C at the 12:00 a.m. (Figure 3-7a).

62

12 18 24 30 36 42 48 54 60 66
TIME (Hr)

0 6 12 18 24 30 36 42 48 54 60 66

i 1 1 1 1 1 1 1 — i r

12 18 24 30 36 42 48 54 60 66

TIME (Hr)

Figure 3-7. Nutrient solution temperature (A) and Oxygen
saturation (B and C), in root chambers with lateral root
systems of Pinus elliottii var. elliottii The roots were
gassed with air ( ■ ) and N2 gas ( □ ) . Each symbol represent
an average with standard error of seventeen root chambers

63

Discussion

I have described a method of measuring ion depletion by
intact roots of mature trees in the field by (i) detailing a
root regeneration system that allows access and minimizes
disturbance of roots, and by (ii) designing and testing a
chamber that effectively encloses the root system under
controlled aeration conditions.

In order to implement the root regeneration system
described here, one must take into account the ability of a
root system to regenerate after damage. In this study I had
an 86% success rate in acquiring new root growth. For Pinus,
lateral roots often extend great distances and are among the
least branched of the important forest species but have
abundant short roots. (Pritchett, 1979).

The laboratory study showed that the chambers were not a
source of P or K and that biotic or abiotic immobilization of
P or K was occurring. All components of the nutrient uptake
system that were in direct contact with the nutrient solution
were made from inert material such as PVC, nalgene tubing,
polyethylene, rubber, and glass. Tygon tubing has been
reported to contain a plastilizer which supports microbial
growth and interferes with ion analysis (Bloom, 1989).
Microorganisms such as Pseudomonas sp. have been shown to
colonize the interior surface of PVC water distribution pipes
(Vess et. al, 1993). However, since there was no change in

64

the P and K solution concentrations over time, I infer that
neither microbial growth, ion sorption to the chamber nor ion
release from the chamber occurred in our system. In previous
studies, microbial growth on surfaces has been reported after
3-7 weeks. Our experiments lasted just five days.

This nutrient uptake system has many advantages for
repetitive measurements of ion depletion. The root system in
each chamber can be subjected to different treatments,
allowing observations of the response of the same root system
to different conditions. For example, in my study, I
illustrated the effect of different gases (air and N2) on P
and K depletion for the same root system. N2 has been commonly
used to deoxygenate solutions resulting in low 02 treatment.
Dissolved oxygen concentrations of <0.5 mg L'1 (<50 pM) could

result in anoxia /hypoxia of root tissue, known as hypoxic
conditions. Nitrogen gas has been used to demonstrate
inhibition of active ion uptake (Hopkins, 1956; Fisher and
Stone, 1990a). Its mode of action is to decrease the oxygen
solution concentration to hypoxic Conditions where active ion
uptake is inhibited or stopped (Steward et al., 1936). I
achieved hypoxic conditions in our N2 gas treatment study (4.2
% oxygen saturation, c. a. =0.35 mg IT1 ) . The net efflux of K
from roots in the N2 gas treatment has been previously
reported (Rosen and Carlson, 1984). Similarly, P depletion
by roots in the N2 gas treatment has been also previously
reported (Topa and Cheeseman, 1993). The difference in K and
P depletion by roots under hypoxic conditions seems to be

65

associated with the ion uptake mechanism. K+ as an
unmetabolised cation is transported across the plasma ■
membrane into the root symplast by a transport mechanism
c^^-^J-eren^' ^rom that of P. For instance K+ ions are
transported actively mainly through the symplast (Lauchli et
al • ' 1973). Although P-phosphate ions are also transported
through the symplast (Clarkson and Sanderson, 1969), starving
roots first take up P-phosphate by diffusion into the
apoplast. After the saturation of the apoplast and when the
steady-state of the external solution-tissue system is
exceeded, the transport proceeds actively (symplastic)
(Ulrich-Eberius et al., 1981).

Estimation of ion uptake rate by depletion experiments
has been criticized due to transient conditions. During the
depletion period, the total composition of the nutrient
solution may vary significantly, inducing an ionic shock
(Bloom, 1989). Moreover as the ion solution concentration
decreases, the internal nutrient status of the roots may
shift, resulting in immediate and long term effects (Clarkson
and Hanson, 1980). Roots with a low nutrient status generally
have a higher capacity and a lower affinity for the limited
nutrient (i.e., higher and lower KJ (Drew et al., 1984).
Therefore, under changing nutrient levels, the response of
the plant is in transition. Measurements conducted under
steady-state conditions avoid some of the uncertainties
associated with transient conditions. Steady-state systems
require a constant nutrient flow system and require

66

monitoring the ion concentration between the solution
entering and leaving the root-chamber. Goutouly and Habib,
(1996) recently described a steady-state nutrient uptake
system in a peach orchard. However, cost and technical
requirements can constrain the use of the same system in
forests. Instead of our Mariotte flask system they used an
electric detector gauge to detect changes in the nutrient
solution level (Honeywell "LL 102007", precision +/- 1 mm).
This device was connected to a pressure balance to measure
root water uptake. Instead of sampling manually from the
nutrient solution, they used an automatic fraction collector.
The complexity of their system only allowed measurement of
nitrate uptake rates from one tree at a time. Peach trees
were 2 m in height, with 3-4 main branches. The whole root
system was split and immersed in a 30 L plastic containers
filled with nutrient solution. Only one tree could be
transported to the nutrient uptake system described above.
Therefore, another advantage of the system I describe is its
relative simplicity and low cost. Forest are usually devoid
of electrical outlets and water faucets commonly found in
managed orchards.

Despite the criticism of depletion studies to measure
root nutrient uptake, measurements in the transient state are
still useful. In practice, the issue of whether steady-state
versus transient measurement conditions should be used in
nutrient uptake by roots depends upon the system and the time
scale involved in the experiment.

67

Fruit trees under managed orchards are subjected to
seasonal fertilizer applications. The focus is to match
nutrient supply from fertilizers with nutrient demand by the
trees. Therefore, root systems from fruit trees are more
likely to be exposed to a more constant supply of nutrients
from the soil solution. Hence steady-state versus transient
measurement conditions could be more useful in nutrient
uptake studies. In comparison, management of pine plantations
is much less intensive with few, if any, fertilizer
applications. Therefore, transient measurement conditions may
represent soil solution conditions in the rhizosphere and the
depletion method described here could be more representative
of pine trees in the field.

In practice, my nutrient uptake system can control
aeration within the chamber and results are reproducible. By
using this methodology, it should be possible to explore
poorly documented aspects of nutrient uptake under field
conditions , such as the nature of the uptake mechanism of
woody fine roots.

CHAPTER 4

PHOSPHORUS AND POTASSIUM DEPLETION BY ROOTS OF FIELD-GROWN
SLASH PINE: AEROBIC AND HYPOXIC CONDITIONS

Introduction

Slash pine ( Pinus elliottii Engelm. var. elliotti) and
loblolly pine (Pinus taeda L. ) are planted extensively on
flatwood soils and coastal savannas of the Southeastern
United States. Forested Spodosols of the Southeastern lower
Coastal Plain typically have perched, fluctuating water
tables. At any single location the saturated zone can range
from near the soil surface to depths > 150 cm during a single
year, and often during a single growing season (Phillips et
al., 1989). Pine roots growing in those soils encounter
shallow water tables which can rise close to the soil surface
on flatwood soils (Comerford et al., 1996). The main problem
^ tree associated with waterlogging is root anoxia.

Anoxia is defined as the absence of 02 in plant tissue, while
hypoxia is defined as low but not zero oxygen in plant tissue
(Drew, 1988). Waterlogging in soils results in reduced soil
conditions which causes severe root tissue hypoxia. However,
some pine trees cope with waterlogged or reduced soil
conditions by strategies of avoidance. In these conifers,
anatomical root modifications promote the internal transport

68

69

of 02 to roots (Armstrong and Read, 1972; Philipson and
Coutts, 1978). An internal gas exchange mechanism between the
atmosphere and oxygen depleted tissues maintains the aerobic
functions of flooded root systems of tree species (Carpenter
and Mitchell, 1980). Roots of seedlings of pond pine ( Pinus
serotina Michx.) grown under 02-deficient conditions were well
ventilated internally as a result of extensive air space
formation from stem lenticels to root tips (Topa and McLeod,
1986a). Roots of loblolly pine seedlings grown under flooded
conditions developed aerenchyma tissue, with large
intercellular spaces present in the phellogen of woody roots.
Also flooded stems exhibited lenticel hypertrophy (Mckevlin
et al . , 1987). Fisher and Stone (1990a), demonstrated
existence of a large volume of interconnected air filled pore
space in the secondary xylem of slash pine taproots and
associated sinker roots.

Low-oxygen concentration in the root environment
strongly inhibits ion uptake. As soil oxygen tension
decreases, the uptake of ions falls, particularly under
hypoxic conditions (Hopkins et al., 1950; Hopkins, 1956;

Drew, 1988). The term hypoxic is used to describe a low 02
treatment that could result in hypoxia of root tissue. The
link between 02 supply and ion transport is principally
through respiration and the generation of ATP to drive
transport (Drew, 1988). Anaerobic metabolism does not
maintain energy metabolism at a level that will drive primary
active transport, presumably by the H+-translocating ATPase in

70

the plasma membrane. Nitrogen gas (N2) has been commonly used
to deoxygenate solutions resulting in dissolved oxygen
concentrations of <50 pM (<1.6 mg L'1 ) to demonstrate
inhibition of active ion uptake for phosphorus (P) (Hopkins,
1956; Topa and McLeod, 1986b) and potassium (K) (Fisher and
Stone, 1990b; Jalil and Carlson, 1993). Its mode of action is
to decrease or deplete the oxygen solution concentration to
levels where active ion uptake is inhibited or stopped
(Steward et al., 1936). Maintenance of ion uptake by roots of
trees subjected to N2 gas treatments depends on the ability of
the tree to maintain an internal gas-exchange mechanism
between the atmosphere and oxygen-depleted tissue. Therefore
it depends on the duration of root acclimation to hypoxic or
anaerobic conditions . It also depends on the ion studied and
initial concentration of the ion.

For K uptake studies, plum roots grown in aerated
solutions with 100 pM K, leaked K into bathing solutions
within 1 hour of changing the solution sparging gas from air
to N2 (Rosen and Carlson, 1984). After 18 h in the
deoxygenated solution (short-term hypoxic conditions), the
same root then resumed net uptake of K immediately after air
sparging resumed. For slash pine roots grown under long-term
hypoxic conditions (5 months), K did not leak into bathing
solutions containing 50 pM K, when changing sparging gas from
air to N2 (Fisher and Stone, 1990b). However net efflux of K
occurred when N2 gas replaced air in enclosures surrounding
the lower stem and basal roots. This effect was reversible

71

and K uptake resumed soon after enclosures were removed or
when N2 gas was temporarily interrupted.

For P uptake studies, short-term hypoxia (1-2 days)
reduced P accumulation by 50% in roots of pond pine
seedlings ( Pinus serotina ) grown under aerobic conditions. In
comparison P influx in roots of pond pine seedlings grown
under long term hypoxic conditions (5.3 weeks) and subjected
to short term hypoxia was 4 times higher that 32P influx of
aerobically grown seedlings (Topa and McLeod, 1986b). In both
cases the initial P concentration was 100 pM. This has
similarly been reported for some herbaceous species (John et
al., 1974). Furthermore earlier studies by Hopkins (1956),
and Larkum and Loughman (1969) suggested that P uptake
sensitivity to hypoxic conditions is related to the level of
P in the solution. Root P uptake of barley seedlings
subjected to short-term hypoxic conditions was reduced 45%
from the aerobic treatment at external P concentration of 100
pM. However, no inhibition of P uptake was observed under
short-term hypoxic conditions at external P concentration of
10 pM. Thus, the anaerobic mechanism for P uptake by roots
becomes more efficient with increasing dilution of external
P, which indicates that the carrier level for P uptake at
concentrations below 10 pM can be maintained solely by energy
from anaerobic metabolism. P concentrations below 10 pM, are
commonly encountered in soil solutions. In contrast, the
sensitivity for K uptake to hypoxic conditions as related to
levels of K in the solution, has not been rigorously tested.

72

Most studies relating K uptake by roots under hypoxic
conditions range between 60-100 pM K in the nutrient
solution; these are K concentrations commonly encountered in
soil-solutions. In all cases K uptake was inhibited when
roots were subjected to hypoxic conditions. The difference in
K and P uptake by roots under either aerobic or hypoxic
conditions seems to be associated with the ion uptake
mechanism. Ion transport across the plasmalemma of epidermal
and cortical cells of plant roots is an energy-dependent
process .

For K, at low external K concentrations (< 100 pM) the
high affinity K uptake system operates against the prevailing
electrochemical potential differences (e.g., 10 pM K+
outside, 80 pM K activity in the cytoplasm) thus an energized
K transport is required (Maathuis and Sanders, 1993).

Although an early report advanced the notion of a close
coupling between K uptake and H + extrusion in plant roots
(Pitman et al., 1975), further studies by Newman et al.,
(1987), and Kochian et al., (1989) have suggested that net H+
efflux is an electrically compensatory response to K influx.

At present the mechanism of energization of K transport can
only be speculated. Mechanisms suggested are carrier-mediated
K proton-antiport ( counter- transport ) or, more likely as 1:1 K
proton-symport, or cotransport (Kochian et al. 1989; Maathuis
and Sanders, 1993). A possible mechanism is a K influx
directly powered by ATP (adenosine triphosphate) with the
involvement of an ATPase (Kochian et al., 1989).

73

For P, more than for K, root uptake has to be a
metabolically driven process. The soil solution immediately
outside the root cells is likely to contain 1 pM P-phosphate
or less, while in the cytoplasm the concentration will be 103
to 10 times greater (Clarkson and Grignon, 1991). Therefore

a steep gradient must be overcome to transport P-phosphate
into the interior of a typical cell. A minimum of 25-40 kJ of
energy is required to transport 1 mole of phosphate which is
roughly equivalent to the maximum free energy released by the
hydrolysis of 1 mole of ATP (50-60 kJ Mol-1) (Clarkson and

Grignon, 1991). Active phosphate influx into the roots does
not appear to be directly related to the H+ efflux pump
discussed for K transport, but rather to an electrogenic
phosphate pump across the plasma membrane. It operates using
steep electrical (potential differences) and chemical (pH
difference) gradients for protons as the driving force
(Dunlop, 1989). For either cation (K+) or anion transport
(phosphate) the plasma membrane H+-ATPase plays a key role in
both the cytoplasmatic pH and the driving force for cation
and anion uptake (Serrano, 1990).

From the point of view of mineral nutrition of trees
under field conditions, it is not the ion transport mechanism
that is important, but the way in which the transport
mechanism is controlled by the tree. Clarkson et al., (1978)
stated that the nutrient-uptake effectiveness of a root
system depends on the differential response of the absorbing
surfaces to environmental variables. Under hypoxic conditions

74

the material referenced above showed that while P uptake by
roots can adjust within relatively wide limits of aeration, K
intake is more sensitive to hypoxic conditions. If we
consider that the fine root system of pine trees growing in
the surface 20—40 cm of a flatwood soil encounter short-term
reduced soil conditions then we must document studies of P
and K uptake by roots of pines trees under hypoxic field
conditions . It is therefore reasonable to ask whether P and K
uptake occurs under short-term reduced soil conditions, and
if so, to what degree. Yet most studies of ion uptake under
hypoxic or low 02 treatment have been carried out on root

systems of trees under greenhouse conditions. Moreover, with
the exception of a few studies with roots of cereals, there
are few reports simultaneously comparing the behavior of
uptake of more than one ion (Hopkins et al., 1950; Larkum and
Loughman, 1969). Until recently, there were no methods to
study ion uptake by root systems of trees under field
conditions (Goutouly and Habib, 1996). Therefore, the purpose
of our study was to investigate the ability and resilience of
P and K absorption by roots of 12 -year-old slash pine under
changing soil aeration. Our objectives were(i) to compare P
and K uptake by lateral roots of slash pine roots subjected
to short term hypoxic treatment, and (ii) to document the
response of K and P uptake by lateral roots of slash pine
following the removal of the hypoxic treatment.

75

Materials and Methods

Site Description

Twelve-year-old slash pine trees (Pinus elliotii Engelm.
var. elliotii ) were selected from a research plot in north
Florida that received complete weed control. A description
of the site is given in Swindel et al. (1988) and Neary et
al. (1990). Weed control was accomplished using a combination
of herbicide and mechanical measures. The soil is a poorly-
drained Pomona fine sand (sandy, siliceous, hyperthermic
Ultic Alaquod). The study site is about 10 km north of
Gainesville, FL (29° 80' N, 82° 20' W) . The climate is warm,
temperate-subtropical with a mean annual precipitation of
1332 mm. Most of the precipitation occurs in summer, with the
least falling in autumn and spring. Mean annual temperature
is 21 °C.

Root Regeneration System

Four lateral roots (1—1.5 cm in diameter) were excavated
at the base of a tree, preserving the fine root system as
much as possible and a root regeneration system was
established as described in Chapter 3. Briefly, each lateral
root was pruned, then a five-cm inside diameter (i.d.) PVC
cap was threaded over each root through a 2.2 cm hole drilled
in the middle of the cap until the cap rested near the base
of the root where it was attached to the tree. Roots were

76

then placed in 50 x 25 x 6 cm black plastic trays and filled
with sieved soil from the A horizon of the study plot. Black
plastic mesh was placed over the tray and covered with pine
litter from the plot. The root system was supplied with a
nutrient solution typical of soil solutions of a Florida
Spodosol (Van Rees and Comerford, 1990). This method was
applied to twenty trees (80 trays) between July 24 to July
28, 1994. Twenty months after the installation of the root
trays twenty intact lateral roots from five trees were gently
uncovered and washed with distilled water from soil
particles. The root regeneration system was evaluated based
on the presence or absence of new root growth. Seventeen

roots from these five trees were used for this study.
These root systems were grown in soil under aerobic
conditions .

Field Nutrient Uptake System Installation

The seventeen intact lateral roots from 5 trees
mentioned above were inserted (while still attached to the
tree) into nutrient-uptake root chambers. The construction
and assembly of the. nutrient uptake root chamber is fully
described in Chapter 3. The root chamber is part of an in
situ nutrient uptake system which includes (a) the gas
system, (b) the root uptake chamber and, (c) the Mariotte
flask system. In short, the root chamber was gassed using
compressed gas tanks with either air (medical standard) or N

gas (99.9%). The Mariotte flask system was used to maintain a

77

constant volume in the root chamber. The root uptake chamber
was placed in a plywood stand for mechanical support.

Together with the Mariotte flask system, they were placed in
a shallow pit. Aeration to each root chamber was checked by
measuring dissolved oxygen in root chambers with a Yellow
Springs Instrument (YSI) portable oxygen meter and a Clark
type membranecovered probe (accuracy 0.1 mg L"1 02 ) .

Depletion Studies
Pre-depletion treatment

The chamber was filled with a nutrient solution
containing 110 pM nitrogen (90 pM as ammonium-N and 20 jjM as
nitrate-N), 65 pM calcium, 10 juM magnesium, 10 pM sulfate-S,
0.24 pM borate-B, 0.20 pM iron, 0.02 jj M manganese, 0.02 juM
zinc, 0.005 pM molybdate-Mo, 0.005 pM copper but lacking P
and K. The nutrient solution was adjusted to pH 4.5. The
roots were continuously gassed with compressed air and kept
in this system for 24 hours. The purpose of the pre-depletion
treatment was to starve the root system from P and K and to
allow roots to recover from any physical manipulation between
treatments. The chamber was covered with a black plastic mesh
to avoid direct sunlight. The chamber was drained previous to
the nutrient depletion study and flushed with distilled

water .

78

Experiment 1: Nutrient depletion study with air

The root chambers were filled with known amounts of
nutrient solution (c.a. 800-1200 mL). Initial P and K

concentrations for the study were 6.25 pM P (ca. 0.20 mg L"1)

and 25.66 pM K (ca. 1.00 ug mL“l), plus other nutrients as
described above. The nutrient solution was adjusted to pH
4.5. Solution samples (8 mL) were collected at 6-hr interval
up to 72 hours. An equal volume of distilled water at pH 4.5
(8 mL) was replaced. Water uptake was measured by calculating
the volume of water replenished by the Mariotte flasks
between sampling intervals. The system was continuously
aerated with pressurized air. Dissolved oxygen saturation,
and temperature were measured for each root chamber at each
sampling interval.

Experiment 2; Nutrient depletion study with N gas

After completion of the previous experiment, all root
chambers were drained and flushed with distilled water. They
were then subjected to the pre-depletion treatment for 24
hours. Afterwards, the root chambers were filled with equal
amounts of nutrient solution (c.a. 800-1200 mL). Initial P
and K concentrations for the study were the same as
previously described. The nutrient solution was adjusted to
pH 4.5. Water (8 mL, pH 4.5) was added to the chamber
following the removal of each sample. Root water uptake was
measured by calculating the volume of water replenished by

79

the Mariotte flasks between sampling intervals. The system
was continuously gassed with N2 gas (ultra-high purity

grade). The nutrient solution was previously purged with N

2

gas for 2 hours before the depletion study experiment.
Dissolved oxygen saturation, and temperature were measured
for each root chamber at each sampling interval.

iment — 3j Nutrient depletion study with air after N gas

After completion of the gas treatment, all root

chambers were drained and flushed with distilled water. They
were then subjected to the pre-depletion treatment for 24
hours. Afterwards, the root chambers were filled with
nutrient solution containing the same amount of P and K
concentrations as in previous experiments. The system was
continuously aerated with pressurized air. Solution sampling,
and water uptake measurements were done similarly as in
Experiment 1. Oxygen saturation and temperature measurements
were also measured for each root chamber in a similar fashion
as described in Experiment 1 and 2.

Experiments 4 , — 5. and 6: Nutrient depletion study with air/ N
gas/ air 2

After completion of the air experiment, all root
chambers were drained and flushed with distilled water. Then,
six of the seventeen root chambers were then subjected to
the pre-depletion treatment for 24 hours. Experiments 4, 5,
and 6 are repeats of experiment 1, 2, and 3, respectively,
with these six roots.

80

Chemical Analyses

Nutrient solution samples were kept on ice until each
experiment was completed. They were analyzed within 24 hours
of the experiment's completion. Concentration of P was
determined by the method of Murphy and Riley (1962).
Potassium concentration was determined by atomic absorption
spectroscopy.

Data Analyses

Periodic P and K uptake were calculated by expressing
the nutrient content at each sampling interval as a fraction
of the initial nutrient concentration. Corrections were made
for the dilution caused by sample withdrawal. Statistical
comparisons for P, K, C>2 saturation, and nutrient solution

temperature were made using a repeated measures analysis (SAS
Institute, 1985). Treatments were the aeration environments
caused by air or N2 gas. The blocks were trees while the
replicates were the roots from each tree. The measurement

unit was the root chamber and the repeated measures were the
time intervals.

81

Results

Oxygen Concentration and Temperature in the Chambers

For both the air and N2 gas treatments the oxygen-
saturation values were not significantly different at
sampling intervals, nor among root chambers. Therefore mean
values of oxygen saturation (%) for each experiment, were
converted to mg L 1 and uM, based on the average nutrient
solution temperature. They are presented in Table 4-1.

The average temperature of solutions gassed with air was
not different from the temperature of those gassed with N2.
Diurnal temperature fluctuations outside of the chamber
influenced the nutrient solution temperature. They were as
low as 18 °C at 6:00 a.m. samplings and as high as 35 °C at
12:00 a.m. (See Figure 3-7A, Chapter 3). Oxygen
concentrations in root chambers gassed with N2 (Experiments 2
and 5) indicated hypoxic conditions (02 < 50 uM), and were
statistically lower than the air treatment (p < 0.001). For
root chambers gassed with air (Experiments 1,3,4 and 6), all
were aerobic (02 > 50 uM); however, during Experiment 4, the
oxygen concentrations were slightly lower than oxygen
concentrations during experiments 1 and 3 (p < 0.001), (Table
4-1). Although it could be argued that Experiment 4 had
different levels of oxygen than experiments 1,3 and 6, the
oxygen concentration is within the aerobic

range, and both K

Table 4 1. Temperature and dissolved oxygen concentration in nutrient solution, durinq
consecutive experiments of P and K depletion by pine roots.

82

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depletion and P depletion were similar to the first aerobic
treatment.

Potassium Depletion with Alternating Aerobic and Hypoxic
Conditions

Potassium concentration changed little after 66 hours
under aerobic conditions in Experiment 1. However, when the
same roots were gassed with N2 (Experiment 2), K depletion

ceased (Figure 4-1). This was true both times the N2 gas was

used (Figure 4-2). The initial K concentrations in chambers
gassed with N2 gas were not significantly different across the
sampling intervals. This effect was reversible and K uptake
resumed after the pine roots were removed from N2 gas and air

was re-introduced (Experiment 3- Air 2). Yet the K depletion
of the pine roots was not as strong as the first experiment
(Air 1 vs. Air 2, Figure 4-1; p < 0.01). While K was depleted
to 35% of the initial K concentration in Experiment 1 (Air
1), K was only depleted about 15% after air was re-
introduced. After experiment 3 (Air 2, after N2 gas), root
chambers were gassed with air again (Air 3, experiment 4),
and K depletion resumed a similar depletion trend to the
first air depletion experiment. The average K depletion in
aerated solution after a second exposure to N2 gas ion (Air 4)
was only 10% (Figure 4-3). This represents about 70%
reduction in K uptake resulting from residual effects of N2
gas exposure.

84

Potassium Depletion Under Hypoxic Conditions

Potassium depletion ceased in the presence of N2 gas in
both experiments where hypoxic conditions were achieved
(Figure 4-2). On both occasions, there was a K efflux from
the pine roots and the K efflux was not significantly
different between the two experiments.

Phosphorus Depletion with Alternating Aerobic and Hvnnvir
Conditions ' "

Phosphorus concentration changed little after 42 hours
(data not shown) when pine roots were gassed with air or N2
gas (Figure 4-4 and 4-5). p depletion in the presence of N2

gas (Experiment 2) was not significantly different from that
in the presence of air. Initial P concentrations were
depleted about 90% when root chambers were gassed with either
air (Experiment 1 and 3) or N2 (Experiment 2). Phosphorus

depletion of pine roots under aerobic conditions was not
different between four experiments ( Experiments 1,3,4, and
6), even after the effect of intervening N2 gas treatment
(Experiment 3 and 6) (Figure 4—5)

Phosphorus Depletion Under Hypoxic Condi t- -i nnc

While P depletion in a N2 environment was not curtailed
in two separate experiments, the amount of P depleted and the
trend of depletion was significantly different (p < 0.001)

85

Figure 4-1. Potassium depletion by seventeen root systems of
Pmus elliottii var . elliottii. under aerobic and hypoxic
conditions. The root chambers were gassed with air ( ■ )
followed by N2 gas ( # ) and then with air again ( □ ). Refer
to experiments 1, 2 and 3 in the text. Each symbol represent
an average with standard error. Values are expressed as
raction of initial concentration in the nutrient uptake
chamber.

86

o

K 0.9""
O
<

O'

LL

0.8-

POTASSIUM

Hypoxic

•

EXP. 2 - N2GAS 1

o

EXP. 5 - N2GAS 2

nr

"Till

i i i i i i

6

12 18 24 30

36 42 48 54 60 66

Time (Hours)

Figure 4-2. Potassium depletion by root systems of Pinus
elliottn var. elliottii . under hypoxic conditions.

Experiment 2; N2 gas 1 ( • ) is the result from seventeen
root chambers Experiment 5; N2 gas 2 (Q ) is the result from
six root chambers, in both cases the root chambers were

Wlth air bef°re and after the N2 gas treatment. Each
ymbol represent an average with standard error bars. Values
are expressed as fraction of initial concentration in the
nutrient uptake chamber.

87

Figure 4-3. Potassium depletion by seventeen root systems of
Pmus elliottii var. elliottii. under series of aerobic
conditions The root chambers were gassed with air ( ■ )
(Experiment 1; air 1), followed by N2 gas (data not shown).
Then root chambers were gassed with air again ( 3 )
(Experiment 3; Air 2). After Experiment 3, six root chambers
were gassed with air ( • ) (Experiment 4; Air 3), followed by
N2 9as (data not shown). Then root chambers were gassed with
air again ( 3 ) (Experiment 6; Air 4). Each symbol represent
an average. Standard error bars are shown for Exp. 1 and
Experiment 3 only. Values are expressed as fraction of
initial concentration in the nutrient uptake chamber.

88

(Figure 4 6). Only 65% of the initial P concentration was
depleted by pine roots subjected to hypoxic conditions for
the second time (Experiment 5). In comparison 90% of the
initial P concentration was depleted by the same pine roots
when subjected to hypoxic conditions for the first time
(Experiment 2). Therefore P depletion decreased about 28%
when the same roots were exposed to hypoxic conditions for
the second time (Experiment 5).

Discussion

Sudden 02 deprivation (hypoxic conditions) resulted in
net efflux of K from roots of trees within 6 hours. This
response has been previously reported by others (Fisher and
Stone, 1990b; Rosen and Carlson, 1984; Jalil and Carlson,
1993). Active membrane transport maintains high K
concentrations inside the cells against electrochemical
gradients that would otherwise cause K diffusion out of the
roots (Cheeseman and Hanson, 1979). With hypoxia, active
transport ceases and K diffuses from the concentrated

r

internal cell solutions into the considerably more dilute
bathing solution. After 72 hours in the deoxygenated
solution, the same roots then resumed depletion of K after
air sparging resumed. This response has also been observed in
seedlings of slash pine (Fisher and Stone, 1990b) and
seedlings of plum (Jalil and Carlson, 1993). it is
attributable to changes in cell energy levels with are

A

89

' Phosphorus depletion by root systems of Pinus
elliottii var. elliottii . under aerobic and hypoxic
conditions. Experiment 2; N2 gas 1 ( • ) is the result from
seventeen root chambers. Experiment 5; N2 gas 2(0) is the
result from six root chambers. In both cases the root
chambers were gassed with air before and after the N, gas
treatment. Each symbol represent an average with standard
error bars. Values are expressed as fraction of initial
concentration in the nutrient uptake chamber.

90

Figure 4-5. Phosphorus depletion by seventeen root systems of
Pinus elliottii var. elliottii . under series of aerobic
conditions The root chambers were gassed with air ( ■ )
(Experiment 1; air 1), followed by N2 gas (data not shown).
Then root chambers were gassed with air again ( □ )
(Experiment 3; Air 2 ) . After Experiment 3, six root chambers
were gassed with air ( • ) (Experiment 4; Air 3), followed
by N2 gas (data not shown). Then root chambers were gassed
with air again ( 3 ) (Experiment 6; Air 4). Each symbol
represent an average. Standard error bars are shown for Exp.

1 and Experiment 3 only. Values are expressed as fraction of
initial concentration in the nutrient uptake chamber.

91

Figure 4-6. Phosphorus depletion by root systems of Pinus
elliottii var. elliottii. under hypoxic conditions.
Experiment 2; N2 gas 1 ( • ) is the result from seventeen
root chambers. Experiment 5; N2 gas 2 ( D ) is the result
from six root chambers. In both cases the root chambers were

™h air before and after the N2 gas treatment. Each
ymbol represent an average with standard error bars. Values
are expressed as fraction of initial concentration in the
nutrient uptake chamber.

92

directly related to 02 availability rather than to more

fundamental damage of cell membranes. The fact that these
pine roots resumed depletion of K after two consecutive
experiments with hypoxic nutrient solution (each lasting 3
days) further suggests a lack of membrane damage. However a
previous treatment with N2 gas (hypoxic conditions) did reduce
K depletion under aerobic conditions by 70%. This response
could be attributed to either a root recovery time due to
membrane depolarization and/or decrease in root 02 supply

after the N2 gas treatment. However evidence supporting either

possibility was beyond the scope of this study. Nevertheless
if one considers that studies with plum seedlings documented
the same level of K depletion both before and after hypoxic
treatment (Jalil and Carlson, 1993). Root systems of
seedlings are easier to saturate with 02 after N2 gas
treatment as compared to root systems of trees . Root
structural differences such as cortical breakdown and
periderm formation due to secondary growth, might account for
a retarded response in root saturation with 0, after the N,
gas treatment in ou,r experiment.

Unlike K, sudden 02 deprivation (hypoxic conditions for 3
days) did not inhibit P uptake of pine roots in the first
hypoxic treatment, but did reduce it by 28% in the second
treatment. Our results from the first hypoxic treatment are
consistent with previous studies by Larkum and Loughman
(1969) which suggested that P uptake sensitivity to hypoxic

93

conditions is related to the level of P in the solution. P
uptake of roots of barley seedlings was not inhibited -under
short term hypoxic conditions at external P concentration of
10 pM. They concluded that P uptake was maintained solely by
energy from anaerobic metabolism. Yet in pine trees grown
under field conditions, 02 movement through roots within
intercellular gas-filled spaces has been shown to account for
some internal root 02 (Fisher and Stone 1990b; Topa and
Mcleod, 1986b), even in root tissues of pine roots grown
under aerobic soil conditions. Nevertheless a root 02
gradient is likely to be found with outer root cortical
tissue experiencing 02 deficiency. If a 02 gradient existed in

the root tissues during the hypoxic treatments, it was still
too low to maintain K depletion by the pine roots in our
experiment .

Implications of p and K Uptake bv Roots of Pines in
Soil Conditions in the Field

Pine root that developed primarily under aerobic
conditions in the surface soil of a common flatwood Spodosol,
should experience shortages of 02, since periodic water table
rise to near the soil surface will induce reduced soil
conditions that results in anaerobiosis . As much as 80% of
the ootal fine root length of slash pine occurs above the 30
cm depth (Gholz et al., 1986; Van Rees and Comerford, 1986;
Adams et al., 1989). Gholz et al.,(1986) observed seasonal

94

water tables reaching the soil surface in both young (7-year-
old) and old (27-year-old) pine stands, with fine roots
showing no major die backs. Therefore they survived the short
term inundation (2 days). Modeling efforts summarizing the
role of belowground dynamics in a Florida pine plantation at
the ecosystem-level, concluded that high water tables often
encountered in these plantations had no significant effect on
root respiration or mortality (Ewel and Gholz, 1991).
Comerford et al, (1996) concluded that soil 15 cm above a
water table will have reduced soil conditions, documenting as
much as 47% of the total area of a flatwood landscape could
have reduced soil conditions at the soil surface for short
time periods. It is, therefore, reasonable to ask to what
degree P and K uptake differences by pine roots under short-
term reduced soil conditions are relevant to forest
plantations of the southeastern United Sates .

Slash pine roots grown in aerobic soil conditions, and
subjected to hypoxic treatments remain functional for P
uptake but not for K. However for either P or K these roots
are still active for both K and P uptake when the hypoxic
soil conditions are removed. These differences in ion
depletion are relevant to Michaelis-Menten kinetics
parameters and the modeling of nutrient uptake by roots. If
one wants to incorporate water table fluctuations when
modeling K uptake one should account for changes in 1^
values. For example I_ for K will be zero under hypoxic
conditions. Most likely these values will also be a function

95

of time since hypoxic conditions were evident. Therefore one
could estimate that a time cycle of aerobic-reduced-aerobic
soil conditions where the reduced phase last 3 days will

result in only 40 to 50 % of the potential K uptake (Figure
4-7).

Besides the physiological mechanism involved in P and K
uptake by pine roots under hypoxic conditions, our findings
are remarkable if we take into account that soil p limits
pine productivity (Neary et al., 1990a). Documenting the
effectiveness of pine roots to acquire P under reduced soil
conditions has been necessary to evaluate mechanisms of
nutrient uptake which regulate the productivity of forest.

Conclusions

In summary, intact slash pine roots grown under aerobic
soil conditions, and from twelve-year-old pine trees,
responded to levels of 02 availability in the nutrient
solution. The response was different for K than for P. Under
hypoxic conditions, K depletion by pine roots ceased and
resulted in net K efflux. Phosphorus depletion was not
eliminated under hypoxic nutrient solution conditions. My
results can be viewed in the context of fluctuating water
tables under field conditions. They suggest that pine roots
grown in aerobic soil conditions of surface horizons are
capable of p depletion even when reduced soil conditions are

present.

96

AEROBIC AEROBIC AEROBIC

E

3

LU

° %

o

o

OH

80-

.1 2 60-1

X LU

s*:

em

20-

AEROBIC

Potassium (b)

HYPOXIC AEROBIC

Figure 4-7. Potassium uptake potential of slash pine roots
when subjected to cycles of aerobic and hypoxic conditions,
(a) Under a time cycle of three consecutive aerobic soil
conditions, (b) Under a time cycle of aerobic-hypoxic-aerobic
soil conditions.

CHAPTER 5

PHOSPHORUS AND POTASSIUM DEPLETION BY WOODY ROOTS OF TWELVE-

YEAR-OLD SLASH PINE TREES

Introduction

There is no question that white roots of trees are
active zones of nutrient absorption (George and Marschner
1996). But of continuing interest to plant physiologists is
the effect that root development (i.e., changing root
morphology from white to brown to woody) may have on a root's
capacity to absorb ions .

A fully developed endodermis in brown roots, which
normally inhibits the flux of water and nutrients through the
apoplasm, can be compromised by short and lateral root
development. The lateral roots leaves a pathway for mass flow
where they exit the pericycle and break the endodermis. When
secondary xylem growth occurs (woody roots) it can cause
corruption of the endodermis with the cortex progressively
sloughing away (Troughton, 1957). A cork tissue with a cork
cambium will eventually develop, becoming another possible
barrier to apoplastic flow.

Nutrient uptake by "woody roots" of trees has been
reported (Chung and Kramer, 1975; Van Rees and Comer ford,
1990). However, the classification of these roots as woody

97

98

was based only on the external root coloration. Citrus sp.
and Pinus taeda L. have both been shown to have brown roots
that absorb P04 (Crider, 1933; Chung and Kramer, 1975). The
work by Chung and Kramer even suggested that an effective ion
barrier existed allowing selective ion absorption. Kramer
(1946) suggested that the selective barrier was either the
cork cambium, the vascular cambium, or both. Uptake of P04 by
brown root of Prunus has also been reported (Atkinson and
Wilson, 1980). Van Rees and Comerford (1990) showed that
brown roots from pine seedlings can absorb K and Rb from
nutrient solutions . Yet all claims of nutrient uptake by
woody roots, based on studies of nutrient uptake by brown
roots, are still questionable. This is because brown roots
have traditionally been considered woody (e.g., Blake and
Hoogenboom, 1988), and are often described as suberized
(Addoms, 1946; Kramer, 1946; Van Rees and Comerford, 1990).
Terms such as "brown root" and "suberized root" are used
imprecisely or incorrectly when used to describe root
morphology and anatomy related to secondary growth (Richards
and Considine, 1981). The brown coloration of roots is a poor
indicator of anatomical attributes that define secondary
growth of loblolly pine roots (nonwoody vs. woody)

(unpublished data). McKenzie and Peterson (1995b) concluded
that roots from pine trees were divided into three
anatomically distinct zones. Beginning at the root tip these
are 1) the white zone, 2) the tannin zone in which the cells
external to the endodermis are dead and their walls are

99

modified by tannin deposits, and 3) the cork zone in which
the process of secondary growth results in the formation of
cork cells. Microscopic investigations of jack pine ( Pinus
banksiana Lamb) (McKenzie and Peterson, 1995a) revealed that
white roots were not colored internally, despite the presence
of suberin in the endodermis. The transition from white to
brown along the root axis was due to the deposition of
tannins in cell walls. Therefore the term "suberized root"
should not be used because all zones of the root contain
suberin, both in the Casparian bands and suberin lamellae. It
is also an inappropriate term for the tannin as the outer
cortical cells are tannified and may not necessarily be
suberized.

because of their abundance, woody roots have the
potential to play a major role in nutrient uptake of trees.
For example, in a twelve-year-old loblolly pine (Pinus taeda
L.) stand, woody fine root tissues constituted from 62 to 80%
of the fine root surface area (SA) available for nutrient
uptake (Comer ford et al., unpublished data). Furthermore, the
research on nutrient uptake by brown root comes principally
from experiments which used either excised roots (e.g. Chung
and Kramer, 1975) or intact roots from tree seedlings grown
under laboratory or greenhouse conditions (e.g. VanRees and
Comerford, 1990). Nothing is known about nutrient absorption
by intact, woody roots of perennial plants under field
conditions. Only a very few attempts have been made to
develop techniques to study the nutrient

uptake of different

100

root types under field conditions. These studies included
nitrogen uptake by long roots of Norway spruce ( Picea abies
(L.) Karst) (Marschner et al. , 1991), and by whole root
systems of peach trees (Prunus persica (L) Batsch) (Goutouly
and Habib, 1996). In both cases the nutrient uptake capacity
of brown or woody root zones was not evaluated.

^roc^uc-:t'.ivi_'ty of southern pine stands of the southeastern
lower coastal plain is limited by nutrient availability
(Neary et al., 1990). Comer ford and Neary (1991) used a
nutrient uptake model to show that, for a semi -mature slash
pine stand, the only way to accurately predict root uptake of
phosphorus (P) was to either adjust mineralization rates or
to limit P uptake to a small percentage of the fine root
system. The latter suggests that a significant portion of the
fine-root (brown-woody) system is not functional in absorbing
P. Consequently, a major question regarding the process of
nutrient uptake by pine trees is to ascertain whether woody
roots function in nutrient uptake. Again, the importance of
this question for southern pine is obvious when one considers
80% of the total fine root length and surface area falls in
the woody category .(Comerford et al., unpublished data).

This study documented P and K depletion of fine roots
for twelve-year-old slash pines growing under field
conditions. The objectives of this study were (i) to compare
P and K depletion between whole fine root systems and woody
fine root systems of slash pine roots and (ii) to compare P
and K depletion between the whole fine root system and woody

fine root systems when gas was used as a metabolic
inhibitor of ion absorption.

101

Materials and Methods

Root System Description

Twelve-year-old slash pine trees (Pinus elliotii Engelm.
var. elliotii) were selected from a research plot in north
Florida that was being treated with complete weed control.

The soil is a poorly-drained Pomona fine sand (sandy,
siliceous, hyperthermic Ultic Alaquod). The study site is
about 10 km north of Gainesville, FL (29° 80' N, 82° 20' W) .

The climate is warm, temperate-subtropical with a mean annual
precipitation of 1332 mm most of the precipitation which
occurs in summer, with the least falling in autumn and
spring. Mean annual temperature is 21 °C.

Four lateral roots (1-1.5 cm in diameter) were
previously excavated at the base of a tree and the fine-root
system was preserved as much as possible. A root regeneration
system was established as described in Chapter 3. Briefly,
lateral roots were pruned, placed in 50 x 25 x 6 cm black
plastic trays, and covered with sieved soil from the A
horizon of the study plot. Black plastic mesh was placed over
the tray and the tray was covered with pine litter from the
plot. The root system was supplied with a nutrient solution
typical of soil solutions of a Florida Spodosol (Van Rees and
Comerford, 1990). Nine months after the installation of the

102

root regeneration system, and prior to initiating these
studies, thirty-six lateral roots from 9 trees were gently
uncovered and washed with distilled water. Thirty-three
lateral roots from these 9 trees were used for the 1995
studies . Twenty months after the installation of the root
trays and prior to the studies, twenty intact lateral roots
from 5 trees were uncovered and washed with distilled water.
Seventeen lateral roots from these 5 trees were used for the
1996 study.

Nutrient Uptake System Installation

The lateral roots were inserted (while still attached to
the tree) into uptake chambers. The construction and assembly
of the nutrient uptake root chamber is fully described in
Chapter 3. The root chamber is part of an in-situ nutrient
uptake system which includes (a) the gas system, (b) the root
uptake chamber and, (c) the Mariotte flask system. The root

chamber was gassed with compressed gas tanks filled with
either air (medical standard) or N2 (99.9%). The Mariotte

flask system was used to maintain a constant solution volume
in the root chamber . Aeration of each root chamber was
monitored by measuring root-chamber dissolved oxygen with a

Yellow Springs Instrument (BSI) portable oxygen meter and a
Clark type membrane-covered probe (accuracy 0.1 pg mL"1 02).

103

Depletion Studies
Pre-depletion treatment

This pre-depletion treatment was performed prior to each
depletion experiment. The chambers were filled with a
nutrient solution containing 110 pM nitrogen (90 pM as
ammonium-N and 20 pM as nitrate-N), 65 pM calcium, 10 pM
magnesium, 10 pM sulfate-S, 0.24 pM borate-B, 0.20 pM iron,
0.02 pM manganese, 0.02 pM zinc, 0.005 pM molybdate-Mo, 0.005
pM copper but lacking P and K . The nutrient solution was
adjusted to pH 4.5. The roots were continuously gassed with
compressed air and kept in this system for 24 hours. The
chamber was covered with a black plastic mesh to avoid direct
sunlight. Prior to the nutrient depletion studies, the
chamber was drained and flushed with distilled water.

Previous to each pre-depletion treatment, a photograph
of every root system was taken. The root systems were
carefully extended on top of a 30 cm x 100 cm clear acrylic
plastic sheet (0.6 cm thick). The acrylic plastic sheet was
marked with a 1 cm x 1 cm grid for reference. The photo was
used to account for any new root growth between depletion
experiments .

To protect roots from dessication, root chambers were
©penned for root manipulation only before sunrise or after
sundown. All root systems were shaded using a black umbrella
and kept moist by continuously spraying with distilled water.

Study #1. p and K depletion bv brown roots: Isolation of
white roots by waxing

104

Experiment l.a: Nutrient depletion study with air:

— — root — systems . Fifteen lateral roots from 4 trees were
used for this experiment. Root chambers were filled with
known amounts of nutrient solution (c.a. 800-1200 mL) .
Initial concentrations of P and K for the study were 6.25 jj M
P (ca. 0.20 pg mL-1) and 25.66 pM K (ca. 1.00 pg mL-1), plus
other nutrients as described in the previous section. The
nutrient solution was adjusted to pH 4.5. Solution samples (8
mL) were collected at 0, 8, 24, 32, 48, and 66 hours. At the
time of sampling an equal volume of distilled water at pH 4.5
(8-mL) was replaced in order to maintain a constant volume.
Water uptake was measured by calculating the volume of water
replenished by the Mariotte flasks between sampling
intervals. The system was continuously aerated with
pressurized air. Dissolved oxygen saturation, and temperature
were measured for each root chamber at each sampling
interval .

Experiment l.b: Nutrient depletion study with air: waxed
roots . After completion of the previous experiment, all root
chambers were drained and flushed with distilled water. From
each tree, 2 root chambers were disassembled in the field.

The white root portion, along with < 1 cm of the adjacent
brown roots, were covered with wax by submerging the roots
for several seconds in melted embedding medium grade paraffin
wax (Oxford Labware, St. Louis MO, USA; ASTM melting point

105

50-54 °C). The purpose of waxing brown roots next to white
roots was to render nonwoody portions inactive in ion. uptake.
After the waxing, the root chambers were re-assembled. Eight
roots were subjected to this treatment. The other 7 roots
were not waxed. Afterwards, all 15 root chambers were
subjected to a pre-depletion treatment. The root chambers
were then filled with nutrient solution containing the P and
K concentrations as previously described. The system was
continuously aerated with pressurized air. Solution sampling
and water uptake measurements were made as in Experiment l.a.
Oxygen saturation and temperature were measured throughout
the experiment.

Experiment l.c: Nutrient depletion study with N gas .

After completion of the previous experiment, all root
chambers were drained, flushed with distilled water, and
subjected to the pre-depletion treatment for 24 hours. The

root chambers were then filled with fresh nutrient solution
( as above ) , but purged with gas for 2 hours prior to the

experiment. A depletion study was run as before, but chambers
were continuously gassed with (ultra-high purity grade)

rather than air. Dissolved oxygen saturation, and temperature
were measured for each root chamber at each sampling
interval .

Experiment 1 . d : — Nutrient depletion study with air after
N^gas. After completion of treatment with N2 gas, all root

chambers were drained, flushed with distilled water, and
subjected to the pre-depletion treatment. A further depletion

106

experiment was run in the manner described for experiment la,
gassing all root chambers with compressed air.

After completion of this experiment, the root system
inside each chamber was cut from the tree at the point where
it entered the chamber. The whole root chamber, with the
intact root system, was transported immediately to the
laboratory for root length measurement and sectioning.

Study #2 (1995) and study #3 (1996 ). P and K depletion by
brown roots: Isolation of white roots by cutting

Experiments 2a-d and 3.a-d were identical to those
described above, with the following exceptions. Fourteen
lateral roots from 4 trees were used for study #2, while 17
laterals from 5 trees were used for study #3. In experiment
2b and 3b, the white roots and their adjacent brown roots
were prevented from absorbing P and K by removing them from
the tree. This was accomplished by cutting them from the
lateral root with a pair of scissors. The cut end was then
sealed with Vaseline. In experiment 2, 9 roots were cut, and

5 left intact. In experiment 3, 11 roots were cut and 6 were
left intact.

Study #4. P and K depletion by whole root systems subjected
to NaOCl solution

Sodium hypochlorite (NaOCl) solution was used for root
surface sterilization to render microorganisms growing on the
root surface inactive in ion inmobilization. Seven lateral
roots from 2 trees were used for this

experiment. The root

107

chambers were filled with known amounts of nutrient solution
(c.a. 800-1200 mL). initial P and K concentrations for the
study were 6.25 gM P (ca. 0.20 mg L-1) and 25.66 K (ca.

1 * 00 uq mL-1), plus other nutrients as described in previous
sections. The system was continuously aerated with
pressurized air. Solution sampling was done similarly as
described in previous experiments . After completion of this
experiment, the whole root system was soaked for 5 minutes in
1% NaOCl solution, then rinsed thoroughly five times with
distilled water and subjected to the pre-depletion treatment.
Afterwards, the root chambers were filled with nutrient
solution containing the same amount of p and K concentrations
as in previous experiments. The system was continuously
aerated with pressurized air. Solution sampling, and water
uptake measurements were similar to the first experiment.

After completion of the above depletion experiment, the
whole root system was rinsed with distilled water and again
subjected to the pre-depletion treatment. Afterwards the
nutrient depletion experiment was repeated.

Laboratory Incubation Studies

Inoculation with root scrapings

After experiment 2 .d. (nutrient depletion study with air
after gas ) root chambers with whole root systems were

detached from the tree and disassembled before being
transported to the lab. The root surface was gently scraped

108

with a wire loop to collected rhizodeposition, including
loose cortical root cells invaded by microorganisms. The
purpose was to collect root surface microoganism to asses
their role in K or P inmobilization. Surface scraping was
done five times per root, for 8 roots and used to inoculate
50 mL of fresh nutrient. The blank consisted of 50 mL of
nutrient solution brought to the field, and subjected to the
same procedure, but without receiving root scrapings. Aseptic
conditions were maintained by immersing the wire loop in an
ethanol solution (70%) between root samples. All samples were
immediately transported to the laboratory. At the laboratory,
two 10 mL subsamples were taken from each 50 mL sample. One
subsample was autoclaved, and the other one received no
treatment. The two subsamples were incubated at 30 °C for 48
hours . Solution samples were taken before and after
incubation for measurement of differences between P and K
concentration before and after incubation.

Inoculation with nutrient solution

After experiment 2.d., the bulk nutrient solution from 7
randomly selected root chambers was collected and transported
to the laboratory where a 12 mL aliquot from each chamber was
added to a 50 mL volumetric flask. This was brought to volume
with freshly made nutrient solution. The purpose was to
collect microoganism from the nutrient solution to asses
their role in K or P inmobilization. Two 20 mL subsamples
were taken from each 50 mL volumetric flask. One subsample

109

was autoclaved, and the other one received no treatment. The
two subsamples were incubated at 30 °C for 48 hours. Solution
samples were taken before and after incubation for
measurement of differences between P and K concentration
before and after incubation.

Ion Mass in the Root Free Space

Upon completion of experiment 3.f., 6 intact root
systems from the uncut treatments were transferred to a walk-
in cooler and soaked in 0.8 L of a nutrient solution (8 °C,
pH 4.5) lacking P and K. Two, 10 mL subsamples were taken
from each flask using a syringe, one immediately after
immersion in the nutrient solution and the other 8 hours
later. Immediately following the cold treatment, roots from
each container were placed in plastic bags and stored in a
freezer for further root length and root section analysis.

Root Measurements

Once in the laboratory, root systems were removed from
the root chambers and separated into coarse and fine-roots.
Coarse-roots were operationally defined as regenerated
lateral roots from which the fine roots regenerated. They
were > 2 mm in diameter (3-5 cm range). Fine roots were < 2
mm in diameter. The waxed root systems were separated into
waxed and non-waxed portions. The waxed portions were
carefully sandwiched between moist pieces of Parafilm
(American Can Company). The whole root systems (never cut or

110

waxed) were separated in white and brown fine roots. The
white roots were also put between layers of Parafilm. Whole
root system is operationally defined as the fine root system
that was always kept intact during the experiment. Root
lengths were calculated from digitized images using a flat-
bed scanner . Root lengths were measured from the digitized
images using the program "Branching" (Berntson, 1992). Root
lengths corresponding to coarse and fine-roots where recorded
separately. Fine-root categories included brown, white, waxed
and cut.

Anatomical Observations of Root Sections

Roots were hand-sectioned in the manner described by
Frohlich (1984). Roots were sandwiched between pieces of
Parafilm and placed on a plastic Petri dish while being
sectioned. Multiple cross-sections were made within 10 mm of
the distal end of the root. Cuts were easily oriented as the
roots were visible through the semi-transparent layers of the
Parafilm. All detached fine roots (brown, white and waxed)
from each root system were sampled. For each detached fine
root, 3 cross sections were selected randomly and observed
under light microscopy. Root developmental stage was
determined using bright-field or polarized light. Roots were
considered nonwoody (primary) or woody (secondary) based on
descriptions developed by McCrady and Comerford (unpublished
data). Briefly, roots were nonwoody if no vascular cambium or
secondary xylem elements were present external to the

Ill

centrally located metaxylem. Roots with secondary xylem
elements located external to the metaxylem were classified as
woody. Root sections were only characterized based on
developmental stage (primary or secondary growth) .

Chemical Analyses

Samples of nutrient solution were kept on ice until each
experiment was completed an analyzed within 24 hours of the
experiment's completion. Inorganic P was measured
colorimetrically by the procedure of Murphy and Riley (1962).
P concentration was not determined for Experiments la-d., as
preliminary studies under greenhouse conditions had
demonstrated interference due to binding of P by the wax
surface. Potassium concentration was determined by atomic
absorption spectroscopy.

Data Analyses

Oxygen saturation, and nutrient solution temnpratnna

Statistical comparisons of 02 saturation and nutrient
solution temperature were made using a repeated measures
analysis (SAS Institute, 1985). Treatments were the aeration
environments caused by air or N2 gas. The blocks were trees
while the replicates were the roots from each tree. The
measurement unit was the chamber (with root) and the repeated

measures were the time intervals between samplings. There was
no difference in time sampled for both 02 saturation and

temperature in the chambers . Comparisons between treatments

were made using Tukey's multiple comparison procedures (Ott,
1988) .

Phosphorus and K uptake and mass flow

Estimates of mass flow influx of P and K were used to
evaluate the maximum amount of each nutrient that could be
acquired by passive uptake. Nutrient influx was calculated as
the product of the mean P or K concentration in solution and
water consumption. "Passive" uptake, as defined here, is used
to describe potential nutrient uptake through the apoplastic
pathway, requiring no metabolic energy. The difference
between total ion uptake and mass flow ion influx is
equivalent to the minimum "active" uptake (Van Rees and
Comerford, 1990). Total uptake was calculated by expressing
the nutrient concentration at each sampling interval as a
fraction of the initial nutrient concentration. Eight lateral
roots in study #1 (where whole lateral root systems were
subjected to waxing), and 11 root systems in study #3 (where
whole lateral roots were subjected to cutting) were compared
for total depletion. Potential uptake was calculated for the
same roots in the manner described earlier.

Phosphorus and K influx

To calculate influx rates of p and K (jjmol cm"1 h"1 ) I

plotted for each time interval. Q. was the total pool of P
or K in solution per unit root length (pmol cm'1), and time

113

was in hours. Depletion curves from studies #1, #2 and #3,
were expressed as n mol cm (QL) for each time interval as
described by Classen and Barber (1974). QL vs. time, was fit
with a non-linear least squares program (SAS Institute, 1985)
to the equation

Ql = Qi^xp (-rt 1/2 ) (4)

where t is time in hours, QL is the amount of P or K in
nutrient solution at a given time (pmol cm"1) , Q. is the
initial amount of P and K in solution (pmol cm"1), exp is the
base of the natural logarithm (i.e. 2.7182), and r is a
constant. The net influx (In) (pmol cm"1 h"1 ) of P and K per

root system is given by -dQL /dt. Influx (In) per unit root
length per unit time is given by:

In = - dQL/dt= -r/(2t1/3) Qi (5)

Influx of P and K were estimated at a fixed nutrient

solution concentration for each root system. For P, the fixed

nutrient concentration was 5.0 x 10"3 ^mol cm'3 P (ca 0.16 mg
-1

L ), while for K was 2.0 x 10"2 pmol cm'3 K (ca. 0.77 mg L *).
These estimates were transformed to natural logarithms to
achieve a normal distribution. When efflux was observed (a
negative value), I transformed as In | In | . Ln (In) data was
compared by a one-way analysis of covariance (SAS Institute,
1985). Treatments were the aeration environments caused by
air or N2 gas and method of isolating roots (cutting or
waxing, vs. uncut). The blocks were trees while replicates
were the roots from each tree. The measurement unit was the

114

chamber (with root). Treatments were looked upon as 2 x 2
factorial [Whole/Brown (due to cutting or waxing) x Air/N-,
gas] plus a Control. The goal was to determine if influx of P
or K was affected by the method of isolating brown roots
(cutting or waxing vs. whole), and if the gassing of the
nutrient solution (Air/N2 gas) affects P or K influx. The
covariant was the nutrient influx of a root system before a
given treatment. The variant analyzed was Ln |ln| of a root
system at a given treatment. The experimental layout is
presented in Table 5-1 for studies 1,2 and 3. When a
covariant was significant and treatment differences were
significant, mean comparisons were made using Least square
means (SAS Institute, 1985). The adjusted treatment means of
the covariance analysis were analyzed further with orthogonal
contrasts comparing "control vs. treatments", "whole vs.
woody (either waxed or cut)", "air vs. N2 gas", and
" (whole/woody ) * ( air/ N2 gas)".

Phosphorus and K uptake. NaQCl solution study

Samples from study #4 were compared in a similar manner
to those described for total P and K uptake in the mass flow
section. Briefly, the nutrient content at each sampling
interval was expressed as a fraction of the initial nutrient
concentration, and its relationship with time was fit to
equation 1. Values of r for individual root systems were
compared for analysis of variance (ANOVA) (SAS Institute,
1985). Treatments were considered to be (1) prior to

Table 5-1. Experimental design of Covariance analysis used for P and K Influx
data obtained from depletion experiments of studies 1,2 and 3.

115

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116

treatment with NaOCl solution, and (2) after the treatment
with NaOCl solution. When treatment differences were

icant , comparisons between treatments were made using
Tukey's multiple comparison procedures (Ott, 1988).

Laboratory incubation studies

For each sample, differences were calculated between P
and/or K concentration before and after incubation.
Differences between the paired measurements were compared
between the autoclaved and non-autoclaved treatments using
paired t-test (SAS Institute, 1985).

Results

Anatomical — Observations and Root System Descriptions

All sections of white roots were primary roots (n= 252),
while all brown root sections from the cutting and waxing
experiments had secondary growth (woody roots; n=738).
Therefore, all roots previously described as brown (wax and
cut experiments) will be referred to as woody fine roots
throughout the following results and discussion sections.

Description of root systems used for the 3 field studies
are presented in Tables 5-2, 5-3 and 5-4. The volume of
nutrient solution in the root chambers varied from 675 mL to
1250 mL, due to differences in root length (tap and fine).

The percentage of woody roots as a portion of the total root
systems ranged from 24% to 100%. The portion of the fine-root

117

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a)

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Description of root systems used in study two, where roots were subjected to
cutting (1995 depletion experiments.)*

118

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to the treatment of isolating brown surface areas. See Text
roots (Lateral root). Fine root < 2 mm in diameter.

119

Refers to the treatment of isolating brown surface areas .
Coarse roots (Lateral root), fine root < 2 mm in diameter
based on external color, f see text for details.

120

Refers to the treatment of isolating brown surface areas.
Coarse roots (Lateral root), fine root < 2 mm in diameter
based on external color, i see text for details.

system that was waxed varied from 24% to 88%, while the
portion that was cut ranged from 7% to 80% of the total,
fine-root length.

121

0, Concentration and Temperature in the Chambers

The average temperature of solutions gassed with air was
not significantly different from the temperature of those
gassed with N2 (Table 5-5 and 5-6). Therefore, mean values of
oxygen saturation (%) for each experiment, were converted to
pM, based on the average nutrient solution temperature.

Results are presented in Tables 5-5 and 5-6. 02 concentrations
in root chambers gassed with N2 indicated hypoxic conditions
(02 < 50 p M), and were significantly lower than the air

treatment (p < 0.001). All root chambers gassed with air were
aerobic (02 > 50 pM) .

Microbial Interferences

There was no evidence of uptake of K or P by
microorganisms during the depletion studies (Table 5-7).
Initially, treatment with 1% NaOCl solution totally inhibited
K uptake (Table 5-7-). However, in a second uptake experiment
following the NaOCl treatment, K uptake resumed with a
significant increase when compared to K uptake before the
NaOCl treatment (Table 5-7). Similarly, P uptake was
initially suppressed after treatment with NaOCl but resumed
with a second uptake experiment (Table 5-7). When root

122

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124

scrapings and nutrient solution from the growth chambers were
incubated with a fresh nutrient solution, there was also no
evidence of microbial uptake for both P and K (data not
shown ) .

Potassium and Phosphorus Influx

For the 6 root systems tested, P and K concentrations in
the root free space were negligible (data not shown).
Therefore, no corrections to the depletion estimates for the
storage of ions in the free space were made. Two methods were
used to determine if active uptake was occurring. The first
estimated the maximum amount of mass flow that could be
accounted for by considering the water influx to the root and
the concentration of the ion in the solution. For K, I
estimated that a maximum of 30% of the total K absorbed by
aerated roots could have been absorbed by mass flow (Figure
5-1). Using the second method I attempted to use N2 to inhibit
the active uptake mechanism. N2 significantly inhibited K

uptake for both whole root systems and woody roots (Tables 5-
8 and 5-9 ) .

Mass flow had a maximum of 25% of the total depletion of
P from the nutrient solution due to non-active uptake
(Figures 5-2, 5-3). in general, N2 did not have a significant
effect on P absorption (Table 5-8 and5-9). However, there was
some evidence that hypoxic conditions did affect P influx but
only for one study and only for the whole root system (Table
5-8). Rates of p and K uptake for woody roots were

Table 5 7. Comparison of constant rate of depletion (r) f and C * for P and K depleted by

pine roots treated with 1% NaOCl. Study four.

125

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126

•

TOTAL WHOLE

■ TOTAL WOODY

o

ACTIVE WHOLE

□ ACTIVE WOODY

Figure 5-1. Calculation of potassium depletion due to mass
flow by eight root systems of Pinus elliottii var. elliottii.
The root chambers were gassed with air. Solid symbols ( • ■ )
represent the total K depleted. Open symbols ( Q CJ )
represent the K depleted not accounted by mass flux (active).
Circles ( # 3 ) represent K depleted by the whole root
system. Squares ( ■ □ ) represent the K depleted by the woody
root systems. Each symbol represent an average with standard
error. Values are expressed as fraction of initial
concentration in the nutrient uptake chamber.

127

similar to rates of ion uptake of whole (woody and nonwoody)
roots (Table 5-9).

Discussion

These results demonstrate that woody fine roots of
twelve-year-old slash pine actively absorb P and K. However,
this conclusion assumes that the root manipulations have not
influenced uptake. Possible criticisms of the methodology are
that (i) some uptake could be ascribed to microorganism
activity on the root surface and that (ii) cutting and waxing
of roots can result in mechanical shock, hence affecting
uptake .

Concern over microbial uptake of P and K during the
depletion experiments was shown to be unfounded. When root
scrapings and nutrient solution from a root chamber study
were added to a nutrient solution, there was no evidence of a
significant microbial effect between autoclaved and non—
autoclaved samples. Likewise, less than 1% of the depleted P
can be explained by potential P immobilization by
microorganisms. Here, I assumed that soluble C released by
roots ranges from 10-100 mg C g*1 root (Newman, 1985) to 143-
455 mg C g root (Lambers, 1987). Using the upper and lower
ends of these values and a pine root wood density of 1 g cm*3
with a root radius of 0.05 cm, then the potential C released
is between 0.01 -0.45 g C cm'1 root. With a C:P ratio of 16:1
in microbial tissue (Stevenson, 1986) and assuming all C
released is used for microbial growth, p

Table 5-8 Covariance analysis for P and K influx (Ln( Inf lux) ) by pine
root systems of studies 1,2 and 3.

128

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• TOTAL WHOLE ■ TOTAL WOODY

O ACTIVE WHOLE □ ACTIVE WOODY

Figure 5-2. Calculation of phosphorus depletion due to mass
flow by eleven root systems of Pinus elliottii var.
elliottii. The root chambers were gassed with air. Solid
symbols ( • ■ ) represent the total P depleted. Open symbols
(Op) represent the P depleted not accounted by mass flux
(active). Circles ( # O ) represent P depleted by the whole
root system. Squares ( ■ □ ) represent the P depleted by the
woody root systems . Each symbol represent an average with
standard error. Values are expressed as fraction of initial
concentration in the nutrient uptake chamber.

131

•

TOTAL WHOLE

■ TOTAL WOODY

o

ACTIVE WHOLE

□ ACTIVE WOODY

Figure 5-3. Calculation of phosphorus depletion due to mass
flow by eleven root systems of Pinus elliottii var.
elliottii. The root chambers were gassed with N2 gas. Solid
symbols ( • ■ ) represent the total P depleted. Open symbols

( -> . ) represent the P depleted not accounted by mass flux

(active). Circles ( • O ) represent P depleted by the whole
root system. Squares ( ■ □ ). represent the P depleted by
woody root systems. Each symbol represent an average with
standard error. Values are expressed as fraction of initial
concentration in the nutrient uptake chamber.

132

immobilization would be between to 2 x 10'11 and 9.1 x 10'10
^jrnol ? cm-1 , less than 1% of the concentrations, even at the
end of the depletion studies. This approach is consistent
with those of Darrah (1993).

Physical manipulation of roots has been shown to affect
subsequent nutrient uptake. For example, just dipping
hydroponically-grown roots in and out of a solution disturbed
P absorption (Gronewald and Hanson, 1980). Bloom (1989)
suggested waiting 24 hours after manipulation before
commencement of measurements in order to allow for recovery
of roots. This suggestion was adapted in these studies.
However, dipping in hot wax and pruning roots is likely to
cause a significant degree of heat and mechanical shock. In
particular, cutting has the potential to open breaks in the
roots and allow for more water influx and hence mass flow.
Root pruning has been shown to initially affect K uptake in
barley (Bloom and Caldwell, 1980), and has influenced both
root respiration and N uptake (Bloom, 1989). In the case of K
uptake, this effect appeared to last for 6 hours after
incision (Bloom and Caldwell, 1988), while for pruning of
corn roots, P uptake was relatively unaffected (Gronewald and
Hanson, 1980). Therefore, it appears that the effect of
physical manipulation is variable and a concern when trying
to estimate ion uptake. The major question addressed in this
study is whether woody fine roots function in uptake and
whether there is evidence that the uptake can be classified
as active. While physical manipulation may affect the actual

133

uptake rate and the magnitude of the effect is unknown, there
is no evidence to suggest that the inhibition of uptake in
the presence of N2 gas will be affected. Therefore, these data
are useful for answering the major questions of the study,
while recognizing that the magnitude of the actual influx
rates of the manipulated roots may be affected. I conclude
then, that woody fine roots of these 12-year-old trees
functioned in P and K uptake in this experiments , and that
they appeared to have active uptake.

This conclusion is consistent with studies of other pine
species, with the major difference being that previous
studies worked with seedlings (Van Rees and Comerford, 1990;
slash pine) or young trees under greenhouse conditions (Chung
and Kramer, 1975; loblolly pine) rather than large trees.
Further, these earlier studies had no evidence to suggest
that roots were woody, only that the roots were brown.

We conclude, that not only do woody roots function in
ion uptake, but that active uptake is implicated. Both the
use of N2 as a metabolic inhibitor and the estimates of the
potential maximum mass flow influx demonstrate than an
oxygen-requiring mechanism for uptake is responsible for the
majority of K uptake. The mass flow evaluation suggested
active uptake of p, while gassing with N2 did not affect
uptake. The results of the N2 treatment could be interpreted
against active uptake of P, but in combination with the
results presented in Chapter 4 and the mass flow evaluation a
better interpretation is that the mechanism of P uptake is

134

insensitive to conditions of low 02. The anatomical pathway,
whereby woody roots absorb P and K, were not addressed, in
this study. They are uncertain and invite further study.

Woody roots have some secondary xylem development. In some of
these roots, the casparian band is still functional. In
others it is disrupted. Consequently there is a range of root
anatomies in woody roots that are likely to be associated
with a range of uptake pathways.

Assuming that woody fine roots actively function in P
and K uptake, then the next question is how important are
these roots to a forest stand? Woody fine roots may
constitute approximately 60-80% of the total fine-root
surface area at this study site (McCrady and Comeroford,
unpublished data). Therefore, the total uptake of nutrients
by the tree would be greatly understimated if this surface
area was not included in uptake models. Estimates of P uptake
previously made for this study site (data not shown), found
that predictions of p using total root length were greater,
by about a factor of 2, than the stand actually absorbed. It
was hypothesized that the lack of uptake by woody roots might
account for this discrepancy. However, the data presented
here suggests that this hypotheses is erroneous, and that
woody roots are indeed likely to contribute to overall
nutrient uptake. Other studies showed that root spatial
pattern, promoting inter-root competition, could reduce
predicted uptake by about 50% (Comerford et al., 1994b)
making this a possible important consideration in modeling of

135

uptake of natural ecosystems and the primary explanation of
uptake overestimates. The description of other important
processes, still need to be adequately evaluated.

In summary, woody, fine-roots of field grown pine trees
absorb P and K, and appear to do so in an active manner.

Since they are a large portion of the total fine-root system,
they are an important source of nutrient uptake by pine trees
and need to be included in estimates of the effective root
surface area.

CHAPTER 6

OVERALL DISCUSSION

Nutrient management of forest plantations in the
southeastern United States is focusing on matching nutrient
suppiy to tree demand. These nutrient management efforts can
be aided through the use of nutrient uptake models . P and K
uptake by field-grown slash pine seedlings have been
successfully predicted using nutrient uptake models (Van Rees
at al. , 1990; Smethurst and Comerford, 1993). However,
nutrient uptake models have overestimated P uptake when used
for mature slash pine plantations. Factors that could account
for overprediction of P uptake by older pine trees growing
under field conditions are (i) the ability to define what is
the functional or effective root absorbing surface; (ii) lack
of knowledge about the relative amounts of different
absorbing surfaces present and the nutrient absorption rates
of each type of surface; (iii) poor understanding of the
ferential response of the absorbing surfaces to
environmental variables; and (iv) the effect of the spatial
distribution of roots at the scale of inter-root competition.

From the above mentioned list, spatial root pattern is
the only factor tested that could account for overprediction
of P uptake by older pine trees growing under field
conditions. A random spatial pattern at a scale of inter-root

136

137

competition can reduce P uptake efficiency by as much as 50%
(Escamilla et al., 1991; Comerford et al., 1994b).

Estimation of different root absorbing surfaces have
documented that under field conditions, brown roots may
contribute over 90% of the fine root system of slash pine
(Pinus elliottii Engelm. var. elliotii) and up to 80% of
those fine brown roots are woody (McCrady and Comerford,
unpublished data). However all claims of nutrient uptake by
woody roots are based on studies of nutrient uptake by brown
roots and they come from experiments which used either
excised roots (e.g. Chung and Kramer, 1975) or intact roots
from tree seedlings grown under greenhouse conditions (e.g.
Van Rees and Comerford, 1990). The brown coloration of roots
is a poor indicator of anatomical attributes that define
secondary growth of loblolly pine roots (nonwoody vs. woody)
(McCrady and Comerford, unpublished data). Nothing is known
about nutrient absorption by intact, woody fine roots of
perennial plants under field conditions. Of continuing
interest to plant physiologists is the effect that root
development (i.e, changing root morphology from white to
brown to woody) may, have on a root's capacity to absorb ions.
Moreover, because of their abundance, woody roots have the
potential to play a major role in nutrient uptake of trees.

Field studies of root uptake tend to be complicated due
to access to plant roots below the soil, plant age and plant
size. This is especially true when considering mature trees.
Not surprisingly, I found only two useful examples of

138

nutrient depletion studies in the field and a handful of
related studies (Marschner et al., 1991; Goutouiy and Habib,
1996; Glavac and Ebben, 1986). Therefore I had to implement a
nutrient uptake system that can be used for trees growing
under field conditions. The design included (i) a root
chamber; (ii) a system for controlling the 02 level of the
root chamber and; (iii) a Mariotte flask system. I used this
system to clarify if woody fine roots of trees, growing under
field conditions, function in the absorption of P and K. I
showed that woody roots actually do function in P and K
uptake, since the hypothesis that woody fine roots do not
play a role in nutrient uptake is refuted.

Under field conditions absorbing surface areas of trees
can be exposed to changing soil environmental variables. An
important characteristic of many forest soils of the
southeastern United States is a seasonaly high water table
that reduces 02 supply to a large portion of the root system.
Pine roots growing in the top 16 cm can account for 50% of
the total fine root system (Van Rees and Comerford, 1986). I
documented that nutrient uptake of slash pine roots was
affected by levels of oxygen availability but that the
response differed for K and P. Under hypoxic nutrient
solution conditions (02 < 50 ^M), K depletion by pine roots
was totally inhibited and even resulted in net efflux of K
from roots. In contrast, P depletion was not inhibited under
hypoxic nutrient solution conditions. Since soils that
support slash pine often have fluctuating water tables, this

139

sensitivity to K is significant. If I also take into account
that soil P limits pine productivity (Neary et al., 1990a)
documenting the effectiveness of pine roots to acquire P and
K under low-02 soil conditions is necessary to evaluate
mechanisms of nutrient uptake which regulate the productivity
of forest.

From my results I can conclude that, in order to improve
predictions of nutrient uptake under field conditions future
research efforts should focus on the inclusion of (i) root
spatial patterns, and (ii) temporal changes in root uptake
kinetics due to changing soil aeration in nutrient uptake
models .

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BIOGRAPHICAL SKETCH

What? tell you my life story? Why?
who would be interested in that?
besides I got a very bad memory.

I am sure I forget a lot of important details .

I mean unimportant details

because I don't think anything very important
ever happened to me.

Of course I can tell you important things,
but what I mean is that,
how can I explain it to you?

It is like I have forgotten everything.

Of course I could tell you that

My name is Jose Armando Escamilla Bencomo. I was born on
May 21, 1960, in Merida, Yucatan, Mexico, the second child of
a family of ten. I graduated from Xmatkuil-Merida High School
in 1978. After graduation, I accepted a scholarship from "The
Pan-American Agriculture School" (Zamorano), Honduras. In
December of 1981, I completed a three year college degree in
Zamorano with honors.

In the Fall of 1982, I transferred to the University of
Florida, Gainesville, USA, and earned a Bachelor of Science
in Agriculture with a soil science major in December of 1983.
In January of 1984, I accepted a position at "The Pan-
American Agriculture School" (Zamorano), Honduras. I worked
there for two years as an instructor in soils and running a
soil testing lab.

With an interest in soil fertility, I entered the
"hidden half", the world of plant roots, in August of 1988,
to pursue graduate studies with Dr. Nick Comerford of the

155

156

University of Florida, Gainesville, FI. I completed my Master
of Science program in August of 1990.

After receiving my MSc . degree, I worked for the Centro
de Investigacion Cientifica de Yucatan (CICY), Mexico, for 3
years. In the summer of 1993, I was re-admitted to the
Graduate School of the University of Florida to work toward
my Ph.D. degree in rhizosphere dynamics of Forest Soils.

Of course I could tell you more,

you know, I realized that actually you can do
what you really want to do,
and that's happiness, don't you agree?

Now it is time to turn the page, right?

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
a dissertation for the degree of Doctor of Philosophy.

Nicholas B. Comer ford, Chair
Professor of Soil and Water
Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

P./Suresh C. Rao
Graduate Research Professor
of Soil and Water
Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Konda R . Reddy
Graduate Research Professor
of Soil and Water
Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

^ Do*jrc/ /H

David M. Sylvia S
Professor of Soil and Water
Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Kimberlyn^illiams
Assistant Professor of
Botany

S->

This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

December, 1997

Dean, College of Agriculture

Dean, Graduate School