1

Investigating various xylem sap

extraction techniques from drought-

stressed Geijera parviflora trees

Miriam Morua

Biology Department California State University Fullerton

800 N. State College Blvd.

Fullerton, CA 92833

mmorua@fullerton.edu

Research Advisor

H. Jochen Schenk, Ph.D., Professor

Plants & H2O Lab

California State University Fullerton

Department of Biological Science

P.O. Box 6850 Fullerton, CA 92834

jschenk@fullerton.edu

Time period of Internship: June 6, 2014 to June 30, 2015

Date report is submitted: June 30, 2015.

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Table of Contents

1. Acknowledgements …………………………………………………………3

2. Executive Summary………...………………………………………………4

3. Project Objectives.……………………………………………………..........5

4. Introduction…………………………………………………………..........6-8

5. Project Approach…………………………………………………….......9-14

6. Project Outcomes……………………………………………………….15-22

7. Conclusions……………………………………………………………..23-25

8. Significance ………………………………………………………………..25

9. References………………………………………………………………….26

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AWKNOWLEDGEMENTS

First off, I would like to thank my project adviser Dr.Jochen Schenk and lab manager, Susana

Espino for helping me during my project. I would also like to thank the undergraduate students,

Lilliana Cano, Roshni Morar, and David Romo for helping me during my experiments. Lastly, I

would like to thank Julie Lappin and the rest of the Watershed Management Resources Initiative

members for giving me the opportunity to conduct research through a USDA- funded project.

Also, for funding conference and seminar trips to allow students to network and learn about

USDA career opportunities.

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EXCECUTIVE SUMMARY

There is a recent criticism on common techniques for xylem sap collection that make the

common assumption that cutting stems under water actually introduces air into the vessels

leaving it susceptible to cavitation and levels of embolisms. Thus affecting the results that could

potentially indicate the stress condition of plants (Wheeler et al., 2013). There are many

techniques that have been used for xylem sap extractions but few make direct comparisons of the

techniques based on analysis of the sap and water status of the plant. The purpose of this project

is to assess the suitability of the sap collection methods for drought-stressed trees. Xylem Sap

was collected from Geijera parviflora trees using two pressurization techniques which mainly

varied in the way sap was collected. The first pressurization technique is done using the

Scholander-Hammel pressure chamber through positive pressure. The second technique was

done using a Vacuum through negative pressure. To determine water stress of the tree, stem

water potentials were measured on site after cutting stem. Xylem sap was collected from both

well-watered and drought-stressed G.parviflora branches. To confirm the validity of the different

techniques, samples were ran across a high sensitive nanotracker to compare the number of

particles in the sample (LM10, NanoSight, LTD) and sap volumes.

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PROJECT OBJECTIVES

The main goal of this study was to investigate whether the extraction techniques altered

concentration of particles in the samples collected and the relationship to stem water potential

(Ψ). Secondly, the goal of this study is to develop better techniques for efficient xylem sap

collection from drought-stressed stems without inducing embolisms or bubble formation at the

cut surface. Specifically, I developed a method for xylem sap extractions that keeps both

artificial bubble formation and contamination of the sap from living wood cells to a minimum.

To compare and confirm the validity of the different techniques, xylem sap samples will be

analyzed using a highly sensitive nanoparticle tracking system to compare the number of

nanoparticles in the sap (LM10, NanoSight, LTD). These nanoparticles include mainly

nanobubbles, as well as small debris that results from contamination from living cells.

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INTRODUCTION

The ongoing drought in California is limiting the state’s water supply and it is likely to

have an effect in horticultural crops grown under irrigation (Schenk, 2014). Various plants and

trees that experience drought conditions are prone to water loss through transpiration. Drought

induces high transpiration to occur and as a result affect the plants ability to transport water due

to the loss of water from the stomata.

Xylem tissues are responsible for transporting water and exchanging solutes from the

roots throughout the plant. The transport of water varies by plant type but in the case of tall trees,

they require the use of gradient transport systems that allow it to overcome gravitational forces.

Gradients in hydrostatic pressure, water potential, and osmotic potential, coupled with gas

exchange through the leaves provide the mechanisms that allow lift of water to the top of trees.

These complex variations of pressure are essential to understanding xylem function in order to

comprehend stress conditions of plants. This is particularly important in plants and trees that are

exposed to drought conditions because soil water deficit results in environmental constriction to

plant growth (Goodger et al., 2005).

Drought stress can cause plants to create embolisms in the xylem when the xylem

pressure declines below a certain negative threshold (Zimmerman, 2002). Trees that are under

periods of low soil water content and high transpiration rates, suffer of negative pressures in the

in the xylem. These negative pressures create tiny bubbles or embolisms in the xylem in which

cavitate the xylem conduits (Zimmerman, 2002). Cavitation frequently occur because of drought

and high transpiration. The resulting embolisms are known to diminish a plant’s capacity to

transport water from the soil to the leaves (Wheeler, 2012). Consequently, embolism reduces the

xylem conductivity required for carbon fixation.

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Embolisms also form when stems are cut for research purposes (Wheeler et al. 2013), and

this can create problems in studies of plant physiology, such as studies of the chemical

composition of xylem sap under drought conditions. Embolisms block xylem and make it very

difficult to extract sap from drought-stressed stems.

Various methods for sap sampling techniques have been developed that overcome the

problems essential for sap collection. Schurr (1998) states that the problem in xylem sap

collection arises during incision which causes entry of air rather than leakage of sap. Most of

these common techniques are destructive because they involve collecting sap from parts of the

plant when they are pressurized the sap is extracted out of the cut end. Schurr (1998) argues that

the tension that is created at the cut surface redistributes the water of the transpiring plant to

different compartments and thus the conditions of water is not the same as before. To account for

this, other sap collection techniques have been developed such as vacuum pressure where it

involves applying a force where it separates the xylem sap from other compartments (Schurr,

1998). For instance, the vacuum extraction technique (Lopez-Portillo et al., 2014) involves

opening vessels because several cuts are made from the apical end of the stems.

By general consensus the Scholander-Hammel pressure chamber method continues to be

the common method used for xylem sap collection. Alexou and Peuke (2013) discuss the usage

of the pressure vessel technique in the field because in transpiring plants the xylem sap is usually

always under tension rather than pressure. In this method gas pressure (N₂ compressed air) is

applied to the cut plant part to compensate the negative pressure in the water column and vessels

to cause sap to flow in the opposite direction.

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Although it is still unclear how plants can transport water under negative pressure in their

hydraulic system, the xylem; it is important to continue studying sap composition in order to

understand stress conditions of plants. Even though it is very difficult to study the xylem

function due to the manipulations that can lead to artifacts (Alexou and Peuke, 2013); formation

and repair of xylem embolisms can potentially determine irrigation for horticultural crops.

As previously mentioned cutting the plant part in air can lead to the creation of artificial

bubbles or artifacts. To avoid these artifacts, the plant part was being cut again under water and

then pressurized. It was hypothesized that cutting under water preserves the hydraulic system

that may have existed when cutting in air. However, according to the recent criticism in Wheeler

et al., 2013, the common techniques for xylem sap collection makes the assumption that cutting

stems under water actually embolizes the stem or creates artificial bubbles. This recent criticism

has made us reevaluate the pressurization techniques. A recent study by Torres-Ruiz et al., 2015

investigated an experimental protocol to avoid the excision artifact suggested by Wheeler et al.,

2013 at the cut surface created when collecting the plant segments. Torres-Ruiz et al., 2015

suggests to cut stems that are twice the vessel length and re-cutting the branch under water 0.2

times the major vessel length. The branch is then rehydrated for 30-minutes and leaves are

wrapped in a plastic bag to prevent transpiration. The branch is then recut again under water

roughly one-two times the major vessel length. Although the Torrez-Ruiz protocol was intended

for hydraulic conductance it can be used for xylem sap extractions to (1) reduce creation of

artificial bubbles at cut surface and (2) successfully collect sap from drought-stressed trees. The

purpose of my research is to develop a method for xylem sap collection that keeps both artificial

bubble formation and contamination of the sap from living wood cells to a minimum.

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PROJECT APPROACH:

Extraction of sap under positive pressure (PP) , using a Scholander pressure chamber and

sap extraction under vacuum or negative pressure (NP) was compared for use on trees grown

under drought stress conditions(Alexou and Peuke, 2013).

Study site

The trees that were sampled were located in front of the Dan Black Hall of California

State University Fullerton in Fullerton, CA (33°52'42.68"N, 117°53'12.72"W). The tree samples

were collected specifically at 9-am, 10-am, Noon, and 2-pm on different days between April to

June 2015. The Australian willow trees are always exposed to full sunlight and induced to

drought-stress. Ideally, the warm dry air prevalent during the summer in southern California

provides favorable situation of drought-conditions. However, there were unexpected weather

patterns during April, May and June where rainfall and cloud cover affected the drought

conditions.

The Australian Willow tree, or Geijera parviflora is planted in the parking lot of the

university because they are fast growing trees with noninvasive roots that grows in full sun and

casts a light shade. Also, these trees have water-filled leaves that are fire-resistant and drought

tolerant trees that survive in moist to dry soil (Gilman et al., 1993). Height of trees sampled

ranged from 25 to 30 feet tall and had a full canopy. Trees that were documented as stressed

were within the parking lot and those well-watered were found in the large outer patches of grass

where trees were more frequently watered due to a sprinkler system (Figure 1).

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Figure 1: A Google Earth image of the study site which included sampled Geijera Parviflora

trees. Branches were obtained from both well-watered and drought-stressed trees. An example of

each is labeled.

SAMPLE PREPARATION FOR BOTH METHODS

Collecting Sample and Re-cutting for Rehydration:

Sample was cut in air at the study site from upper canopy from both north and south side

of the tree. Stems collected had minimal branching and mean length of branch was 147-cm long.

The mean major vessel length (MVL) of G.parviflora was determined to be 100-cm long using

the external port of the Scholander-pressure chamber (Model 1000, PMS, Albany, OR). The

diameter of the stem varied depending on the extraction technique. The average basal part of the

stem diameter collected for the positive pressure technique was 5 to 7-mm. While the diameter

collected for the negative pressure technique ranged approximately between 5 to 15-mm in

diameter. Trees that were well-watered had more foliage and darker green leaves compared to

the stressed branches which were a lighter green. Branches were cut in air at the site were

returned to the lab which located in Dan Black Hall less than 300-ft away. In addition, stems

were cut and placed in Ziploc’s for stem water potential measurements. Branches were

Stressed

Watered

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immediately placed in a container filled with deionized water and leaves were bagged to prevent

transpiration. Basal end of the stem was recut under water approximately 2-cm using pruners.

Stems were allowed to rehydrate under water for about 30-minutes. Duration of time to rehydrate

was determined based on prior hydraulic conductance experiments (Torres-Ruiz et al., 2015).

EXPERIMENT 1- SCHOLANDER-PRESSURE CHAMBER

I. Stem Water Potential

While the branches rehydrated, stem water potential was measured to assess the water

status of the branch cut at the site. Using the Scholander pressure chamber stem was pressurized

until water was observed at the top of the cut surface. The tree was considered stressed if the

stem water potential ranged between -3.5 to -2.5 mPA. The tree was considered as well-watered

if the stem water potential ranged from -2 to -0.5 mPA. Stressed branch where sap was collected

from was labeled as sample stress (SS) and sap collected from a well-watered branch was labeled

as sample watered (SW).

II. Positive Pressure Sap Extraction

In this experiment, 17 stems were pressurized using the pressure chamber (Model 1000,

PMS, Albany, OR) or collected with Technique P. After the rehydration period the branch was

re-cut twice at the basal end (bottom) close to 20 to 40-cm in length depending on original size

of branch. These two cuts should be at least twice the MVL. For example, mean length of

branches of this study was 147-cm long and for these experiments 20-cm to 40-cm were cut,

leaving a little over of 100-cm.

A third cut was done under water at the top using a sharp blade to make a fine cut. Bark

was removed roughly on the top 0.5-cm under water to expose the xylem tissue. The xylem was

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then washed with deionized water that was delivered from a high pressure flosser (WP-100 Ultra

Water Flosser, Waterpik Inc., Fort Collings, CO, USA) to remove excess tissue, cell debris, and

potential contaminants. It was then re-washed after attachment of tygon tubing to the exposed

xylem tissue. A control was taken for each sample (n=17) by adding 1-mL of nanopure water

inside the tygon and latex tube attachment and it was left for one-minute. Stem was attached to a

pressure chamber gasket which contained a beaker filled with acid fuschin inside the chamber.

Gas pressure (using Nitrogen compressed air) was then applied to the stem at a low flow of 0.3

mPA (Figure 2). Sap was collected from the tygon tubing using a Pasteur pipette and transferred

to a eppendorf tube.

EXPERIMENT 2- VACCUM EXTRACTION

I. Negative Pressure Extraction

For this experiment, 17 stems were pressurized using a vacuum or with technique N.

After the rehydration period the stem was re-cut twice at the distal end. The surface of the xylem

core at the distal end of the stem was shaved using a blade to make a sharp fine cut. The distal

end of the stem was debarked approximately 0.5-cm to expose the xylem tissue. This end was

washed with a high-pressure jet stream of deionized water to remove excess tissue (WP-100

Ultra Water Flosser, Waterpik Inc., Fort Collings, CO, USA). The surface was re-washed after

attachment of the tygon and latex tubing to remove other potential contaminants and excess

phloem. A control was taken for each sample by adding 1-mL of nanopure water inside the tygon

tubing and it was collected after one-minute. The tygon tube was removed leaving the brown

latex enforcer. The branch was then fitted into another tygon tube connected to the vacuum

apparatus (Figure 2). The Vacuum apparatus was connected to a rubber stopper which contained

pipette tips that delivered the sap at a low rate into a 500-mL Erlenmeyer side-arm flask that

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contained a small test tube inside a larger one (Figure 2). The stopper sealed the flask and the

side-arm was connected to a vacuum. Lastly, parafilm was used to seal the vacuum filtration

apparatus. The stem was pressurized with the vacuum air for 30-seconds. An initial first cut was

made at the apical end of the branch approximately 4-cm in length. This following with several

successive cuts approximately 2-cm in length. The consecutive cuts were made to all the side

branches of the stem until sap was observed in the test tube. Once sap was observed, cuts were

made every 1-minute to allow all of sap to drop slowly.

The consecutive cuts are hypothesized to open up the vessels at each cut to allow the sap

to be extracted as it is pressurized with the vacuum force. Cuts were consecutively made until the

stem reached the tygon tubing. The vacuum apparatus was disconnected after sample was

collected in the test tube. Using Pasteur pipettes, 1-mL of the sap collected was transferred to

eppendorf tubes.

\

\

Figure 2: Images demonstrate the different techniques used in this project. Sap was

extracted using Technique P with the Scholander pressure chamber (left). In addition,

pressure chamber was used to measure Ψ potential. The second technique used was to

extract sap from a vacuum using a vacuum filtration apparatus or Technique N (right).

Technique P

Technique N

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PROJECT APPROACH: ANALYSIS

I. Nanoparticle Tracking Analysis

Samples collected were immediately analyzed for mean particle size (nanometers) and

concentration of particles/frame using the Nanoparticle tracking analysis with the LM10

microscope (NanoSight Ltd., Minton Park, Amesbury, Wiltshire, UK) proposed by Saveyn et al.,

2010. One sample per branch was analyzed along with the control for contamination analysis. A

total of 0.5 ml of the control and the sap sample were transferred using infusion syringes

connected to the LM10 chamber via the NanoSight syringe pump. The control and sap samples

were exposed to a blue laser (λ = 405-nm) to visualize the Nano particles in the microscope

connected to a Marlin camera. These images were processed using the Nanosight NTA 2.3

Analytical Software which produced 5 batches or frames (Images A-D). These frames were

averaged to produce a mean particle size (nm) value and concentration value (particles/frame).

This concentration value was assessed for contamination particles in the xylem sap to determine

efficacy of technique. Following analysis samples were immediately freeze dried with liquid

nitrogen and stored in a freezer for permanent storage. This was to reduce enzymatic activity for

future analysis procedures.

II. Data Analysis

Linear regression model was performed to assess the relationship between concentration

(particles/frame) and covariate, stem water potential (Ψ) using the R-studio. Linear Regression

statistical output was also used to determine the R₂ value to assess if stem water potential is a

predictor for number of particles found in the sample extracted. A boxplot was also conducted to

compare the means between the techniques N (NP) and P (PP) for each conditions; stress (S) and

well-watered (W). Comparison was further analyzed by sample; control (C) and sap collection

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(S). Furthermore, variation was also compared to validate findings. The Tukey Kramer

procedure was used to make multiple comparisons between techniques and conditions at 95%

familywise confidence intervals.

PROJECT OUTCOMES

I. Imaging from the Nanoparticle Tracking Analysis Software

The images illustrated in the following pages are to demonstrate the Nanoparticle tracker

analysis through the Nanosight NTA 2.3 Analytical Software performed on May 24th, 2015.

The concentration (particles/frame) was produced by the software by taking the average of

five batches via a 30-second analysis of the sample. A value of mean particle size (nm) is

also produced by the software. Panel A demonstrates each batch concentration taken in 30-

second intervals (n=5) which provides the particle size distribution of the sample (Images A-

D). The concentration value was assessed to determine the contamination from the controls

and the xylem sap samples collected from each Technique. Panel B demonstrates an example

of a batch taken to demonstrate a frame or video of particles in each sample: control and sap.

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NANOPARTICLE TRACKING ANALYSIS

A. Images comparing Technique P to N for Control Sample collected from a Watered Stem

A

Illustration of control sample collected from a watered branch through technique P. Few particles are

observed to demonstrate minimum contamination (concentration = 1.98 particles/frame)

A

B

Illustration of control sample collected from a watered branch through Technique N where more

particles were observed (concentration = 20.26 particles/frame)

B

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B. Images comparing Technique P to N for Sap Samples collected from Watered Stems

Illustration of sap sample collected from a watered branch using Technique P where high

contamination was observed (concentration =67.99 particles/frame).

Illustration demonstrates sap sample collected from a watered stem using Technique N. Large

particles are observed in the video frame with fewer particles compared to technique P

(Concentration=53.8 particles/frame).

A B

B

A

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C. Images comparing Technique P to N for control samples colected from stressed stems.

Illustration of control sample collected from a drought-stressed stem using Technique P where no

particles were observed thus no contamination (concentration = 1.21 particles/frame).

Illustration of control sample collected from a drought-stressed stem using Technique N a few

particles were observed thus little to no contamination (concentration = 6.79 particles/frame).

Illustration of control sample collected from a drought-stressed stem using Technique N where no

particles were observed thus no contamination (concentration = 1.21 particles/frame).

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D. Images comparing Technique P to N for Sap samples colected from stressed stems

Illustration of control sample collected from a drought-stressed stem using Technique P large

particles were observed thus high contamination (concentration = 82.78 particles/frame).

Illustration of control sample collected from a drought-stressed stem using Technique N many particles

were observed in sample to demonstrate high contamination (concentration = 82.76 particles/frame).

Although, not many inferences can be made since concentration was similar to technique P.

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II. DATA ANALYSIS

No linear relationship was observed through regression analysis using R-studio software. No

statistical differences were observed through linear regression models. It was observed that sap

samples collected from branches that had a low stem water potential (Ψ potential) had the highest

mean particle size (nm) compared to samples with high Ψ potential (Figure 3). In addition, the

concentration of particles was found to be higher in the watered sample (low Ψ potential)

compared to the stressed (high Ψ potential) (R₂=0.02, Figure 4). Although, no statistical evidence

could support the relationship between stem water potential and concentration it is important to

note more particles are found with less stressed branches.

Sap collected with technique P had higher concentration of particles compared to technique

N specifically for the stressed samples with high Ψ potential (Figure 5). However, sap collected

from watered stems using technique N had a higher concentration of particles (Figure 5). Two

sample t-tests and multiple comparisons using the bonferri procedure were conducted to

determine a significant difference between technique P and N. No statistical evidence was found

when comparing stressed sap samples between techniques nor with watered samples.

A boxplot was performed to compare concentration means between techniques and

conditions. A large spread and variability within the means of the samples collected was

observed (Figure 6). Boxplot also shows outliers present in the samples collected with technique

P for the watered control and stressed sample. The Tukey Kramer procedure that accounts for

95% familywise confidence intervals showed no significant differences between the techniques.

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Figure 3: Relationship between stem water potential (mPA) and mean particle size (nm) was

compared in order to assess whether Ψ potential affected the mean particle size in the samples

collected. Sap samples that were collected from branches that were well-watered had the highest

mean particle size (nm). No statistical evidence was found when linear regression models were

analyzed (Regression, R₂=0.01, p=0.369).

Figure 4: Stem water potential was measured to assess the water status of the branch cut. Well-

watered varied between -1 to -2 and drought-stressed varied between -2.3 to -3 (mPA) . The

concentration of particles is higher in the well-watered samples compared to the stressed

samples. No statistical difference was found between the relationship of stem water potential and

concentration (particles/frame) (Regression, R₂=0.02, p=0.687).

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Figure 5: Concentration (particles/frame) was compared across all samples collected and

analyzed with the Nanoparticle tracking. Sap collected with technique P was observed to have

the highest concentration of particles for the stressed branches compared to technique N.

Watered stems, technique N had a higher number of concentration.

Figure 6: Boxplot shows large spread and variability within the means of the samples collected.

In addition, there are outliers present in the samples collected with technique P for the watered

control and stressed sample. The Tukey Kramer procedure that accounts for 95% familywise

confidence intervals showed no significant differences between the techniques.

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10

20

30

40

50

60

70

PPCS NPCS PPCW NPCW PPSS NPSS PPSW NPSW

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CONCLUSIONS

Although recent criticisms suggest that cutting under water creates artificial bubbles or

embolizes the stem, research conducted in this study provides evidence that resolves this

problem by minimizing the creation of bubbles (Alexou et al., 2013; Wheeler et al., 2013). The

protocol suggested by Torres-Ruiz et al., 2014, was considered to account for this problem. This

protocol suggests recutting the stem under water at the basal end of the stem after rehydration

would allow the stem to recover and tension would be released.

The first cut in air at the site would essentially introduce air bubbles into the xylem

vessels because the xylem is under tension. Initial re-cutting under water approximately 0.2-cm

or at the surface of the cut can also create artificial bubbles as suggested by Wheeler et al., 2013

because the xylem remains under tension. However, by letting the stem rehydrate in water for a

short period of time will let the stem recover and allow hydraulic continuity. The second re-cut

under water removes part of the stem-or-in theory a vessel that contains many of the initial

bubbles created. Additionally, this also should allow for water to enter the xylem, thus releasing

the tension. The last re-cut under water hypothetically will remove more bubbles because the

tension has already been released under water. Following this protocol along with any

pressurization technique (positive or negative pressure) should resolve the creation of artifacts

suggested by Wheeler (2014). Additionally, would allow to resolve the problem of destructive

techniques that result in small volumes of sap. To validate argument the samples collected using

the Torres-Ruiz protocol coupled with the various techniques, positive pressure and negative

pressure; the samples were run against a highly sensitive Nanoparticle tracking device. Because

the criticism of these techniques relies on creation of artificial bubbles the nanotracker was used

to track the number of particles found in the sample. Although it is unknown of the composition

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of these nanoparticles within the xylem, it is a useful technique to compare these techniques to

because it indicates the levels of concentration and contamination.

There was no statistical evidence found when comparing between the techniques for each

condition using multiple comparisons test (p > 0.05). No linear regression relationships were

found when comparing the stem water potential and concentration rates (Figure 3 & 4).

Although, linear regression model showed a weak relationship, this does not mean that the

parameters are unrelated. It was concluded that samples using the positive pressure technique

contained more particles compared to negative pressure. Sap samples collected with drought-

stressed stems or a high water potential contained more nanoparticles (Figure 5). Since drought-

stressed trees are usually under more tension than those that are well-watered it can be expected

to find more particles. However, results also indicated a higher level of particles for samples

extracted from well-watered stems or have a low water potential using the negative pressure

technique (N). No statistical evidence was found when comparing stressed sap samples between

techniques nor with watered samples using paired t-tests and multiple comparisons tests:

pairwise and Tukey-Kramer procedure.

Pressurization techniques are by far more useful than other sap collection techniques that

are non-destructive due to the large amounts of volumes extracted from stems or branches. In

conclusion, it was found that both methods tested provided sufficient amounts of sap for

comprehensive analysis for stems under different stress. It was concluded that the negative

pressure method using the vacuum infiltration apparatus allows for more sap to be collected

compared to the positive pressure using the Scholander pressure chamber. More sap was

collected overall throughout the study using technique N (NPSS= 6.25-mL, NPSW=6.10-mL)

for each sample compared to technique P (PPSS=2.15, PPSW=1.86). It is recommended that

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both techniques can be used for xylem sap collection because there were no traces of differences

between nanoparticles in samples

SIGNIFICANCE OF STUDY

The cohesion-tension theory states that the xylem sap is under tension whenever the plant

is transpiring. Under low soil water availability or high transpirational demand, negative

pressures in the xylem or bubbles are formed and pulled in the conduits. Cavitation can occur

and blocks the hydraulic system of the plant which limits photosynthesis. Cavitation is known to

frequently occur in plants as drought conditions continue due to the high transpiration (Wheeler

2013; Schenk, 2013; Zimmerman 2002). It is important to understand this phenomenon to

understand the xylem function in order to comprehend stress conditions of plants.

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Alexou M., Peuke A.D. (2013) Methods for Xylem Sap Collection. Maathuis M.F. Plant Mineral

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Environmental Horticulture Department, University of Florida.

Goodger J.Q.D., Sharp R.E., Marsh E.L. (2005) “Relationships between xylem sap constituents

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sampling techniques”. Journal of Experimental Botany. 56. 2389-2400.

Lopez-Portillo J, Ewers FW, Angeles G. 2005. Sap salinity effects on xylem conductivity in two

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