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thesis entitled

Water Transport and Xylem Structure.in Lonicera

presented by

Shau-ting Chiu

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Doctoral degree in Botany & Plant Pathology

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WATER TRANSPORT AND XYLEM STRUCTURE IN LONICERA

BY
Shau-ting Chiu

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1992

JKBSTTUKEP
WATER TRANSPORT AND XYLEM STRUCTURE IN LQNIQEBA
BY

Shau-ting Chiu

Wood structure and function in Lonicera was investigated by
experimental and theoretical approaches. The mean measured K5 (-
volumetric flow rate / pressure gradient) in successively shortened
segments gradually increased by a total of 7% when shortened from 20 to
2 cm with more variation in results from shorter stem segments. A
computer simulation of the vessel ends in the stem segments showed an
even distribution of vessel ends when random start points were used.
Segments shorter than the median and mean vessel length (0.5-1 cm) had
a 20% drop in measured K5, possibly reflecting an uneven distribution
of vessel ends in the original segments.

Computer simulations were used to show that random placement of
vessel ends could explain the observed vessel length frequency
distributions in plants. At very low numbers of vessel ends, the
longest vessels had a very high frequency but with higher numbers of
vessel ends there were many short vessels and few long ones. Uniformly
distributed vessel ends in rows within vessel columns appear to favor a
Poisson or positively skewed distribution with many short vessels. The
normal and binomial distributions of vessel ends within columns
simulated situations where more vessel ends are placed at a node or
other junctions. At the lower numbers of vessel ends, the vessel

length distributions then showed a bimodal or multi-modal pattern.

In different growth forms of temperate honeysuckles (Lonicera
spp.), all three species had many narrow vessels and relatively few
wide ones, with the measured Kg about 24 to 55% of the theoretical Kg
predicted by Poiseuille's law. The twiner had the narrowest stem xylem
diameters, suggesting the greater maximum vessel diameter hydraulically
compensated for narrow stems. Conversely, the free-standing shrub, L.
maackii, had the greatest annular increment of xylem but the least
percent conductive xylem implying that a great portion of the wood was
involved with mechanical support. The scrambler, L. sempervirens had
low maximum vessel diameter, high Huber values (- xylem area / leaf
area), and low specific conductivities (- measured K5 / xylem area),
much like the shrub. The greatest vessel frequency (- vessels / xylem
area) occurred in the scrambler (901 vessels nmfz), the highest thus

far recorded in vines, which could result in lower mechanical strength

of the wood.

ACKNOWLEDGEMENTS

I wish to express my gratitude to Dr. Frank Ewers, my major
professor, and the members of my committee, Dr. Kenneth L. Poff, Dr.
Peter G. Murphy, Dr. Gene R. Safir, Dr. James A. Flores and Dr. John H.
Beaman for their guidance. I also thank my department and the computer
center at Michigan State University for providing the computer
facilities and consulting, and Dr. Gerard T. Donnelly who was the
Curator of Woody Plants on the Michigan State University campus for
allowing me to sample from the collection.

At Michigan State University, I am grateful to the Department of
Botany and Plant Pathology for financial support as a graduate teaching
assistant and for all the other hard-to-specify support from faculty,
staff and friends. I am deeply honored for and wish to acknowledge the
receipt of the William G. Fields Award for Teaching, 1989.

A special thanks goes to Dr. Jeffery A. Elhai, Dr. Wan-Ling Chiu
and Mrs. Shirley A. Owens who have helped and encouraged me throughout
my graduate study in Michigan State University. My brother, relatives,
teachers and friends who have supported and helped me during my

academic journey also have my sincere and unpublished thanks.

iv

TABLE OF CONTENTS

Page
LIST OF TABLES ............................................... .vii
LIST OF FIGURES .............................................. viii
GENERAL INTRODUCTION ............................................ 1
CHAPTER 1 THE EFFECT OF SEGMENT LENGTH ON CONDUCTANCE
MEASUREMENTS IN LONICERA
ERANGRANTISSIMA .................................... 8
Introduction .............................................. 8
Materials and Methods .................................... 10
The effect of stem segment length on Kh ................ lO
Vessel length measurements ............................. 13
Computer simulation of vessel distribution ............. 17
Results .................................................. 17
Kg measurements ........................................ l7
Vessel length measurements ............................. 19
Computer simulation .................................... 23
Discussion ............................................... 23
CHAPTER 2 A MODEL OF VESSEL LENGTH IN PLANTS ................ 26
Introduction ............................................. 26
Materials and Methods .................................... 29
Results .................................................. 35
Discussion ............................................... 41
CHAPTER 3 XYLEM STRUCTURE AND WATER TRANSPORT IN A TWINER,
A SCRAMBLER, AND A SHRUB OF LONICERA
(CAPRIFOLIACEAE) .................................. 48
Introduction ............................................. 48
Materials and Methods .................................... 50
Plant materials ........................................ 50
Measured Kg ............................................ 51
Anatomical study ....................................... 52
Xylem area and leaf surface area ....................... 53
Huber value ............................................ 53
Moisture content of wood ............................... 54
Statistical analysis ................................... 54

TABLE OF CONTENTS (continued)

Page
CHAPTER 3 (continued)

Results .................................................. 54
Maceration ............................................. 55
Transections ........................................... 56
Wood moisture content .................................. 63
Measured and theoretical Kb ............................ 65
Huber value, specific conductivity, LSC ................ 65

Discussion ............................................... 70

CONCLUSIONS .................................................... 76
BIBLIOGRAPHY ................................................... 81

vi

LIST OF TABLES

Table Page

1. Range in cell lumen diameters (pm) of vessels,

tracheids and fibers of the twiner (L. japonica),

scrambler (L. sempervirens) and shrub (L. maackii) ....... 55
2. Range in cell wall thickness (pm) of vessels, tracheids

and fibers of the twiner (L. japonica), scrambler (L.

sempervirens) and shrub (L. maackii) ..................... 56
3. Quantitative features (mean i standard error) of xylem

in the twiner (L. japonica), scrambler (L.

sempervirens) and shrub (L. maackii) ..................... 64

vii

Figure
1.1.

1.2.

1.3.

1.4.

1.5.

1.6.

LIST OF FIGURES

Page
Modified Sperry apparatus to remove emboli and
measure hydraulic conductance in isolated stem
segments ............................................... 11
Vessel columns in a 3-dimensional image (left) and a
schematic representation (right) of a portion in a
computer matrix. The entire matrix was 1000 columns
wide and 200 mm long. Asterisks indicate the vessel
ends which are vessel elements with only one
perforation plate ...................................... 14
Flow chart describing the computer simulations of
the positioning of vessel ends. The placement of the
first vessel end in each vessel column is determined
from random selection using a uniform distribution.
The successive vessel ends were placed at the vessel
length interval ........................................ 15
Relationship of hydraulic conductance per pressure
gradient (Kg) and segment length. The control
segments were 20 cm long. The experimental segments
were out every 15 min. Control n - 10 and
experimental n - 20. Bars - i standard error .......... 18
Frequency distributions of vessel length from six
stems of the shrub Lonicera fragrantissima by the
paint-infused method. All showed the Poisson
distribution. Arrows indicate maximum vessel
lengths. Dotted lines were subdivisions under 2 cm
in length .............................................. 20
Correlation of vessel end numbers to segment length
from computer simulating data based on random
placement of starting point of vessel ends in each
column with assigned vessel lengths. r - 1.00 ......... 22

viii

Figure
2.1.

2.

2.

2.4.

2.

2.

3.

5.

LIST OF FIGURES (continued)

Vessel column including vessel A with seven vessel
elements and six perforations, and vessel B with
three vessel elements and two perforations. Arrows
indicate the vessel ends. 0n the left is a 3-
dimensional image showing simple perforation plates.
0n the right is a schematic representation of a
portion of a column (rows 449-463) in a computer
matrix ..............................................

Flow chart describing the computer simulations of
the positioning of vessel ends in a matrix. The
column numbers were chosen from a uniform
distribution and row numbers within the column were
chosen from uniform, normal or binomial
distributions of random numbers .....................

Probability for the occurrence of vessel ends at
certain row numbers within the vessel column,
calculated from the uniform, normal or binomial
distribution ........................................

Frequency distributions of vessel length for
different numbers (n) of vessels in the matrix.

Each graph is from one representative simulation.
Vessel ends were placed within vessel columns from
random numbers selected from uniform, normal or
binomial distributions. Arrows indicate the longest
vessels .............................................

Frequency distribution of vessel length for 500 and
6500 vessels in the matrix with a 1:1 combination of
two types of random distributions. Vessel ends were
placed within vessel columns from random numbers
with half selected from a uniform distribution and
the other half selected from normal or binomial
distributions. Arrows indicate the longest

vessels .............................................

ix

Page

...30

...31

...33

...36

...38

Figure
2.

2.

3.

3.

3.

3.

6.

7.

1.

2-4.

5.

6.

LIST OF FIGURES (continued)

The relationship between length of a hypothetical
stem with a node at the 500 position and the vessel
end number per stem length. Results are shown for
when uniform, normal or binomial distributions were
used to simulate the placement of vessel ends in
columns .............................................

Frequency distributions of vessel length for narrow,
medium and wide vessels of two woody vines, Vitis
rotundifolia and Saritaea magnifica (modified from
Ewers and Fisher, 1989b) ............................

Percentage of total theoretical K5 (open circles, o)
for each class of vessel diameter and the frequency
distributions of vessel diameter (histograms) in
current year and four-year-old stems of a twiner
Lonicera japonica, a scrambler L. sempervireng, and
a shrub L. maackii. Each graph is from one
representative stem. Arrows indicate the largest
vessel diameter in each sample ......................

Transverse sections of the current year stems (a)
and the older stems (b) of the twiner Lonicera

japonica (2), the scrambler L. sempervirens (3) and
the shrub L. maackii (4). Bar - 100 um .............

Comparison of vessel diameter among different growth
forms: a twiner Lonicera japonica, a scrambler L.
sempervirens and a shrub L. maackii. Bars indicate
i standard error. Asterisks indicate statistically
significant difference between species ..............

Measured hydraulic conductance per pressure gradient
(measured Kb) compared to the theoretical K5
determined by Poiseuille's law in current year stems
(a) and older stems (b) of the twiner L. japonica,
the scrambler L. semDervirens and the shrub L.
maackii .............................................

Page

...40

...44

...57

...60

...62

...66

Figure
3.7.

LIST OF FIGURES (continued)

 

Page
Comparison of means of Huber value, specific
conductivity and leaf specific conductivity for the
stems of the twiner Lonicera japonica, the scrambler
L. semDervirens and the shrub L. maackii. The
probability of F value in the statistic analysis of
variance among species were 0.0001 (HV), 0.0003
(SC), 0.0147 (LSC) for current year stems and 0.0524
(HV) for older stems. Bars indicate i standard
error. Asterisks indicate statistically significant
difference between species ............................. 68

xi

GENERAL INTRODUCTION

Water exerts a direct or indirect controlling influence on all
living organisms on earth. As most woody plants exist in a terrestrial
environment, standing free in a nonsupportive medium and not bathed in
water, support and transport tissues are necessary adaptions. Xylem,
the type of vascular tissue that has functions of water and mineral
transport, mechanical support and storage of water and nutrients, has
the tremendous variation in the dimensions of its cells (elements) and
in the length of the transport pathways. There are well known
correlations between xylem anatomy and species distribution in nature
(Carlquist, 1975, 1985, 1988; Liang and Bass, 1990). In large trees
and lianas (=woody vines), which have great transport distances, the
xylem transport system can limit the water potential gradient through
the plant, gas exchange and transpiration efficiency throughout the
crown, the geographic distribution and even the maximum height that a
particular species can achieve (Tyree, Caldwell and Dainty, 1975;
Zimmermann, 1978a; Tyree et al. 1983; Ewers and Zimmermann, l984a,b;
Tyree, 1988; Ewers, Fisher and Chiu, 1988; Tyree and Ewers, 1991). It
has been noted that comparing vines, in which mechanical demands on the
xylem are minimal, to closely related trees or shrubs should limit some
of the inherent sources of variation and give insights into the
transport and support functions of xylem (Ewers et al., 1988, 1990;

Ewers, Fisher and Fichtner, 1991; Gartner, 1991b).

2

The genus Lonicera L., honeysuckle, in the Caprifoliaceae,
includes twiners, scramblers and shrubs, and is mainly distributed in
cooler regions of the Northern Hemisphere (Ohwi, 1965; Rehder, 1967;
Gleason, 1968; Bailey, 1977; Li et al., 1978; Gleason and Cronquist,
1991). It is well known that vines are very unevenly distributed
geographically. The great majority of woody vines are restricted to
tropical forests. Temperate woody vines have extremely low diversity
in species per ha and density in individuals per ha (Gentry, 1991).
Four species of Lonicera in the same climatic zone but with different
growth forms were chosen for the present study of wood structure and
function.

Lonicera japonica Thunb. is a twiner, a vine whose shoots
spirally twine around a support. It was introduced into the United
States in 1806 as an ornamental and to be used for erosion control
(Sasek, 1983). Since then, it has escaped from cultivation and become
naturalized throughout much of the eastern United States (Leatherman,
1955; Wyman, 1969; Sasek, 1983). This species has been widely reported
as a highly successful weedy exotic vine in the southeastern United
States (Leatherman, 1955; Cartner, Teramura and Forseth, 1989; Sasek
and Strain, 1991). As it covers over herbaceous vegetation and kills
young trees, natural successional patterns are significantly altered
(Oosting, 1956). However, it does not escape from cultivation as far
north as Michigan and Wisconsin (Leatherman, 1955).

Lonicera sempervirens L., is native to the eastern United States
where it ranges as far north as central Michigan. It is a scrambler, a
vine that is initially self-supporting but eventually falls over to be

supported by an external host plant or object. While shoots of L.

3
japonica are indeterminate, with yellow flowers in the axils of the
leaves, L. sempervirens has determinate shoots that produce a terminal
inflorescence of orange-red flowers (Bailey, 1977; Gleason and
Cronquist, 1991). The terminal inflorescences are large enough to put
additional mechanical demands on the stem relative to what occurs in L.
japonica. Although L. sempervirens is a common species in the
southeast, it is not as ubiquitous as L. japonica nor is it considered
a weed (Sasek, 1983).

Lonicera maackii (Rupr.) Maxim. is a tall shrub with dense
branching and vigorous growth. It is an exotic species that was
introduced to the United States in 1860 (Wyman, 1969). It escaped and
became established in the eastern United States including Michigan. It
is found most commonly in the understory of forests. Its flowers are
produced in pairs on the axillary peduncles, in a manner similar to L.
japonica inflorescences. However, it is a free-standing growth form
with full mechanical self-support.

Lonicera fragrantissima Lind. & Paxt. is an ornamental shrub
native to China with dense branching and flowers borne in leaf axils on
the previous year's twig growth (Wyman, 1969; Bailey, 1977). While it
is also free—standing and mechanically self-supporting as L. maackii,
its stem internodes have a solid pith instead of the hollow pith in the
other three Lonicera species (Bailey, 1977). The solid pith allows for
conductance measurements even in very short stem segments lacking
nodes.

In the present study, water transport pathways in Lonicera
species were examined by comparison of vessel dimensions, xylem

properties, leaf areas, and xylem areas. For measuring water flow in

4

tracheary elements, xylem transport efficiency is often expressed as
the measured hydraulic conductance per pressure gradient (measured K5)
(Tyree and Ewers, 1991). For quantifying the movement of water through
tracheary elements, the Hagen-Poiseuille Law is valuable in that it
describes the relationship between four quantities: (1) volume flow,
(2) capillary (vessel) radius, (3) number of capillaries of each
diameter, and (4) the pressure gradient. Therefore, it provides a key
link between xylem structure and transport physiology.

The measured Kh, supplied leaf area, and xylem transverse area of
a stem are also useful in determining the hydraulic architecture and
the mechanical support properties. Since the leaf specific
conductivity (LSC) is equal to measured K5 divided by the supplied leaf
area of the stem, the pressure gradient in the stem (dP/dx) could be
predicted as the average evaporative flux density (E) divided by the
LSC. Specific conductivity (- measured K5 / xylem area) shows the
water transport efficiency relative to the xylem area. Huber value
(axylem area / leaf area) or the "amount" of wood per leaf, relates to
mechanical properties. These three properties are related as follows:
LSC - Huber value - Specific conductivity (Ewers and Zimmermann,
1984a,b). This relationship provides a link of hydraulic and
mechanical functions of xylem. Since the free-standing shrub has
mechanical self-support but not long distance water transport, one can
expect Huber values to be greatest and specific conductivity to be
lowest in the free-standing shrub.

Since long distance transport in most angiosperms, in the
gymnospermous plants of the Gnetales, and in ferns of Marsilea occurs

through vessels of the xylem, the effect of structural variables on

5
conduction is very important for understanding the water transport
efficiency. Most early studies of the structural effect on conduction
were in tracheids (Petty, 1974; Bolton, 1976; Gibson, Calkin and Nobel,
1985; Calkin, Gibson and Nobel, 1986; Schulte, Gibson and Nobel, 1987),
and vessel elements (Robson and Bolton, 1986; Bolton and Robson, 1988;
Schulte, Gibson and Nobel, 1989). Vessels are composed of a series of
vessel elements (vessel members), that are nonliving at functional
maturity, and that are stacked end to end in a column (Fig. 1.2, 2.1).
Perforations at the end walls interconnect the vessel elements of a
vessel. Since vessels are of finite length, water must eventually move
from vessel to vessel or from a vessel to a tracheid through pits in
the cell walls. A pit offers more resistance to flow more than does a
perforation. Although vessels can also vary in length even within a
single plant from less than 1 mm to several m (Ewers and Fisher, 1989b;
Ewers et al., 1990), the factors that control vessel length in a plant
are not understood. Because vessel ends may be a major source of the
resistance in the water transport pathway in vessel-bearing plants, the
positioning of vessel ends among vessel elements may be very important
to water transport efficiency. If a vessel is 1 m long, it means the
distance between vessel ends in a column is l m.

While vessel diameter is easily determined in transverse
sections, vessel lengths cannot be measured with conventional
microscopic techniques. There are at least two major approaches to
quantify vessel length, the compressed-gas method (Handley, 1936;
Greenidge, 1952; Scholander, 1958; Zimmermann and Jeje, 1981; Sperry et
a1. 1987) and the latex paint-infusion method (Zimmermann and Jeje,

1981; Salleo, LoGillo and Siracusano, 1984). The concept of the

6
multitude of vessel lengths in wood was previously approached by
estimating "maximum and minimum vessel length" (Greenidge, 1952),
"average short vessel length" (Scholander, 1958), and vessel-length
distribution (Skene and Balodis, 1968; Zimmermann and Jeje, 1981; Ewers
and Fisher, 1989a, b). Cinematographic analysis can supply precise
information on the layout of the vessel network in wood and the nature
of the vessel ends (Zimmermann, 1971a, 1978b) but it is not practical
when large sample sizes are needed.

There is some evidence that vessel ends and tracheids tend to
occur abundantly at nodes where leaves attach to the stem (Salleo et
al., 1984) and at the junctions between stem and branch, root and
shoot, and main root and lateral root (Zimmermann and Potter, 1982;
Zimmermann, 1983; McCully and Canny, 1988; Aloni and Griffith, 1991).
Whether the observed frequency distributions of vessel length such as
Poisson (Skene and Balodis, 1968; Zimmermann and Jeje, 1981; Ewers and
‘Fisher, 1989a, b; Ewers, Fisher and Chiu, 1990), bimodal or multimodal
patterns (Zimmermann and Jeje, 1981; Sperry et al., 1987; Ewers and
Fisher, 1989a, b; Ewers et al., 1990) could be explained by a random
placement of vessel ends is uncertain. If vessel ends are not evenly
distributed along the stem, are there changes of measured K5 with the
change of stem segment length?

In Chapter 1, I develop experimental and theoretical approaches
to determine the effect of stem segment length on measurements of xylem
transport efficiency. In Chapter 2, computer simulations are used to
determine whether random placement of vessel ends could explain the
observed vessel length frequency distribution in plants. In Chapter 3,

various wood anatomical and physiological features were compared in a

twiner, a scrambler, and a shrub species of Lonicera. From these
studies, the xylem structure in vessel—bearing plants can be
interpreted comprehensively in terms of function, adaption and

evolution.

CHAPTER 1

THE EFFECT OF SEGMENT LENGTH 0N CONDUCTANCE MEASUREMENTS

IN LONICERA FRANGRANTISSIMA

INTRODUCTION

A vessel is a series of vessel elements (members) that are
stacked end to end, with a primary function of water conduction (Fig.
1.2). While vessel and tracheid diameter are widely recognized as
important for models of xylem transport according to the Hagen-
Poiseuille law for ideal capillaries (Zimmermann, 1983; Gibson, Calkin
and Nobel, 1985; Ewers and Fisher, 1989a; Chiu and Ewers, 1990, 1991;
Chapter 3), neither vessels nor tracheids are ideal capillaries of
infinite length. Measured Kb (hydraulic conductance per pressure
gradient) is usually reported to be less than 50% of the theoretical Kb
(predicted by the Hagen-Poiseuille law) (Zimmermann, 1971b, 1983;
Ewers, Fisher and Chiu, 1989; Chiu and Ewers, 1990, 1991; Chapter 3)
but exceptions show results close to 100% of the theoretical Kh
(Berger, 1931; Dimond, 1966; Giordano et al., 1978; Schulte, Gibson and
Nobel, 1989). Perforation plates and tremendous within-plant variation
in vessel length provide more conductive complexities in vessels than
in tracheids or vessel elements (Robson and Bolton, 1986; Schulte,
Gibson and Nobel, 1989). Most reports suggest that the discrepancy

between measured K5 and theoretical Kb arises from the limited length

9
of vessels (Zimmermann, 1971b; Ewers et al., 1989; Ewers, Fisher and
Fichtner, 1991). Since vessels are of finite length, somewhere along
the vessel lateral transport must occur between the vessel and an
adjacent conduit. In vessels lateral transport can occur through any
of the pits of all the vessel elements. It is not known if the pits of
a vessel end, which is a vessel element with only one perforation
plate, differ in porosity from the pits of ordinary vessel elements
which have two perforation plates. Resistance to water flow in vessels
can be classified as lumen resistance, perforation plate resistance
(Schulte et al., 1989; Bolton and Petty, 1977) and pit resistance.

The positioning of vessel ends could influence K5. A vessel end
is a vessel element with only one perforation (Fig. 1.2). In the
present paper I make empirical vessel length measurements with the
paint infusion method (Zimmermann and Jeje, 1981; Ewers and Fisher,
1989a) and use this data for computer simulation to describe possible
positioning of vessel ends in the stem segments. The simulation used
random placement of the first vessel end in a column (Fig.1.2) and
followed by equal spacing of vessel ends.

Measurements of K3 in isolated stem segments often ignore the
effect of segment length. If segments are shorter than most of the
vessels, measured K5 might not be representative of Kfi of longer
segments. It is also possible that shortening segments will remove air
emboli that are trapped within vessels. The present study was designed
to quantify the effect of segment length on K5 measurements by

repeatedly shortening the transport distance in isolated stem segments

of Lonicera fragrantissima Lind. & Paxt.

10
MATERIALS AND METHODS

The effect of stem segment length on K5

Five sets of two control and four experimental one-year-old stem
segments of the shrub L. fragrantissima, growing on the campus of
Michigan State University were randomly selected for measurement of Kb.
A preliminary examination of vessel length indicated that the longest
vessel in one-year-old stems of L. fragrantissima was about 16 cm. The
20 cm long unbranched segments with the proximal (basal) end 5 cm away
from the junction to the major axis were cut under water, kept
submerged, defoliated with shears, trimmed at both ends with a fresh
razor blade, tightly fitted with clear vinyl tubing at the proximal
end, and vacuumed for 20 min at 70 kPa to remove air embolisms that may
have been introduced at the cut surface during handling. The stems
were perfused with a 150 mmole-m'3 acetic acid (pH—5) solution (Calkin,
Gibson and Nobel, 1986), filtered through the 0.2 pm Gelman membrane
filter (Fig. 1.1), under 150 to 175 kPa pressure for 15 min to remove
emboli (Sperry, Donnelly and Tyree, 1988; Chapter 3). After the
segments were perfused, the drain volumetric flow rate was measured
with a micropipet and stopwatch and the initial Kg was then determined
as the flow rate divided by the pressure gradient at 7 kPa pressure
induced by the secondary supply reservoir (Fig. 1.1). In stem xylem
the measured K5 is generally the same regardless of the pressure or
tension used in the laboratory (Tyree and Zimmermann, 1971; Edwards and
Jarvis, 1982; Dryden and Van Alfen, 1983). We used only a pressure
head of 7 kPa since this was convenient with our apparatus. The distal

2 cm of experimental segments were repeatedly cut with fresh razor

blades but the controls remained 20 cm long. After each cut, both

11

Fig. 1.1. Modified Sperry apparatus to remove emboli and measure

hydraulic conductance in isolated stem segments.

A=

compressed air pressure;

three-way stopcock;

pressure chamber regulating the pressure in the system;

captive air tank (high pressure reservoir of degassed solution);
0.2 u m filter;

reservoir with only gravipressure gradient;

stem segments fitted in tubing system;

micropipets measuring the drain flow rate;

two—way stopcock for releasing the system pressure.

13
control and experimental segments were perfused at high pressure and Kg
was remeasured at 7 kPa. After the experimental stems were cut to 2 cm

long, repeated measurements of 1 cm and 0.5 cm segments were made.

Vessel lengths measurements

Paint-infusion method --- After the repeated measurements of K5,
two control segments from each set were used for the measurement of
vessel length. A 1% (v/v) green latex solution containing 0.2 to 5 pm
size pigmented particles was fed into the segments at the proximal
(basal) end under 0.2 MPa pressure. The latex emulsion was allowed to
pass through the stem until flow completely stopped, which took up to 3
days in some cases.

The 20 cm segments were cut at 2 cm intervals from the distal
end. The proximal 2 cm segments were cut at 0.5 cm and 1 cm intervals.
The segments were stored in a vertical position with the surface on
which vessel counts would be made facing down on a glass surface. The
paint-filled vessels, including partially filled ones, were counted
with a dissecting microscope. In some cases, shaving of 0.5 mm off the
transverse stem surfaces was necessary to remove surface paint and thus

provide a clean and sharp image.

Calculation of the vessel length frequency distribution ---

 

Since we used uneven stem lengths, the frequency distribution of vessel
length was calculated by an equation modified from previous reports
based on even stem lengths (Zimmermann and Jeje, 1981; Ewers and
Fisher, 1989a). The raw vessel count represents the number of vessels

continuous from the infused proximal point. In the successive cutting

l4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*
*- —- +-at5mm
* *
*
*
*
_*.__ ..._.. ..
*
* _
* _
*_
* *
*
*_ _
__ __*
— —— —- - «atZOmm

 

 

 

 

 

Column 895 896- 897

Fig. 1.2. Vessel columns in a 3-dimensional image (left) and a
schematic representation (right) of a portion in a computer matrix.
The entire matrix was 1000 columns wide and 200 mm long. Asterisks

indicate the vessel ends which are vessel elements with only one

perforation plate.

15

Fig. 1.3. Flow chart describing the computer simulations of the
positioning of vessel ends. The placement of the first vessel end
in each vessel column is determined from random selection using a
uniform distribution. The successive vessel ends were placed at

the vessel length interval.

16

Simulation matrix

1000 vessel columns

I" —————————————— “I

W

 

>”-200 mm

 

 

 

 

 

.4!

 

Column 1 ' Column 1000

Fixed vessel length,
random start point

1000 vessel columns

 

 

 

 

, 3*"200 mm

 

 

 

 

..al

Column 1 Column 1000

17
segments, the total number of vessels (N) in each length class is given

by (Skene and Balodis, 1968; Zimmermann and Jeje, 1981):

N _ ( Cn-1_Cn_ Cn-Cn+1)X
’1 X;-A%_1 .X - n ;

n+1 n

 

where C is the raw vessel count; X is the distance from the proximal
end; and n - 0,1,2,--- indicating the position of successive cutting

planes along the segment.

COmputer simulation of vessel distribution

A bundle of hypothetical vessel columns (Fig. 1.2) represented
the computer simulation imitating the empirical frequency distribution
of vessel length. A matrix was set up to represent 1000 vertical
columns of vessel elements, each column 200 mm long (Fig. 1.3). The
empirically derived frequency distribution of vessel length in L.
fragrantissima calculated as shown above was used to determine the
number of vessel columns for a particular vessel length. A uniform
distribution of random numbers, from 1 mm to the vessel length of each
length class, was used to assign the placement of the first vessel end
in the vessel column. The successive vessel ends were placed at the
intervals of the vessel length in the vessel column. The numbers of

vessel ends were then counted from zero to various lengths.

RESULTS
Kg.measurements
Throughout the high pressure perfusions and the K5 measurements
in 3 hrs, the measured K5 of controls was reduced by 5% of the initial

Kb, 1.98 i s.e.0.34 -10'“’nfi-MPa'Lsf1 (Fig. 1.4). The mean initial Kh

18

 

 

 

 

 

 

1
I: " Control
f ~ $0.22
2 oo = i L i 3
E Time (hrs)
hp.
0
be m o 1 434+ 1

 

Experimental
r’=0.13

A l A l A . A I A I A I A I A L A l A
v i v v V ' v ' v ' v ' v ‘ v j v v v

0
20181614121086420

Segment length (cm)

A
v

 

 

 

Fig. 1.4. Relationship of hydraulic conductance per pressure gradient
(K5) and segment length. The control segments were 20 cm long. The
experimental segments were cut every 15 min. Control n = 10 and

experimental n - 20. Bars - i standard error.

19
of experimental segments was 1.94 i s.e. 0.22 -10'“’nfi-MPa'L:§1. Based
on repeated measurements after successive shortening of the segments,
the mean measured K5 of the experimental segments gradually increased
by as much as 7% in 2 cm segments, but dropped by about 20% when
segments were shorter than 2 cm (Fig. 1.4). The variation of measured
K5 in shorter segments was larger than in longer segments, as shown by
the standard errors (Fig. 1 4). The increase of K5 from 20 cm

shortened to 2 cm was 0.39% of the initial K5-cmfik

However, within 2
cm, the mean measured K5 dropped to 22% of the initial at the 1 cm
point and then increased 3.3% at the 0.5 cm point (Fig. 1.4). The mean
decrease in K5 from 2 to 1 cm segment lengths was highly significant
based on the Spearman's nonparametric coefficient of rank (rs - 0.853)
(Steel and Torrie, 1980). The regression line of all points showed a
trend of decreasing K5 from 20 cm to 0.5 cm segments with a slope of
-0.66% initial K5-cm"1 (Fig. 1.4). Eventually, the measured K5 of 0.5

cm long segments was about 10% lower than what was expected from the

regression line.

Vessel length measurements

The vessel lengths followed Poisson distributions with the
highest frequency of the shortest vessels and bimodal distributions
with mostly short vessels but minor peaks located at lengths of 1-2 cm
were also found (Fig. 1.5). The maximum vessel lengths in sampled
segments ranged from 6 to 14 cm (Fig. 1.5). While the highest
frequency always occurred at the shortest vessel class where it could

be higher than 90% (Fig. 1.4), the mean vessel lengths ranged from 0.6

to 1.2 cm and the median vessel lengths ranged from 0.1 to 0.5 cm.

20

Fig. 1.5. Frequency distributions of vessel length from six stems
of the shrub Lonicera fragrantissima by the paint—infused method.
All showed the Poisson distribution. Arrows indicate maximum
vessel lengths. Dotted lines are subdivisions under 2 cm in

length.

 

 

21

18
16
16
16

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

w A:
1'4 1'4 ll4 1'4
1 1 1 1
v Av A.
112 [I2 1'2 1'2
(I (I 1 1|
7 A. A v
llw I'm l I” I'm
L w .fi
1 9'8 LIB 1'8 1'8
ALD Iv .6 ..6 llv ..6
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14 I4 414 ,T4
7 Ar
4 2 a 2
u .
o .
fluv “I;
QIIA '1
o “on“...fiquuo no: 0 o onwduwmomJMuO -
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5 w

 

Vessel length (cm)

22

 

slope: 0- 5230
0 — 6652
A— 7070
A - 7024
D- 6103
I - 6386

d

Number of vessel ends (103)
s '8’

 

 

 

Segment length (cm)

Fig. 1.6. Correlation of vessel end numbers to segment length from
computer simulating data based on random placement of starting point of

vessel ends in each column with assigned vessel lengths. r2 - 1.00.

23
More than 70% of the vessels were shorter than 2 cm. The valley in the
frequency distributions at 0.5 to 1 cm nearly matched the drop in the

K5 measurement at these lengths (Fig. 1.4, 1.5).

COmputer simulations

Based on the simulations for the vessel distributions in six
segments of L. fragrantissima, the vessel end numbers proportionally
increased as the segment length increased (Fig. 1.6). The slopes of
vessel end numbers to segment length (Fig. 1.6) varied depending on the
frequency of the shortest vessels; the more short vessels the higher
vessel end numbers-cufl. All the r3 values for the regression were

1.00. The mean vessel end number°c:m'1 was 6750 (s.e. = 386).

DISCUSSION

It has been found that plants of a wide range of growth forms
(trees, shrubs, vines, herbs) have many short vessels and few long ones
(Zimmermann and Jeje, 1981; Ewers and Fisher, 1989b; Ewers, Fisher and
Chiu, 1990). While many studies on measured K5 suggest using segment
lengths longer than the maximum vessel length (Zimmermann, 1978b;
Zimmermann, 1983; Ewers and Fisher, 1989b), in some species such as
many vines and ring-porous trees, where vessels can be many m in
length, it may be impractical to include the longest vessels in the
segments. The present results indicate that shorter segments will have
greater variation of measured K5 than longer ones. The segment length
(cm) affected about 0.66%-c:m’1 of measured K5 when segments were longer
than median vessel length.

The repeated high pressure perfusions did not result in increases

24
in K5, suggesting that the initial 15 min, 175 kPa perfusion was
sufficient to remove all air emboli. The slight decrease in K5
overtime in controls may be due to partial clogging of the pit
membranes, despite the use of a 0.2 pm filters. Another possibility is
that swelling of pit membranes slightly reduced the size of pores in
pit membranes.

When the segment lengths were near the shortest vessel lengths,
the measured K5 was abruptly reduced. This could have been due to
either uneven distributions of vessel ends or artificially low K5
readings resulting from a great impact of turbulence at the point where
sap flowed out of the segment. The very short segments should not have
disrupted laminar flow. A capillary 24 pm in diameter (maximum vessel
diameter) and 0.5 cm long would have a Renolds' number of 0.065 at the
flow rates derived from Poiseuille's law for a capillary at the
pressure gradient we used, much below the value of 2000 for turbulent
flow, and even below the critical value for short capillaries defined
as R5'-0.8 L/r (Siau, 1984), which in this case would be 328. However,
the effect of nonlinear flow could occur where fluids enter a pit
opening (Bolton and Petty, 1975). Since the L. fragrantissima segments
contained many short vessels, the effect of pits on nonlinear flow
might show up more in the K5 readings in short segments since long
vessels with linear flow would be artificially shortened and their
impact lessened. Additional kinetic-energy losses can also occur in
curved tubes of changing diameters (Scheidegger, 1974). If vessel ends
are tapered, short vessels would have a greater impact of changing
lumen diameters than long ones.

From our computer simulations, the uniform distribution of the

25
start points of vessel ends with fixed vessel lengths always results in
a constant ratio of vessel end number to segment length. If lateral
flow through pits is similar in each element, including vessel ends, a
uniform distribution of vessel ends would predict a constant K5 with
decreases in segment length. However, the drop in K5 when the segments
were shorter than 2 cm suggests a possible uneven distribution of
vessel ends near the infused end. This is consistent with the vessel
length frequency distributions, which follow bimodal and multimodal
patterns, as predicted for nonuniform distributions (Chapter 2). A
high frequency of vessel ends could be at 0.5 to 1 cm. The K5 reading
would then be correct but not equal to that for a longer segment.

Although Zimmermann (1983) has suggested that vessel endings are
a significant source of resistance to water flow, it is difficult to
determine this in stem segments without an experimental method to
remove pit membranes. Another problem is in determining whether vessel
ends are equally or more porous than ordinary vessel elements. The
minor increases in K5 from 20 cm to 2 cm segments but a drop of K5
within 2 cm segments in L. fragrantissima implies that the vessel end
resistance could exist, but there may be other significant resistances
that influence K5 readings in very short segments.

To conclude, the segment length affected about 0.39%-c.m’1 on
measured K5 in L. fragrantissima. It may not be important to select a
segment length longer than the maximum vessel length. However, in very
short segments there can be irregularities in K5 readings, due to the
nonuniform distribution of vessel ends (Chapter 2) or due to possible

non-linear flow. For measured K5, a suggested segment length would be

at least longer than the median vessel length.

CEHUHTERLIB

A MODEL OF VESSEL LENGTH IN PLANTS

INTRODUCTION

Long distance water transport in most angiosperms, in the
gymnospermous plants of the Gnetales, and in ferns of the genus
Marsilea occurs through vessels of the xylem. Vessels are composed of
a series of cells, vessel elements (vessel members), that are nonliving
at functional maturity, and that are stacked end to end in columns
(Fig. 2.1). Perforations at the end walls interconnect the vessel
elements of a vessel. Since vessels are of finite length, water must
eventually move from vessel to vessel or from a vessel to a tracheid
through pits in the cell walls. A pit offers more resistance to flow
more than does a perforation. A vessel end differs from a normal
vessel element in having only one perforation plate. Because vessel
ends may be a major source of the resistance in water transport pathway
in vessel-bearing plants, the positioning of vessel ends among vessel
elements may be very important to water transport efficiency. Vessels
can vary in length even within a single plant from less than 1 mm to
several m (Ewers and Fisher, 1989b; Ewers, Fisher and Chiu, 1990). If
a vessel is 1 m long, it means the distance between vessel ends in a

column is l m. In the present paper I refer to vessel length in terms

of the number of vessel elements composing a vessel including the two

26

27
vessel ends (Fig. 2.1).

The functional significance of vessel length may be related to
trade-offs between safety and efficiency. There is increasing evidence
that the xylem of plants is frequently subjected to dysfunction due to
air embolism (Tyree and Sperry, 1989; Ewers and Cruiziat, 1991; Tyree
and Ewers, 1991). By the air-seeding hypothesis, water stress-induced
embolism is caused by pores in pit membranes that can allow for air
entry. Increasing vessel length would increase the danger of water-
stress induced embolism due to increasing pit numbers and lateral
contact areas. Increasing vessel length also increases the risk of
freezing-induced embolism (Ewers, 1985). Short vessels may also be
safer water conductors than long ones due to greater
compartmentalizaion of embolisms (Zimmermann, 1983; Carlquist, 1988).
Vascular "segmentation", which separates plant parts by zones with
great number of vessel endings, may be adaptations to drought and cold
temperatures (Aloni and Griffith, 1991).

Based on improved methods to calculate the frequency of vessel
length distribution (Skene and Balodis, 1968; Milburn and Covey-Crump,
1971; Zimmermann and Jeje, 1978), it has been found that vessels from
plants of a wide range of growth forms (trees, shrubs, vines, herbs)
and a wide range of taxonomic affiliations have many short vessels and
few long ones. This is true regardless of the maximum vessel length in
a species. Distribution patterns include the Poisson distribution
(Skene and Balodis, 1968; Zimmermann and Jeje, 1981; Ewers and Fisher,
l989a,b; Ewers, Fisher and Chiu, 1990) and the positively skewed normal
distribution (Ewers and Fisher, 1989a,b). Some stems show a bimodal or

multi-modal frequency distribution of vessel length (Zimmermann and

28
Jeje, 1981; Sperry et al., 1987; Ewers and Fisher, 1989a,b; Ewers et
al., 1990). The mechanism(s) resulting in these patterns have not been
investigated.

The length and diameter of vessels may be inversely correlated
with auxin concentration when the vessels are being formed (Aloni,
1987, 1991; Aloni and Zimmermann, 1983). Vessel ends may be more
frequent at nodes than internodes (Salleo, Lo Gullo and Siracusano,
1984) and tend to occur abundantly at the junctions between stem and
branch, root and shoot, and main root and lateral root (Zimmermann and
Potter, 1982; Zimmermann, 1983; McCully and Canny, 1988; Aloni and
Griffith, 1991). However, factors controlling vessel lengths have been
little studied.

In the present paper, we use computer simulations to determine
whether the strongly skewed normal vessel length distribution patterns,
as well as bi- or multi-modal patterns, can be explained merely by the
random placement of vessel ends among the vessel elements. Within
simulations, the placement of vessel ends was based on the generation
of random numbers. It should be noted that random placement does not
necessarily mean that all numbers have equal chance of being selected.
There are several probability distributions that are applicable in
computer modeling and simulation (Martin, 1968; Knuth, 1981). I used a
uniform distribution to simulate the situation with no influence of
nodes or junctions in the stem, a normal distribution for a more
frequent occurrence of vessel ends at nodes or junctions, and a
binomial distribution for the large impact of nodes and junctions. In
the uniform distribution, there is an equal chance of selecting any

value. The normal distribution, which describes many measurement

29
phenomena, is also frequently applied in computer simulation. In the
normal distribution, numbers have an unequal chance of being selected.
In the binomial distribution 1 used, there would be no vessel ends in
the middle portions of the internodes. Both uniform and normal
distributions are continuous distributions whereas the binomial
distribution is discrete. In addition to varying the type of random

selection, I varied the numbers of vessel ends in each simulation.

MATERIALS AND METHODS

A hypothetical vessel column imitated a series of vessels, each
vessel determined by two vessel ends (Fig. 2.1). A matrix was set up
to represent 100 vertical columns of vessel elements, each column 1000
vessel elements (rows) long (Fig. 2.2). The random placement of vessel
ends within the matrix determined vessel lengths, with 100 vessel ends
already assumed at row 1 and row 1000. The maximum possible vessel
length was thus 1000 vessel elements. Random numbers, from (1, l) to
(100, 1000) for (column, row), were selected to assign the placement of
vessel ends in the matrix. The column numbers were always chosen from
a uniform distribution of random numbers. However, the row numbers
within the column were chosen in three different ways, using uniform,
normal and binomial distributions in the generation of random numbers.
The probability of the selection of row numbers is shown in Fig. 2.3.

The random numbers generated from a uniform distribution, from 1 to
1000, all had an equal probability of occurrence, defined as él’ where

n is the number of possible positions for vessel ends in a vessel
column (Martin, 1968; Newman and Odell, 1971; International

Mathematical and Statistics Libraries, 1989); in this study n=1000.

 

30

449

450

 

451

452

453

454

455

456

457

 

458

459

460

 

 

461

462

463

 

b_—_—_‘P_—_—_d

Vessel A

Vessel B

Fig. 2.1. Vessel column including vessel A with seven vessel elements

and six perforations, and vessel B with three vessel elements and two

perforations. Arrows indicate the vessel ends.

0n the left is a 3-

dimensional image showing simple perforation plates. 0n the right is a

schematic representation of a portion of a column (rows 449-463) in a

computer matrix.

31

Fig. 2.2. Flow chart describing the computer simulations of the
positioning of vessel ends in a matrix. The column numbers were
chosen from a uniform distribution and row numbers within the

column were chosen from uniform, normal or binomial distributions

of random numbers.

32

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0.03

 

0.02 --

0.01 --
.. ._ binomial

Probability

uniform normal

0.00 -g-r"":'“‘:} :L'~~:~“‘-'--fi
0 500 1000
Position of vessel end

 

 

 

 

Fig. 2.3. Probability for the occurrence of vessel ends at certain

position within the vessel column, calculated from the uniform, normal

or binomial distribution.

34
For the normal distribution of random number generation (Martin, 1968;
Kinderman and Ramage, 1976; International Mathematical and Statistics

Libraries, 1989), the probability function was

_ (x—g)’
P-F(X)- 1 e 2“: ;
a 2n

 

where X is a particular position of a vessel end in the vessel columns;
u is the mean; 0 is the standard deviation; and e is the base for
natural logarithms. The probability function for the binomial
distribution of random number generation (Martin, 1968;
Kachitvichyanukul, 1982; International Mathematical and Statistics

Libraries, 1989) was

P - F(X) - (—§)p"(1-p)’*"‘

where X is a particular position of a vessel end in the vessel column;
n is the number of Bernoulli trials; and p is the probability of
success on each trial, which is 0.5 in this case (Fig. 2.3).

The total number of vessel ends varied with the simulation, n -
100, 400, 1600 and 6400. Vessel ends were randomly placed with the row
numbers determined by uniform, normal and binomial distribution of
random numbers (Fig. 2.3). In addition, combinations with half of the
random numbers generated from the uniform distribution and the other
half from the normal or binomial distribution were tested with n = 400
and 6400 vessel ends. At least three trials for each type of
simulation were examined.

Each vessel length was determined as the number of elements

between vessel ends plus the two ends. Stem length refers to the
length of hypothetical stems with 100 vessel columns from O to 1000

elements long, with a node at row position 500. The number of vessel

35

ends per stem length was separately calculated.

RESULTS

For each of the three replicates of a particular simulation, the
results were quite similar. The graphs chosen for Figs. 2.4 and 2.5
were those that appeared to be the most intermediate of the three. The
computer simulations demonstrated skewed non-normal and bi- or multi—
modal distributions of vessel length. The maximum vessel length was
1000 elements long at simulations of 200 and 500 vessels. As the
number of vessels increased, the frequency distribution of vessel
length tended to skew into the classes of shorter vessels. Maximum
vessel length also decreased (Fig. 2.4, 2.5). With greater number of
vessel ends, there were many more short than long vessels.

When row selection was from random numbers generated from a
uniform distribution, a Poisson distribution of the frequency of vessel
length occurred in the populations of 500 vessels or more, with the
highest frequency occurring in the shortest vessel classes (Fig. 2.4).
However, for distributions involving only 200 vessels, there were at
least two peaks of frequencies in the vessel length distribution.
Within these distributions, the highest frequency occurred in the
maximum vessel lengths.

When row selection was from random numbers generated from a
normal distribution, the simulations resulted in a bimodal to multi-
modal distribution of vessel length frequency. When the number of
vessels was 500 or more, the frequency distribution of vessel length

was bimodal, with likely two joined Poisson distributions (Fig. 2.4).

However, a multi-modal distribution of vessel length frequency occurred

36

Fig. 2.4. Frequency distributions of vessel length for different
numbers (n) of vessels in the matrix. Each graph is from one
representative simulation. Vessel ends were placed within vessel
columns from random numbers selected from uniform, normal or

binomial distributions. Arrows indicate the longest vessels.

37

 

 

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38

 

 

length (elements)

a son 1000
IN?
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fixmu
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Vessel
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ov"'aéo" who
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“

0

u

0
an”

n

n

n

0 71‘: : i c ‘ :
0 600 1000

 

Frequency distribution of vessel length for 500 and 6500

vessels in the matrix with a 1:1 combination of two types of random

distributions.

Vessel ends were placed within vessel columns from

random numbers with half selected from a uniform distribution and the

other half selected from normal or binomial distributions.

indicate the longest vessels.

Arrows

39
in the population of 200 vessels. As the number of vessels increased
in the simulation, the highest peak shifted from the longest vessel
class to an intermediate and then the shortest vessel class. The
highest peak of 1000 vessel elements class in the smallest vessel
population disappeared in populations of 1700 vessels or more.

When row selection was from random numbers generated from a
binomial distribution, a multi—modal frequency distribution occurred at
n - 200, with the highest frequency at middle classes. With more than
500 vessels, there was a bimodal distribution (Fig. 2 4). The
different modal peaks were more discrete when row selection was from a
binomial distribution rather than from a uniform or normal
distribution. With the binomial distribution of random numbers, the
frequencies of certain vessel classes were always zero (Fig. 2.4).

With greater numbers of vessel ends there were many more short than
long vessels.

When row selection was from random numbers generated from a
uniform combined with normal or uniform combined with binomial
distribution, the frequency distribution of vessel length was bimodal
with two joined Poisson distributions when there were 500 vessels, with
more narrow than wide vessels. Only one Poisson curve with many narrow
vessels occurred when the vessel number was increased (Fig. 2 5).

In the simulations of the uniform distribution of vessel ends
within the columns, there was a nearly constant value of vessel end
number per stem length throughout the stem (Fig. 2.6). However, for
the normal and binomial distributions of vessel ends there was a
threshold around the middle point of the stem (Fig. 2.6). When row

selection was from random numbers generated from a normal distribution,

40

 

15
‘ 0—0 uniform
4’ A—A normal

“ D—D binomial

q

 

-.
9

A

 

 

Vessel and count / stern length
(elements‘l)

o H H H H H h 500 1000
Stem length (elements)

Fig. 2.6. The relationship between length of a hypothetical stem with
a node at the 500 position and the vessel and number per stem length.
Results are shown for when uniform, normal or binomial distributions

were used to simulate the placement of vessel ends in columns.

41
a gradual increase of vessel end number per segment length occurred
after the threshold. The increase followed a left side of convex
parabolic curve whose optimum was at estimated one fifth of stem length
away from the end point (1000 elements) (Fig. 2.6). When row selection
was from random numbers generated from a binomial distribution, a
dramatic increase of vessel end number per segment length occurred at
the threshold near the middle point of segments (Fig. 2.6). A concave
curve following the threshold in the binomial distribution showed an

abrupt decrease to the end of the stem (Fig. 2.6).

DISCUSSION

The computer simulations suggest that a random distribution of
vessel ends results in predictable non-normal patterns that closely
resemble empirical results for Lonicera fragrantissima (Chapter 1) and
almost all woody plants (Zimmermann and Jeje, 1981; Salleo et al.,
1984; Sperry et al., 1987; Ewers and Fisher, l989a,b; Ewers et al.,
1990). Uniformly distributed vessel ends within columns favors a
Poisson or positively skewed distribution with many short vessels. The
normal and binomial distributions of vessel ends would appear to favor
a bimodal or multi-modal frequency distribution of vessel length.
However, the particular pattern depends on the number of vessel ends.
With greater numbers of vessel ends, more skewed frequency
distributions of vessel length occurred. The skewed distribution had
mostly short vessels and few long ones.

From our simulations, the uniform distribution of vessel ends in
the rows imitated the situation in an unbranched axis without the

influence of nodes. The binomial and normal distribution of the vessel

42
ends in the rows results in a greater number of vessel ends in the zone
centered at the 500 position in the vessel columns. In plants, a
greater number of vessel ends occurs at nodes (Salleo et al., 1984) and
at junctions between stem and branch, root and shoot, and main root and
lateral root (Zimmermann and Potter, 1982; Zimmermann, 1983; McCully
and Canny, 1988; Aloni and Griffith, 1991). The combinations of
uniform with binomial, and uniform with normal distributions may
simulate a situation where only a portion of the axis is dominated by
the node or junction. For instance, with secondary growth in the stem,
the impact of nodes may be lessened.

Applying auxin to plants results in the production of shorter
vessels (Aloni and Zimmermann, 1983). In our simulations, increasing
auxin concentrations could be imitated merely by increasing the number
of vessel ends. At nodes, there may be localized high auxin
concentrations, resulting in a greater number of vessel ends (Aloni and
Zimmermann, 1983; Aloni, 1991). The vessel length frequency
distribution patterns are not consistent with developmental mechanisms
that involve inhibition of vessel end formation in elements neighboring
vessel ends. Such inhibition would result in more equal spacing of
vessel elements in a column, and in normal rather than skewed or
bimodal vessel length distribution patterns. The greater number of
vessel ends at nodes or at junctions thus requires developmental
control by the plant. However, the factors that determine whether a
particular element becomes a vessel end remain unknown.

The computer simulations predict that the frequency distributions

of vessel length will show a skewed or Poisson frequency when there is

a great number of vessel ends. This frequency pattern mostly occurs in

43
those species with short and narrow vessels, such as diffuse-porous
woody plants (Zimmermann and Jeje, 1981). Some researchers have
proposed that in the species with wide vessels, random positioning of
vessel ends is nearly achieved for the short vessels but not for the
long ones (Zimmermann and Jeje, 1981). Our computer simulations with
small numbers of vessel ends, especially those from nonuniform
distributions of vessel ends, resulted in bimodal or multi-modal
frequency distributions of vessel length. This is much as occurs in
wide—vesseled species such as vines and ring-porous woody plants. Long
vessels have a small number of vessel ends, presumably allowing for
increased transport efficiency.

The interrelationship between vessel diameter and vessel length
should not be ignored. Higher auxin concentrations result in narrower
as well as shorter vessels (Aloni and Zimmermann, 1983, 1984; Aloni,
1991). There are intra-stem (Ewers and Fisher, 1989b) as well as
inter—species (Ewers et al., 1990) correlations between vessel length
and vessel diameter. Since both vessel length and vessel diameter
could limit conductivity, when natural selection favors more efficient
vessels, both vessel length and vessel diameter should increase over
evolutionary time.

It appears that even within a plant, the population of wide
vessel elements will tend to contain fewer vessel ends than the narrow
elements (Fisher, 1970; Ewers and Fisher, 1989b). The frequency

distributions of vessel length of two woody vines, Vitis rotundifolia

 

Michx. and Saritaea magnifica Dug., were reproduced from previous
experiments (Ewers and Fisher, 1989b) in Fig. 2.7. Within a stem, the

vessel length frequency distributions were multi-modal in V.

44

Fig. 2.7. Frequency distributions of vessel length for narrow,
medium and wide vessels of two woody vines, Vitis rotundifolia and

Saritaeg magnifica (modified from Ewers and Fisher, l989b).

45

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Itls Saritaea
100-- 100--
. . 1; ,
~ Wlde ~___ wlde
501* 50‘-
., .. .
2L " —-i—1—L[—_|
O0 100 200 O 10 20
1oo-_ 1oo-__
7,, medium ~ medium
% 501* % 5040'
OH I ii I . I . I . E—l - . 3% o f h
0 100 200 0 10 20
100; 100--
v narrow " HOFFOW
50" 50‘-
00 j 2 5—! i c : c 4 : t O” ‘ .l l. :
O 100 200 O 10 20

 

Vessel length (cm)

46
rotundifolia or bimodal in S. magnifica for wide vessels, but bimodal
or Poisson for narrow vessels. The frequency distributions for the
medium diameter vessels were intermediate (Fig. 2.7). For each vessel
diameter type the greatest length frequency occurred in the shortest
vessel class except in the wide vessel population of y. rotundifolia
(Fig. 2.7). For wide vessels of V. rotundifolia there was a plateau in
the short vessel classes, a valley in the intermediate classes, and a
small peak in the longest vessel class. The above patterns could be
explained by a random selection of the location of vessel ends, with a
great, medium and small number of vessel ends in the narrow, medium and
wide vessels.

Why would plants have more than one type of vessel? Perhaps
vessels of various dimensions and mechanical properties evolved to meet
the sometimes conflicting demands for water transport, water storage
and mechanical support (Carlquist, 1988). Short and narrow vessels may
be better for mechanical support, lateral transport and storage whereas
long and wide vessels are efficient in axial transport but more
vulnerable to dysfunction. This could result in a bimodal frequency
distribution of vessel length by a combination of many narrow vessels
with a great number of vessel ends and relatively few wide vessels with
a small number of vessel ends. The combined result is a major peak in
the short classes and a minor peak in the long classes (Zimmermann and
Jeje, 1981; Sperry et al., 1987; Ewers and Fisher, l989a, b; Ewers et
al., 1990).

In the xylem, the elements with two vessel ends would be
classified not as vessel elements but as vascular tracheids (Carlquist,

1988), which are elements that are stacked end to end but which lack

47
perforations. By the simulations, a great number of vessel ends would
result in the "reinvention" of these tracheids, which intergrade with
narrow vessel elements in morphology, position and function (Carlquist,
1988). Carlquist (1988) suggests that vascular tracheids and short,
narrow vessels act as an auxiliary transport system for when large
vessels become embolized.

These are the first computer simulations of vessel length
distributions. The simulations suggest that varying the number of
randomly distributed vessel ends can explain most of the variation in
vessel length frequency distributions that occur in nature. The common
occurrence of vessel ends at nodes or junctions is simulated by random
selection from binomial and normal distributions. The developmental
control mechanism for vessel end placement is not known but is
influenced by auxin concentration and does not result in equal spacing

of vessel ends.

CBUUHPEFLIB

XYLEM STRUCTURE AND WATER TRANSPORT IN A TWINER, A SCRAMBLER, AND A

SHRUB OF LONICERA (CAPRIFOLIACEAE)

INTRODUCTION

In plants, differences among taxa in features of xylem structure
are often assumed to be of adaptive significance (Carlquist and
Hoekman, 1985). However, the interpretation of descriptive, ecological
and physiological features of wood structure has been "slow and
difficult" (Carlquist, 1988). Xylem functions include transport of
water and minerals, mechanical support of the plant body, and storage
of water and nutrients (Ewers and Cruiziat, 1991; Ewers, Fisher and
Fichtner, 1991). Since in vines, the plant gets mechanical support
from an external source, it is not surprising that vines differ from
free standing growth forms in the mechanical properties of the stems
(Gartner et al., 1990; Gartner, 1991a,c; Ewers and Fisher, 1991).
There are few studies of xylem structure and function that compare
vines to closely related free-standing growth forms. Ewers et a1.
(1988, 1990) compared tropical tree, shrub and liana (-woody vine)
species of Bauhinia. Gartner (1991b) compared lianas to shrubs within
the western North American species, Toxicodendrog diversilobum (T. &
G.) Greene. The present study investigated wood structure and function

in different species of temperate honeysuckles (Lonicera spp.).

48

49

In recent years more attention has focused on quantifying and
modeling the movement of water through plants (Waisel et al., 1972;
Pickard, 1981; Fiscus, 1983; Gibson et al., 1985; Radcliffe et al.,
1986; Schulte and Gibson, 1988; Tyree, 1988). For modeling water flow
in tracheary elements, xylem transport efficiency is often expressed as
the measured hydraulic conductance per pressure gradient (measured K5)
(Zimmerman, 1983; Tyree and Ewers, 1991). Theoretical hydraulic
conductance (theoretical K5), which relates xylem anatomy to the ideal
efficiency of water transport by the Hagen-Poiseuille equation,
modified for elliptical cross area (Calkin et al., 1986), is calculated

by the following formula:

A 2 .
n2 81 b1 . 251in in m"-MPa‘1'S'1 EQ- (1)

thoeretlcal K5 - 128n af+bf

where a1 and D1 are the major and minor axes of elliptical cross area
of the capillary in m; 1 - 1, 2,---,n; and n is the viscosity of the
fluid (10-9 MPa-s for water at 20 °C).

To determine how effective a stem is at supplying its leaves with
water, one needs to consider not only measured K5 and theoretical K5,
but also the leaf area that the stem segment supplies. Leaf specific
conductivity (LSC), measured K5 divided by supplied leaf area
(Zimmermann, 1978), has been used to determine "hydraulic dominance" of
the main stem over lateral branches (Tyree et al., 1983; Ewers and
Zimmermann, 1984a,b; Ewers et al., 1989) and to compare two temperate
trees to a tropical tree (Tyree et al., 1991). However, it has seldom

been used to compare different growth forms (Gartner, 1991b). Specific

conductivity (- measured K5 / xylem area) is also useful in comparing

50
different taxa because it relates water transport efficiency to the
xylem area. Because xylem of a shrub contributes more in mechanical
self-support than that of a liana (Gartner, 1991c), one might expect
that a shrub would devote a greater percentage of its xylem tissue to
mechanical support and thus have lower specific conductivity than a
liana. Huber value (= xylem area / leaf area) is related more directly
to mechanical properties than hydraulic properties of stems. However,
producing more wood per leaf area is also one way of enhancing
transport (Ewers and Zimmermann, l984a,b). For a given stem segment,
the relationship between these values is as follows (Ewers and

Zimmermann, l984a,b):

LSC - Huber value - Specific conductivity Eq. (2)

I expected Huber values to be greatest and specific conductivity to be

lowest in the free-standing shrub.

MATERIALS AND METHODS
Plant materials
I examined cultivated specimens of the twiner L. japonica Thunb.,
the scrambler L. sempervirens L., and the free-standing shrub L.
maackii (Rupr.) Maxim. A twiner is a vine whose shoots spirally twine
around a support. A scrambler is a vine that is initially self-
supporting but eventually falls over to be supported by an external

host plant or object. The scrambler, L. sempervirens, has straight

stems that do not twine and remain erect until they become too long and

heavy to support their own weight. By the end of their first year, the

51

stems normally fall upon the soil or adjacent support. Plants of L.
gempervirens (up to four years old) were grown outdoors in pots at MSU
and in the ground at Flushing, Michigan. Two outdoor individuals died
in 1990 due to winter freezing. Because L. sempervirens is not hardy
enough to survive some winters in Michigan, the potted plants were
sheltered in the greenhouse for the winter. Plants of L. japonica and
L. maackii (both up to seven years old) were grown outdoors on the
Michigan State University (MSU) campus.

Specimens were collected between May and September during 1988 to
1991. Stem segments of mostly 15 cm length, but 10 cm for densely
branched specimens, were labeled, a sketch of the branch architecture
was then made, and leaves distal to labeled segments were collected for
the measurement of leaf area. Fifty-five segments from 8 plants of L.
japonica, 60 segments from 4 plants of L. sempervirens, and 51 segments
from 5 plants of L. maackii were used for all the measurements
described below, except for vessel frequency, mean and median vessel
diameter and theoretical K5 for which 20 (L. japonica), 17 (L.
sempervirens), and 19 (L. maackii) stem segments were used. For wood

macerations, two stem segments were sampled per species.

Measured K5

Stem segments were collected early in the morning and cut under
water to prevent the introduction of air embolism. The cut surfaces
were shaved smooth with a fresh razor blade, fitted with vinyl tubing
at the proximal end, and the stems with tubing were submerged in water

and evacuated at 0.07 MPa for 15 min to remove superficial air bubbles.

A 150 mmole-m'3 acetic acid (pH-5) solution (Calkin et al., 1986),

52
filtered through 0.2 pm Gelman membrane filter (Sperry et al., 1987),
was perfused through the segments under a gravity gradient with a
maximum pressure head of 4.1 kPa. Volumetric flow rate was determined
with a pipet and stopwatch. Measured K5‘was calculated as the measured
volumetric flow rate (m3-s‘1) divided by the pressure gradient (MPa-m’l)

(Tyree and Ewers, 1991).

Anatomical study

Following the measurements of K5, a filtered 0.5% safranin
solution was perfused through the stem segments for 5 h to demarcate
the conducting xylem (Ewers and Zimmermann, l984a,b). The middle of
each stem segment was transversely sectioned with a sliding microtome.
The 20 pm sections were dehydrated through an ethanol-xylene series and
mounted in permount. Kodachrome slides of the sections were made with
a Nikon SMZ-lO photomicroscope and the images projected on a large
sheet of paper for measuring the vessel diameters (Ewers and Fisher,
1989a), and vessel frequency which is the number of vessels per
transverse xylem area (Wheeler, Baas and Gasson, 1989). In the young
stems, every vessel seen in a transverse section was measured. In
stems older than two years, all the vessels in randomly selected
sectors were measured. Each sector had vascular rays for marginal
boundaries and the pith and the vascular cambium as its inner and outer
boundary. Measurements from the sectors were extrapolated to the total
xylem area for calculation of theoretical K5 from Eq. 1.

Wood macerations were necessary to learn cellular characteristics

to distinguish narrow vessels from fibers or tracheids, since in

transverse sectional view narrow vessels appeared similar to tracheids.

53
A 5 mm long segment adjacent to the sectioned region was used for
macerations. All tissues outside the cambium were removed and the wood
containing pith was cut into longitudinal slivers. The material was
treated with Jeffrey's solution (10% chromic acid : 10% nitric acid -
1:1 vzv) for 36 h in a 60 °C oven until the material was soft to the
touch (Johansen, 1940). The tissue was washed in water, stained with
0.5% aqueous safranin, dehydrated in an ethanol-xylene series, and
mounted in permount. Cell wall thickness and lumen diameter of vessel
elements, tracheids and fibers, which were determined at the middle of
the long axis of each cell, were measured directly with an ocular
micrometer under a Zeiss compound microscope. For each segment, cell

populations were randomly sampled from the macerated materials.

Xylem area and leaf surface area

Xylem transverse areas were determined from the 20 um sections.
The conducting xylem area was identified based on the presence of
safranin dye in the vessel walls. The conducting and nonconducting
transverse areas of xylem were separately traced with a camera lucida
attached to a Nicon SMZ-lO microscope. The area of paper cutouts was
measured with a Li-Cor 3000 portable area meter. Stem xylem diameter
was determined as the mean of four diameters measured from the traced
drawings. One-sided leaf surface areas of all the leaves distal to the

stem segments were directly measured with the area meter.

Huber value
After measurements of leaf area, leaves were dried in a 100 °C

oven for at least four days until their dry weight remained constant.

54
Leaf weight was determined to the nearest 0.01 g with a balance. Both
(a) leaf area based and (b) leaf weight based Huber value were
calculated as the xylem transverse area of the stem segment divided by
(a) the leaf area or (b) the oven dried weight of leaves distal to the

stem segment.

Moisture content of wood

Stem segments from 3 to 6 cm long abutted to those used for
measured K5 were collected. The longer segments were needed for
narrower stems. Immediately after the bark was removed, the fresh
weight was determined to the nearest 0.01 g with a balance and fresh
volume was measured to the nearest 0.05 ml by displacement of water
upon submerging the specimen in a slender graduated cylinder. The
moisture content of fresh wood was calculated by the following equation
(Stewart, 1967; Pallardy, Pereira and Parker, 1991):

W’ _ —l--0.6S fresh weight of wood

8 DB Where DB = Eq. (3)
fresh volume of wood

 

Statistical analysis

Differences among taxa and differences between current year (one-
year-old) and older (older than one year) stems were evaluated from the
SAS General Linear Model (GLM) procedure for analysis of variance with
unequal sample numbers and Duncan's test for multiple comparisons

(Steel and Torrie, 1980; Peterson, 1985).

RESULTS
Analysis of variance indicated that there were statistically

significant differences between current year stems and older stems in

55
most examined features for all three species. Therefore, the
transectional and physiological data below are presented separately for

current year and older stems.

Maceration
A comparison of cell lumen diameter (Table 1) and wall thickness
(Table 2) from the macerated xylem indicated that vessels had generally

wider cell lumens and thinner walls than the tracheids and fibers.

Table 1. Range in cell lumen diameters (pm) of vessels,
tracheids and fibers of the twiner (L. japonica),

scrambler (L. gempervireng) and shrub (L. maackii)

 

 

Growth forms Vessels Tracheids Fibers
n-l70 n—70 n-220

twiner 7.6 - 103.0 . 3.8 - 17.6 2.5 - 11.3

scrambler 10.1 - 52.9 7.6 - 15.1 1.3 - 11.3

shrub 7.6 - 58.0 5.0 - 13.9 1.3 - 10.1

 

However, the very narrow vessels were similar to the tracheids
except for the presence of perforations. To exclude the tracheids
and fibers in vessel measurements from wood transections, below,

we included only cells (vessels) that had walls thinner than 2.5

56
um and lumens more than 18 Um in twiners or more than 15 um in

scramblers and shrubs.

Table 2. Range in cell wall thickness (pm) of vessels,
tracheids and fibers of the twiner (L. jagonica),

scrambler (L. sempervirens) and shrub (L. maackii)

 

 

 

Growth forms Vessels Tracheids Fibers
n=170 n=70 n-240
twiner 0.6 - 2.5 1.3 - 5.0 3.2 - 5.7
scrambler 0.6 - 2.5 2.5 - 3.8 2.5 - 5.0
shrub 0.6 - 2.5 1.9 - 4.4 2.5 - 6.3
Transections

Vessel diameter frequency distributions are shown for one
representative current-year and one four-year-old stem of the twiner,
scrambler and shrub (Fig. 3.1). Except in the older stems of twiners,
the frequency distribution of the vessel diameters tended to the
Poisson distribution (Steel and Torrie, 1980) with a high percentage
of the narrow vessels (Fig. 3.1). The pattern for the older stems of
twiners was a positively skewed normal distribution with many more

narrow than wide vessels, and a pronounced tail to the wide vessel

57

Fig. 3.1. Percentage of total theoretical K5 (open circles, o)
for each class of vessel diameter and the frequency
distributions of vessel diameter (histograms) in current year
and four—year-old stems of a twiner Lonicera japonica, a
scrambler L. sempervirens, and a shrub L. maackii. Each graph
is from one representative stem. Arrows indicate the largest

vessel diameter in each sample.

58

Current year stems Older stems
twiner

 

 

 

o ' 50 160
scrambler

40' o 40 o
l . n=202 n=465

 

 

 

 

 

 

 

o ' 5b 160 0 5'0 160
shrub
0
4o 40
n=171 ° n=170
O
o
A 20 ° 20
1 l
0
00 5b 160 06' ' ' 5b 160

Vessel diameter (,um)

59

diameter (Fig. 3.1). For all the distribution patterns, a great
portion of total theoretical K5 resulted from the wider vessels. While
only 1.1% (twiners), 15.3% (scramblers), 12.2% (shrubs) of the total
number of vessels in the current year stems and 0.6% (twiners), 4.7%
(scramblers), 8.9% (shrubs) in the older stems were grouped in the
highest two classes of vessel diameter in each sample, these classes
contributed 30.5% (twiners), 62.7% (scramblers), 50.6% (shrubs) of the
total theoretical K5 of current year stems and 27.7% (twiners), 25.5%
(scramblers), 46.9% (shrubs) of that of older stems. Conversely, the
narrow vessels were frequent but contributed little to the theoretical
K5 (Fig. 3.1). When stained vessels and tracheids below the 18 um
(twiner) and 15 um (scrambler and shrub) threshold were included, they
increased theoretical KL by less than 1% (data not shown).

Qualitative features of wood anatomy can be seen in Figs. 2-4.
Based on the examination of vessel diameter in 56 stem segments, the
twiner had the greatest maximum vessel diameter (Fig. 3.5) but also
many small vessels (Figs. 3.1, 3.2a, 3.2b). As stems aged, the outer
growth rings of xylem contained the widest vessels (Fig. 3.2b). The
means of maximum vessel diameter for twiners, 65.8 um in current year
stems and 94.7 pm in older stems, were significantly greater than those
of the other growth forms (Fig. 3.5). Stems of the scrambler contained
relatively narrow vessels (Figs. 3.1, 3.3a, 3.3b). In this species,
for each growth ring the widest vessels occurred at about the fifth to
seventh cell layer outside the previous year's late wood (Fig. 3.3b).
Unlike for the twiner, when the stems became older, the maximum vessel
diameter did not increase by much (Figs. 3.1, 3.3b, 3.5). In shrubs

(Figs. 3.4a, 3.4b), going from earlywood to latewood, vessel diameter

60

Fig. 3.2-4. Transverse sections of the current year stems (a)
and the older stems (b) of the twiner Lonicera japonica (2), the
scrambler L. sempervirens (3) and the shrub L. maackii (4). Bar

= 100 um.

..0 .‘.

.0.o.0....

 

62

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100 *
[In median i
" [111 mean
A 80-- Emaximum
E
3 ' l*
a 60--
.+J
0E3 ..
.9 4o-- 1 I I
T’ l
'6 n-
8 l
a, 20“ ~
> .- ~
0- L J _ l
L. j. L. s. L. m. L. j. L. s. L. m.
Current year stems Older stems

Fig. 3.5. Comparison of vessel diameter among different growth forms:

a twiner Lonicera jgpgnica, a scrambler L. sgmpervirens and a shrub L.

maackii. Bars indicate i standard error. Asterisks indicate the

statistically significant difference between species.

63
consistently decreased and the number of tracheids and fibers
consistently increased. As with the scrambler, when the stems became
older, the diameter of maximum vessels increased little in the older
growth rings (Figs. 3.4b, 3.5). The anatomical characters of the
scrambler were generally similar to those of the shrub. However, in
the scramblers, there was a wider distribution area of moderate vessel
diameters throughout the growth ring (Figs. 3.3, 3.4), a greater number
of very small vessels (Fig. 3.1), a smaller xylem area and a higher
percentage of stem conductive xylem area (Table 3). For all growth
forms, the median vessel diameter was usually lower than the mean
vessel diameter (Fig. 3.5). The difference between median and mean
vessel diameter was greatest in the twiners (Fig. 3.5).

The shrub had greater stem xylem diameters than the twiner and
the scrambler species. Likewise, the xylem diameter increment and
total xylem area increment per year were greatest in shrubs (Table 3).
The ratio of conductive xylem area to total xylem area (percent
conductive xylem area) was lowest in the shrubs (Table 3). The
greatest vessel frequency (vessels nmfz) occurred in scramblers,
especially in current year stems, and the least occurred in twiners,

especially in older stems (Table 3).

WOOd moisture content In the current year stems, the shrub had the
highest moisture content of fresh wood and the twiner had the lowest
(Table 3). However, the decline of moisture content of fresh wood from
current year to older stems was greatest in the shrubs and least in the

twiners (Table 3). For the older stems, there was no statistically

significant difference in moisture content among the different species.

64

 

 

 

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Measured and theoretical K5 The relationship between measured Kh and
theoretical K5 was linear and correlated in all three species. The r
values of the regression lines of measured K5 over theoretical K5
ranged from 0.72 to 0.98 (Fig. 3.6). The mean ratios of measured Kb
over theoretical K5 were 34.2% (twiner), 45.3% (scrambler), 54.6%
(shrub) in the current year stems (Fig. 3.6a) and 25.2%, 35.0%, 24.3%
in the older stems (Fig. 3.6b). The percentages were not statistically

different among species.

Huber value, specific conductivity, LSC The leaf area based Huber
value was greatest in the current year stems of scramblers and least in
the older stems of twiners (Fig. 3.7). In current year stems,
scramblers significantly differed from twiners and shrubs in this
parameter, but twiners and shrubs were not distinguishable from each
other. For older stems, twiners were distinguishable from scramblers
and shrubs but scramblers and shrubs were not statistically different
from each other. In addition, leaf weight based Huber value was much
higher in current year stems of the scrambler than in the twiner and
the shrub. However, in older stems, leaf weight based Huber value was
lowest in the shrub (Table 3). Mean specific conductivity was more
than twice as large as in twiners than the other growth forms, but the
difference was statistically significant only in current year stems
(Fig. 3.7). The LSCs were not statistically different from one another
except the current year stems of shrubs were significantly lower in LSC
than the other species (Fig. 3.7). The scramblers had the lowest

measured Kh and smallest supplied leaf area.

66

Fig. 3.6. Measured hydraulic conductance per pressure gradient
(measured Kb) compared to the theoretical Kh determined by
Poiseuille's law in current year stems (a) and older stems (b)
of the twiner L. jagonica, the scrambler L. sempervirens and the

shrub L. maackii.

67

 

o 0— twiner 3.0.90

A . -- scrambler
_5 D I ..... shrub

 
  

 

 

log (measured Kh) (m4.MPo-1.s"1)

 

 

 

—1o -9 -a -7 -e —5

log (thoereticol Kh) (m4.MPo-1.s"1)

68

Fig. 3.7. Comparison of means of Huber value, specific
conductivity and leaf specific conductivity for the stems of the
twiner Lonicera japonica, the scrambler L. sempervirens and the
shrub L. maackii. The probability of F value in the statistic
analysis of variance among species were 0.0001 (HV), 0.0003
(SC), 0.0147 (LSC) for current year stems and 0.0524 (HV) for
older stems. Bars indicate i standard error. Asterisks

indicate statistically significant difference between species.

69

 

 

+

 

g

 

+
*N\\\\\\\\\

 

m current year stems

(:1 older stems

 

 

“‘.““““1“I“““

 

 

 

 

 

 

 

 

 

 

 

 

 

P ’ p ’ ’ ’ b I b 0 ’ ’ D I i, ’ ’

 

 

Anlo— V on...» Lena:

‘ ‘ ‘ ‘ ‘ ‘ 4‘ . ‘ ‘ ‘ ‘ . 1 1‘ ‘ ‘ ‘

w m m o
9:. .73.: .«E also .0 .m 9:87.13 .«5 To; .0 .m 4

70

Discussion

The widest vessels of the twiner, L. japonica, appear to
hydraulically compensate for narrow stems. The wide vessels enhanced
the theoretical and measured Kg, and hence the specific conductivity,
such that the LSCs were similar in the narrow-stemmed twiner as in the
scrambler and shrub species. Conversely, the widest stems and greatest
xylem area occurred in the shrub, L. maackii. This implies that a
greater portion of the wood was involved with mechanical support in
this species. This is consistent with results for trees, shrubs and
lianas of Bauhinia (Ewers et al., 1990) and for shrubs and lianas of I.
diversilobum (Gartner, 1991b).

The vessel diameters, Huber values, and specific conductivities
of the scrambler, L. sempervirens, were surprising in that the
scrambler appeared to be more like the free-standing shrub, L. maackii,
than the twiner, L. japonica. In fact, the current year stems of the
scrambler had the highest Huber values based both on leaf area and leaf
weight. The scrambler thus had the most wood per leaf area and per
leaf weight, suggesting that of the three species it had the greatest
mechanical support for its leaves. However, the relatively high Huber
values in the scrambler do not by themselves entirely reflect the
mechanical support of their leaves. The extremely high vessel density
in L. sempervirens may tend to weaken the wood, making the production
of more wood necessary for minimal mechanical support. The large
terminal inflorescences of the scrambler, compared to the small axial
inflorescences of the twiner and the shrub, implies that the xylem of

scrambler might be overbuilt to provide mechanical support and

71

structural stability in the maintenance of flowering and fruiting,
especially in the current year stems. For both the scrambler and the
twiner, there was a trend of decrease in leaf area based Huber value
from current year stems to older stems. It could be expected,
especially in the scrambler, that older stems would have greater
external support and less influence of inflorescences or fruits than
the current year stems, allowing for lower Huber values in older stems.

The terms "ring-porous" and "diffuse-porous", as they are
normally defined (Panshin and Zeeuw, 1980), do not apply well to
Lonicera wood. Carlquist (1988) proposed at least 13 types of ring to
semi-ring porous woods, based on the origin and function of growth
rings (Carlquist, 1988). L. japonica and L. sempervirens have "type
10" growth rings in which maximum vessel diameter is deferred until
after the beginning of the growth ring. L. japonica has rings of
markedly wide vessels but L. sempervirens does not. The occurrence of
maximum vessel diameter not at the beginning of the earlywood also
implies that growth commences during cool winter months when soil
moisture is available but peak transpiration does not occur until some
weeks after initiation of the growth ring (Carlquist, 1988). L.
maackii has "type 7" growth rings in which vessels are wider in
earlywood than in latewood. It thus has markedly enhanced earlywood
conductive capacity (Carlquist, 1988).

The maximum vessel diameters of the twiner L. japonica and the
scrambler L. sempervirens are close to the lower end of the range for
vines in general (Ewers et al., 1990). Narrow vessels are held to

increase safety against freezing-induced xylem embolism (Carlquist,

1985; Ewers, 1985). Temperate lianas such as L. japonica and L.

72
sempervirens might have evolved or retained relatively narrow vessels
and low maximum vessel diameter due to exposure to annual periods of
freezing weather. L. japonica is evergreen except when cultivated as
far north as Michigan, where its leaves start to turn brown before
April. Narrow vessels, which are more resistant to freezing induced
embolism, are compatible with the evergreen strategy which allows for a

longer growing season. Temperate liana species of Vitis have extremely

 

wide vessels, but in these species the wide embolized vessels are
refilled by the high root pressures in the spring (Sperry et al.,
1987).

The minimum vessel diameters in Lonicera, of 7.6 um, are not
surprising given that vessels as narrow as 4 pm were reported for the
lianas Macfadyena unguis-cati (L.) A. Gentry, Derris scandens (Roxb.)
Benth., and Serjania polyphylla (L.) Radlk. (Ewers et a1. 1990). In
tropical lianas, the narrower vessels in stems, although quite
numerous, contribute an insignificant amount to the theoretical Kg
(Ewers and Fisher, l989a). Likewise, the exclusion of the narrowest
vessels in our sampling of Lonicera dropped the theoretical Kfi by less
than 1%. However, ignored narrow vessels would affect measurements of
the vessel frequency, mean and median vessel diameters, and
physiological functions such as water storage and lateral transport.

The mean vessel frequency of the three Lonicera species ranged
from 561 to 901 vessels nmfz. Despite the fact that I excluded the
narrowest vessels, this is much higher than reported for 35 species of
Caprifoliaceae of Japan. For instance, the vessel frequency for L.
maackii was reported as only 230 (Ogata, 1988). One possibility is

that Ogata ignored the narrower vessels, which I found to resemble

73
tracheids in transverse view. The highest vessel frequency previously
recorded within the Caprifoliaceae was 737-741 in Zabelia mosanensis
(Ogata, 1991). Carlquist (1988) claimed that vessel frequencies above
500 were unusual for plants in general, but these high frequencies had
been found in plants of notably cold or dry habitats (Miller, 1975;
Michener, 1981,1983; Carlquist and Hoekman, 1985). Those reported
above 1000 in shrubs are 1350 in Romneya racemosa Harv. of the
Papaveraceae (Carlquist and Hoekman, 1985) and 2673 in a species of
Cassiope of the Ericaceae (Wallace, 1986). In vines, 901 vessels-mm’2
in current year stems of L. sempervirens may be the highest vessel
frequency thus far recorded, compared to 8 to 90 in 25 vine species of
the Bignoniaceae (Gasson and Dobbins, 1991), 24.1 in yigis girdiana
Munson of the Vitaceae and 425 in Clematis lasiantha Nutt. of the
Ranunculaceae (Carlquist and Hoekman, 1985).

In my studies, the measured Kh'was about 24 to 55% of the
theoretical K5, with values not significantly different in the
different growth forms. This is similar to what most researchers have
found in plants in general. However, Berger (1931) reported a value of
100% for three species of vines. Zimmermann (1983) suggested this high
ratio might be due to long vessels that behaved more like ideal
capillaries than did short vessels.

Why is the measured Kh not equal to 100 % of theoretical K5? The
Hagen-Poiseuille equation, which is based on an ideal capillary, is not
adjusted for changing vessel diameter along the vessel length, and does
not consider resistance of the vessel ends, the pits and the
perforation plates along the pathway of conduction. Furthermore, it

assumes non-turbulent flow. From the present report, and the study on

74
the liana Bauhinia fassoglensis (Ewers et al., 1989), it does not
appear that liana vessels behave differently than in other growth
forms.

With LSC and maximum transpiration rates (E), one can predict the
maximum pressure gradients (dP/dx - E / LSC) that should occur in stems
(Zimmermann, 1978a; Ewers and Cruiziat, 1991; Tyree and Ewers, 1991;
Ewers et al., 1991). The LSC of the twiner, L. japonica, is much lower
than in other vines (Ewers et al., 1991; Gartner, 1991b; North, 1992),
and E is near the upper end of the range (Ewers, 1985; Bell, Forseth
and Teramura, 1988; Ewers et al., 1991; Gartner, 1991b; North, 1992).
The mean LSC for L. japonica was also lower than that for
dicotyledonous trees and most conifers (Ewers, 1985) except Thule
occidentalis (Tyree et al., 1991). I found a maximum E value for L.
japonica of 1.5-10'71mtnf1-sq'(unpublished) in the range Bell et a1.
(1988) reported. This is higher than reported in most other vines
(Gartner, 1991b; North, 1992). The predicted pressure-potential
gradient 0.75 MPa m”; in L. japonica is also higher than in the other
reported vine species. It implies that stems of L. japonica must be
subjected to larger water potential gradients to allow for their
evaporative flux. The stomates in this species appear to close at
water potentials of about -2.0 MPa (Bell et al., 1988). In the 3 m
long specimens used in the present study, a drop in water potential of
2.25 MPa would occur along the stem at maximum E. The efficiency of
the xylem conductive system could thus limit water supply to the
leaves.

The twiner, L. japonica, has been widely reported as a highly

successful weedy exotic vine in the southeastern United States

75
(Leatherman, 1955; Cartner et al., 1989; Sasek and Strain, 1991).
Narrow stems of this vine species may demand wider, more efficient
vessels than in the congeneric shrub or scrambler species in order to
overcome long distances of water transport and high transpiration rates
due to full sun exposure. Since the scrambler and shrub species of
Lonicera that I examined are not forest canopy competitors, they may
not have been exposed to selective pressure for the evolution of wide
vessels. Narrow vessels, although less efficient in transport, may be
more resistant to dysfunction and selected for in understory species

and species with shorter transport distances.

CONCLUBIONS

In this dissertation, the methodological, theoretical and
comparative studies provide a better understanding of xylem structure
and function and allow for some ecological and evolutionary
interpretations. Through the experimental and theoretical approaches
to investigate the effect of segment length on measurements of water
transport efficiency, I found that stem segment length affected about
0.39%-cm’1 of measured K5 in L. fragrantissima. Most early studies
(Zimmermann, 1978, 1983) suggested that selecting a proper segment
length for the K5 measurements in a species is necessary due to the
variation in vessel length. From my results, it may not be important
to select a segment length longer than the maximum vessel length. Due
to a greater impact of unevenly distributed vessel ends, changing
vessel lumen diameter, or the effect of pits on non linear flow (Bolton
and Petty, 1975), K5 readings in the very short segments of L.
fragrantissima may not represent those in the longer segments. For
measured K5, a suggested segment length would be longer than the median
vessel length.

The first computer simulations of vessel length suggest that
varying the number of randomly distributed vessel ends can explain most
of the variation in vessel length frequency distributions that occur in
nature. Vessel ends at a node or a junction are simulated by random

selection from binomial and normal distributions. The case with

76

77
multiple nodes could be simulated in the future. Because a
considerable percentage of vessels can end at the nodes (Salleo et al.,
1984), vessel ends may be an important segmentation in the water
conducting system of plants (Zimmermann and Jeje, 1981; Salleo and Lo
Gullo, 1986; Aloni and Griffith, 1991). The developmental control
mechanism for vessel end placement is not known but is influenced by
auxin concentration and does not result in equal spacing of vessel
ends. The distribution of narrow vessels may be predominated by the
uniform random pattern, whereas the distribution of wide vessels may be
influenced by the normal and binomial random patterns.

In the relationship of xylem structure and function, all three
growth forms of temperate honeysuckles (Lonicera spp.) had many narrow
vessels and relatively few wide ones, with the measured K5 (flow rate /
pressure gradient) about 24 to 55% of the theoretical K5 predicted by
Poiseuille's law. Only the twiner, L. japonica, had some vessels
greater than 50 um in diameter. The twiner had the narrowest stem
xylem diameters, suggesting that the greater maximum vessel diameter
hydraulically compensated for narrow stems. Conversely, the free-
standing shrub, L. maackii, had the greatest annular increment of xylem
but the least percent conductive xylem implying that a great portion of
the wood was involved with mechanical support. The scrambler, L.
sempervirens had low maximum vessel diameter, high Huber values (-
xylem area / leaf area), and low specific conductivities (- measured K5
/ xylem area), much like the shrub. The greatest vessel frequency
occurred in the scrambler (901 vessels'nmfz), the highest thus far

recorded in vines, which could result in lower mechanical strength of

the wood. The lowest Huber value and highest specific conductivity

78

occurred in the twiner, suggesting little self-support but relatively
efficient water conduction. Future studies can involve physical
studies of wood strength and flexibility for the different xylem types.
LSC (- measured Kg / leaf area) and maximum vessel diameter of Lonicera
vines were close to the lower end of the range for vines in general.
The narrow vessels in the shrub and scrambler may be related to low
transpiration rates and short transport distances. At least for the
twiner, which grows in the canopy, maximum transpiration rates and
predicted stem pressure gradients were at the upper end of the range of
published results in vines. Since the xylem pressure gradients were at
a magnitude to limit transpiration and hence, assimilation, there would
be selective pressure to evolve wider vessels in the future.

Investigation difficulties and inherent complexities about vessel
ends in regard to their structure, distribution and control of their
occurrence, leave an open area for further studies. I was not able to
quantify the effect of vessel ends per se on transport efficiency. One
of the difficulties is in not knowing if vessel ends behave differently
than ordinary vessel elements in their water transport through pits.
If the pitting in vessel ends is much more porous and thus offers much
less resistance than in other vessel elements, the combined effect of
lumen resistance and vessel end resistance on water transport
efficiency could be modeled as in an electric circuit in series. The
lateral transport through ordinary elements could be ignored, and the
effect of vessel length on transport efficiency could be quantified.
Conversely, if the porosity of vessel ends is similar to ordinary
vessel elements, the multitude of major transport pathways through the

vessel system is more complex and more difficult to model given the

79
tremendous variation in vessel lengths. The flow would have to be
modeled after multiple electric circuits both in parallel and in
series, with a multitude of lengths of the transport pathways within
the xylem. The most complex situation would involve significant
lateral flow by ordinary vessel elements, but greater porosity in
vessel ends than in ordinary elements. I assume the perforations offer
less resistance. A good experimental system might be herbaceous vines,
with limited secondary growth. Such plants would have long vessels but
perhaps many vessel ends at the nodes. In woody plants secondary
growth swamps out the effect of nodes.

For modeling water flow with related xylem structure or
understanding ecological strategies under water stress, the impact of
embolism on xylem conductance may be important. Further investigations
can also focus on the role of vessel ends at the segmented zones which
have been called 'safety zones', with little documentation. Do these
safety zones limit the spread of embolism? Are they hydraulic
constrictions? Whether the high frequency of vessel ends at nodes or
other junctions is physiologically or ecologically significant is still
unclear.

Studying xylem structure, Kb, LSC, specific conductivity, Huber
values and transpiration in different species and in different growth
forms can always supply valuable information due to different transport
distances and hydraulic demands. Comparisons of the hydraulic
architecture of adjacent understory and canopy species would be
interesting. With broader surveys of hydraulic architecture in plants,
including field measurements of water transport, we can learn more

about the importance of the variation in xylem structure that occurs in

nature .

80

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