1

Testing for Xylem Vulnerability to Embolism

and Embolism Repair in Citrus species

An undergraduate Thesis

Presented to the Faculty of the Southern California Ecosystems Research Program

Department of Biological Science

California State University, Fullerton

Emily Nguyen Wieber

Approved by

Dr. H. Jochen Schenk (Thesis Advisor) Date

Dr. Darren Sandquist (SCERP Director) Date

Dr. Bill Hoese (SCERP co-Director) Date

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General Introduction

In vascular plants, xylem is responsible for transporting water and inorganic and organic

solutes, termed xylem sap. Xylem vessels are composed of tracheids, vessel elements, and

parenchyma. Constant leaves’ evaporation generate a negative pressure gradient that draw the

water from the soil to the root to the leaves. The soil has higher water potential than the roots

because the roots contain higher concentration of solutes. Based on diffusion principle, water

flows from high to low water potential, thus water flow from the soil into the roots. Hydrogen

bonds links water molecules together; water molecules adhere to the hydrophilic of cell walls.

The cohesion and adhesion of water molecules are assisted with capillary action pull water

upwards against gravity (Joly and Dixon, 1894). Since leaves constantly evaporate water; there

is always tension, negative pressure on the surface of the leaves. As the water is pulled, many

menisci are formed in small diameter xylem such as capillary action. This tension imposes a

strong pressure on the xylem, demanding for more water. If the soil is dry, there is not enough

water to supply water demand; air is drawn into the xylem through the small spores between the

conduits, call pit membranes. Hacke and Sperry (2001) referred to this process as air seeding.

Xylem embolisms can impede water flow through the xylem, and if not removed, can lead to leaf

and branch mortality. Embolisms formation can be detected by measuring hydraulic

conductivity of stems before and after hydrating stems to fill all conduits with water. A lower

hydraulic conductivity before hydration indicates embolism formation. A higher resistance to

embolism formation indicates a high tolerance to water deficiency (Hacke & Sperry 2001;

Melcher et al. 2003; Poggi et al. 2007).

As water resources for irrigation of crops and fruit trees are becoming scarce in many

regions, there is a high need to study the drought resistance mechanisms of fruit species,

including Citrus varieties. Growing Citrus in Southern California, a Mediterranean climate,

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requires efficient irrigation to maintain satisfactory tree growth and fruit development. It has

been hypothesized that the vulnerability to embolism formation may be an important factor for

determining the timing and amounts of irrigation, especially when irrigation water is scarce

(Poggi et al. 2007). The main objective of my research was to determine the vulnerability of

Citrus species to embolism formation and test for the capability to repair embolisms, with the

intent to contribute to more effective irrigation scheduling for Citrus in California. I measured

Citrus vulnerability to embolisms and tested different hydration methods to obtain maximum

hydraulic conductance as a baseline for all measurements. The best hydration method was then

used to hydrate Citrus stems throughout the subsequent study.

Another set of experiments was designed to test for the ability of Citrus to form and

repair xylem embolisms. It is commonly observed that embolisms form during daytime while

the hydraulic systems experience maximum transpiration and the strongest negative pressure.

Embolism repair in the past was thought to take place only in the absence of transpiration and

while xylem tension is low. However, recent studies have indicated that embolisms can be

repaired during transpiration and under negative pressure (Clearwater and Goldstein 2005). The

physiological conditions under which embolisms form and/ or are repaired were documented by

measurements of leaf stomata conductance, branch pressure potentials, and stem hydraulic

conductance. Three 24-hour experiments were conducted to measure the degree of embolism

formation and repair in fall 2011, and spring and summer of 2012.

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Part I: Measuring Xylem Vulnerability in Three Citrus Species

Abstract

This study documented xylem vulnerability of Citrus x sinensis (Valencia orange), Citrus

x paradisi (star ruby red grapefruit), and Citrus x reticulata (tangerine) under drought conditions.

During water shortages, a better understanding of drought tolerance mechanisms in citrus would

enable to improve water conservation. Physiological mechanisms of drought resistance were

investigated by measuring the formation of xylem embolisms, also known as air bubbles that

prevent water flow through vascular tissue. Four methods were compared to determine the best

technique for hydrating the stems in order to achieve maximum hydraulic conductivity through

the xylem: 1) high pressure flushing for one hour at room temperature, 2) sixteen-hour vacuum

treatment of submerged stems at room temperature, 3) low pressure flushing at 12oC for 16

hours, and 4) one-hour vacuum treatment of submerged stems followed by one-hour high

pressure flushing at room temperature. Based on previous results and studies, we hypothesized

that submergence under a vacuum would be the best method of stem hydration because it would

effectively remove trapped air molecules from pit membranes. Based on differences in fruit

sizes, we hypothesized that grapefruit would be the most vulnerable to embolism formation.

High-pressure flushing was a widely used standard method that produced the lowest hydraulic

conductivity, whereas, long-term vacuum and long-term low pressure flushing both caused the

highest hydraulic conductivities. The three Citrus species were not significantly different in their

vulnerability to embolism formation. In the future, a long-term study would allow for a better

understanding of plant hydraulic conductivity in drought-prone regions.

Introduction

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Citrus plants are widely grown in tropical and Mediterranean climates. The genus

originates from South East Asia (Monrovia, 2010). Citrus farming was once the main fruit crop

in California (Cooley et al. 2009). Due to California’s Mediterranean climate, there is a high

demand for irrigation dedicated to citrus production, which could result in adverse economic and

possibly ecological effects. Orange County was once responsible for producing large amounts of

high quality Citrus. With the advancements being made in agricultural farming and urban sprawl,

many of these farming communities have moved to rural areas. Yet, a large-scale citrus

production still persists in the adjacent counties.

In order to lessen the economic and environmental impact and to increase water

conservation it is necessary to examine drought tolerance of citrus trees. More knowledge

regarding drought tolerance mechanisms of Citrus could lead to more efficient Citrus selection of

varieties for dry-summer climates and could possibly increase water conservation via improved

irrigation scheduling. Therefore, it is important to investigate the physiological mechanism of

citrus adaptation to a drought prone environment (Poggi et al. 2007).

One crucial physiological aspect of drought resistance in plants is their vulnerability to

form xylem embolisms (Hacke & Sperry 2001; Melcher et al. 2003; Poggi et al. 2007). A tree

under normal conditions utilizes negative xylem pressure to draw water from the ground into the

leaves, replenishing water lost to transpiration and transportation of nutrients. Under drought

conditions, a limitation of soil water availability can cause the formation of xylem embolisms, as

air is drawn into the xylem through small pores called pit membranes, a process known as air

seeding (Hacke & Sperry 2001). Embolisms impede the flow of water through the xylem and, if

not removed, can cause leaves to die off or lead to the death of the plant. The process of forming

embolisms, often referred to incorrectly as cavitation, occurs when negative pressure in the

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xylem drops below a certain threshold. In many studies of plant species, a lower vulnerability

towards forming embolisms indicates a high

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tolerance to water deficiency (Poggi et al. 2007). Yet, there are not many studies of xylem

vulnerability for fruit species and only one study of one citrus variety (Poggi et al. 2007).

Therefore, the purpose of this study was to document the xylem vulnerability of three Citrus

species: Citrus x. sinensis (Valencia orange), Citrus x paradisi (Star Ruby Red grapefruit), and

Citrus x. reticulata (Tangerin).

Xylem vulnerability can be measured by determining how much positive air pressure is

required to push air laterally into the xylem of detached stems (Sperry & Saliendra 1994). If

embolisms are formed in the xylem conduits, the hydraulic conductivity of the stems decreases.

A xylem vulnerability curve documents the percent loss of hydraulic conductivity (PLC) as a

function of pressure. Measuring xylem vulnerability curves of three citrus species allows gaining

insights into drought tolerance mechanisms in Citrus and may aid in developing improved

irrigation schedules and water conservation in California.

Three Citrus species were studied Citrus x sinensis (Valencia Orange), Citrus x paradisi

(Star Ruby Red Grapefruit), and Citrus x reticulata (Tangerin). Stems of these three species were

collected from the Fullerton Arboretum in California. The purpose of this study was to document

xylem vulnerability in three citrus species C. x sinensis, C. x paradisi, and C. x reticulata. To do

this, maximum hydraulic conductance of fully hydrated stems had to be measured as a baseline

for the measurements. A recent study has found that the standard method of hydrating stems by

flushing them with water under positive pressure may not always result in full hydration (Espino

& Schenk, 2010). Therefore, the first step of this research project was to determine a standard

method to hydrate Citrus stems. This method was then used for all measurements of xylem

vulnerability.

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Four stem hydration methods were tested, including short term high pressure flushing

(T1), long term vacuum (T2), overnight low temperature, low pressure flushing (T3), and a

combination of high pressure flushing and short term vacuum (T4). Based on findings by Espino

& Schenk (2010), it was hypothesized that the best method to hydrate citrus stems would be to

submerge them in an under-water vacuum for 16 hours. The long term vacuum was predicted to

out-compete the other methods due to its ability to generate a long-lasting pressure differential

that allows for efficient diffusion of air molecules from embolized vessels through pit

membranes into water-filled cells.

Based on published research regarding irrigation demands (FAO 2010) and on differences in

fruit sizes, it was hypothesized that C. x paradisi would be the most vulnerable to embolism

formation. This was based on the assumption that a higher demand for water corresponded to a

higher vulnerability to embolism formation.

Materials and Methods

Collecting stems samples

Stems of three Citrus species: Citrus x sinensis (Valencia orange), Citrus x paradisis

(Star Ruby Red Grape Fruit), and Citrus x reticulata (Tangerin) were collected from the

Fullerton Arboretum, CA. Fresh stems with diameters between 2.3 mm and 5.0 mm and length

of 25 to 40 cm for stem hydration experiments and longer than 80 cm for xylem vulnerability

measurements were collected. Stems were cut and placed in a bucket of water which was

transported to the plants and H2O lab at Cal State Fullerton; green stems with minimal or no

branching were selected. In the lab, stems were submerged in a container containing de-ionized

(DI) water. For stem hydration experiments, stems were successively cut down to 15cm lengths

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on both ends of the stems, cutting 1 cm at a time. The bark at the proximal end of the stems was

removed.

Hydraulic conductivity measurements

Hydraulic conductivity was measured as described by Espino and Schenk (2010). A

XYL’EM embolism meter (Bronkhorst, Montigny les Cormeilles, France) was used to measure

the flow rate of water through the stems. Stems were connected from their proximal ends to a

manifold, which was connected via tubing to the XYL’EM. Water used for measurements and

for hydrating stems was degassed using a membrane contactor degassing unit (Liqui-Cel mini-

module 1.7 x 5.5, Membrana, Charlotte, NC, USA) and passed through a 0.2 μm filter (model

Polycap AS 75, Whatman Inc., Piscataway, NJ). Initial flow rate of water moving into stems was

measured in the absence of a pressure differential, (Fo at 0 kPa). Stems were then measured

under a pressure differential of 6 kPa which was generated by raising the water supply relative to

the stems. Once the flow rate was stable, typically a duration of one to five minutes, the flow rate

was recorded as Fp. Hydraulic conductivity, k was determined as flow rate at 6 kPa, Fp minus

flow rate at 0 kPa pressure divided by the pressure differential, delta p: k = (Fp - Fo)/ delta p.

Specific hydraulic conductivity (ks) of a stem with certain length L and area A was

calculated as flow rate at 6 kPa minus flow rate at 0 kPa times length and divided by the

differential pressure and by area: ks= (Fp – Fo) L/ delta p A.

All hydrating conductivities reported in this study include a temperature correction to

20◦C to allow for changes in water viscosity with temperature.

Hydration treatments

Citrus stems of all three species were hydrated using four treatments to determine the

optimal hydration technique; treatment 1: Ten stems were flushed with DI, degassed water for 1

10

hour at 150kPa using the high-pressure chamber in the XYL’EM apparatus. This is the standard

hydration technique used in almost all xylem vulnerability studies (Espino & Schenk, 2010).

Treatment 2: Ten stems were submerged under DI water for 16 hours in a vacuum chamber

under 3 kPa absolute pressure, generated using a vacuum diaphragm pump (model DAA-

V715A-EB, Gast, Benton Harbor, MI, USA). This treatment was recommended by Espino and

Schenk (2010) as to avoid the bubble formation that can be caused by high pressure flushing.

Treatment 3: Ten stems were connected to one manifold and were flushed under low pressure

with DI, degassed water that was cooled to 12oC before entering the stems. Cooling increases gas

solubility in water (Mercury et al. 2003). Flushing was done for 16 hours at a rate of 10g hr- for

10 connected stems (i.e.,approximately 1 g hr- per stem). Treatment 4: Ten stems were

submerged under DI water in a vacuum chamber for 1 hour under 3 kPa absolute pressure. This

treatment would first remove air from open vessels at both ends of the stems, which could help to

avoid bubble formation under the subsequent flushing under high pressure.

Xylem Vulnerability Curves

Xylem vulnerability curves are measurements of the percent loss of hydraulic

conductance (PLC, relative to the maximum conductance, kmax) as a function of xylem

pressure.

The air injection method (Sperry and Saliendra, 1994; Meltcher et al., 2003) was used to

measure xylem vulnerability in Citrus. For Valencia Orange stems with 100 cm length, vessel

length of the stems were determined by gradually cutting 1 cm until the bubbles being released

were observed. The vessel length of an orange stem was 60 cm, as was the vessel length for

grapefruit and tangerine. Stems were notched 1 cm apart on two sides of the stem in order to

allow for air entry into the xylem. Notches were made by cutting at an angle directly into the

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xylem, only into the section that was subjected to air pressure; then 65 cm to 100 cm long Citrus

stems were hydrated as described in treatment 2. After hydration, they were inserted into

cavitation chambers (PMS Instruments, Albany, OR) with both ends protruding. The cavitation

chambers were connected to a pressure chamber (model 1000, PMS instruments), which was

used to raise the air pressure inside the cavitation chambers successively from 0 to a maximum

of 10 MPa. After each successive increase in pressure, the pressure was held for 5 minutes then

reduced to 0.1 MPa for about 1 to 2 minutes (Sperry and Saliendra, 1994), which was then

reduced to an ambient pressure for measurements of hydraulic conductance using the XYL’EM

apparatus. Percent loss of conductance (PLC) was calculated as:

where a PLC of zero % indicates completely filled xylem and a PLC of 100% indicate a

completely embolized xylem. For all xylem vulnerability curves, kmax was defined as the ks

measured after application of 0.25 MPa pressure to correct for cavitation fatigue of the xylem

(Jacobsen et al. 2007).

Data Analysis

Hydration treatments were compared among the three Citrus species by ANOVA using

the statistical software SYSTAT (version 13, SYSTAT Software Inc., San Jose, CA). Xylem

vulnerability curves were analyzed using nonlinear regression of PLC vs. air pressure. Logistic

dose response curves were fitted to the data using the software TableCurve 2D (version 5.01,

SYSTAT Software Inc., San Jose, CA), and the pressures at which PLC reached 50% (Ψ50) was

determined from these regressions.

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Results

The long-term vacuum treatment (T2), long-term low-pressure and cold flushing

treatment (T3) were equally effective in removing embolisms from Citrus vessels (Fig. 1).

However, the vacuum treatment was superior to high-pressure flushing, whether combined with

vacuum or not. The maximum specific hydraulic conductivity of stems after the long-term

vacuum treatment was on average about twice as high as achieved after one hour of high-

pressure flushing (Fig. 1), suggesting that the vacuum was much more effective for removing air

bubbles from stems. Maximum vessel lengths determined by the air injection method was 60 cm

for the Valencia orange, 60 cm for the Star Ruby Red Grapefruit, and 60 cm for the tangerine.

Based on these findings on the length of stems, 100 to 120 cm were used for xylem vulnerability

measurements were not significantly different in their vulnerability to xylem embolism

formation (Fig. 2), as differences between calculated Ψ50 values (= pressure at 50% PLC) were

not statistically significant. The Valencia orange, Ruby Red grapefruit and tangerine were not

significantly different in their vulnerability to xylem embolism formation (Fig. 2), as differences

between calculated Ψ50 values (= pressure at 50% PLC) were not statistically significant. This

was largely caused by the very high variability of conductivities in response to air injection into

the stems, as indicated by the large scatter of points on all graphs of Fig.2.

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Discussion

As hypothesized the best method for hydrating Citrus stems was the sixteen hour

submerged vacuum treatment (T2). This method proved to be the most effective technique to

remove air from the xylem. This method stands out the most among other treatments in

providing a long-lasting partial pressure gradient between the gas phase in the vacuum chamber

and the gas phase in embolized vessels, which drives the movement of air molecules from

vessels through pit membranes into water-filled vessels and ultimately through the water’s

surface in the vacuum pump. As Espino and Schenk (2010) found previously, submergence

under vacuum effectively removed air from vessels, while high-pressure flushing was found to

be the least effective in removing air from vessels. High-pressure flushing, the widely used

standard method produced the lowest hydraulic conductivity, whereas, long-term vacuum and

long-term low pressure flushing both caused the highest hydraulic conductivities. As air removal

from wood occurs by way of diffusion, a relatively slow process, the longer the time, the more

efficient the removal of air will be. The second vacuum treatment, being the only one to be

significantly different from treatment 1 and, was also more feasible to implement as a standard

method than treatment 3, was adopted as the method of choice for hydrating Citrus stems.

The second hypothesis, that grapefruit would be the most vulnerable to embolism

formation than orange and tangerine, was not supported. The three Citrus species were not

significantly different in their vulnerability to embolism formation, although there appeared to be

a trend that showed that Valencia oranges were the most resistant species and the tangerines

were the most vulnerable. The inability to detect statistical differences between the

vulnerabilities of the three species was entirely due to large variances within species, resulting in

large standard errors (Fig. 2). The widely distributed data points suggests that the air injection

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method, as applied, did not result in highly reliable and reproductive measurements of xylem

vulnerability. The xylem vulnerability curves of all three species did not go up to 100% PLC

over the range of pressures measured. Poggi et al. (2007) documented a pattern of xylem

cavitation for Clementine that was similar to that of the three Citrus species studied here. These

authors suggested that xylem conduits in Citrus may be divided into more vulnerable and less

vulnerable “compartments”, but could not find evidence for their species to support this

hypothesis.

For future works, it would be constructive to study the xylem’s anatomical structure of

the three Citrus species. The anatomical xylem analysis of the three Citrus species would

examine any difference in xylem straits that have been associated with vulnerability to embolism

formation in previous studies, including vessel diameters and the area of pits connecting vessels

(Hacke et al. 2009). Such measurements could be used to test the xylem compartments

hypothesis of Poggi et al. (2007), for example if vessels turned out to be present in two distinct

diameter classes. Also, the three Citrus species turned out to be anatomically similar, which

would explain why the three Citrus species were not statistically different in their vulnerability to

embolism formation.

As a follow up study on the findings of the research reported here, I would like to

conduct a study of the effects of Citrus root grafting on hydraulic traits of Citrus. Root grafting is

currently utilized within the agricultural field to increase the yield and control the longitudinal

growth of Citrus trees. Citrus farmers use the practice of grafting one species of Citrus trees to a

developed root system of a secondary citrus species to shorten the growth rate which it will take

a species to develop from a seedling to an adult tree capable of producing fruit. Another useful

and relevant study for the future could be on the effects of grafting on the formation of

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embolisms within the trees’ roots and stems by comparing grafted and un-grafted trees. This

would allow me to test the hypothesis that root grafting leads to the development of more

embolism due to a hydraulic mismatch between roots and stems. Steppe et al. (2006) reported

evidence for such a hydraulic mismatch in orange trees by observing peculiar stomatal

oscillations that may have been due to an insufficient hydraulic conductance of the root system.

In the future, I would like to expand my study within the area of water usage for citrus

species and work with Citrus farmers to develop and test root irrigation systems either above or

below ground and generate improved irrigation schedules to allow for effective water

conservation within drought prone areas, which would ultimately lower costs in yield production

for the farmer. I believe that this would facilitate a thriving agricultural economy here in

California that could persist in the face of climate change. Moreover, lower cost production of

locally grown produce would be more attractive to local area consumers within a targeted

community and would allow for a competitive job growth of small agricultural farmers with the

potential of creating job opportunities in a given area.

In conclusion, this study has resulted in a new standard method to hydrate stems for

measurements of xylem vulnerability curves, which will now be tested on other plant species.

The research on xylem vulnerability in Citrus has remained inconclusive, but will continue to

develop better experimental protocols and increase the reliability of the technique.

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Literature Cited

Cooley, H., Christian-Smith, J., Gleick, P. 2009. Sustaining California Agriculture in an

Uncertain Future. Pacific Institute, July Report, 1-81.

Espino, S. and Schenk, H. J. 2010. Mind the bubbles: Achieving stable measurements of

maximum hydraulic conductivity through woody plant samples. Journal of Experimental

Botany. Vol, 62, 1119-1132.

FAO 2010. Crop water information; Citrus. Available from

http://www.fao.org/nr/water/cropinfo_citrus.html.

Hacke, Uwe., Sperry, John. 2001. Functional and ecological xylem anatomy. Urban and Fischer

Verlag. Vol. 4/2, 97-115.

Hacke, U. G., A. L. Jacobsen, and R. B. Pratt. 2009. Xylem function of arid-land shrubs from

California, USA: an ecological and evolutionary analysis. Plant, Cell & Environment.

Vol. 32, 1324-1333.

Mercury, L., M. Azaroual, H. Zeyen, and Y. Tardy. 2003. Thermodynamic properties of

solutions in metastable systems under negative or positive pressures. Geochimica et

Cosmochimica Acta. Vol. 67, 1769-1785.

Monrovia [Internet]. [cited 2010 Oct 10]. Campbell Valencia Orange. Available from

http://www.monrovia.com/plant-catalog/plants/789/campbell-valencia -orange.php

Poggi, I., Polidori, J., Gandoin, J., Paolacci, V., Battini, M., Albertini, M., Ameglio, T., and

Cochard, H. 2007. Stomatal regulation and xylem cavitation in Clementine (Citrus

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clementia Hort) under drought conditions. Journal of Horticultural Science &

Biotechnology, Vol 82, 845-858.

Sperry, J. S., Donnely, J. R., and Tyree, M. T. 1988. A Method for measuring hydraulic

conductivity and embolism in xylem. Plant, Cell and Environment. Vol 11, 35-40.

Sperry, J. S. and N. Z. Saliendra. 1994. Intra- and inter-plant variation in xylem cavitation in

Betula occidentalis. Plant, Cell and Environment 17:1233-1241.

Steppe, K., S. Dzikit, R. Lemeur, and J. R. Milford. 2006. Stomatal oscillations in orange trees

under natural climatic conditions. Annals of Botany, Vol. 92, 831-83.

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Part II: Xylem Embolism Repair in Valencia Orange, Citrus x sinensis

Abstract

Plants normally transport water under negative pressure, which make their hydraulic

systems, the xylem, vulnerable to embolism formation if there is a water shortage in the soil. Air

can be drawn into xylem conduits through pit membranes, and the resulting embolisms impede

water flow, which can lead to branch and leaf mortality. Recent studies have shown that in some

species, embolisms form and are repaired diurnally during transpiration and even under negative

xylem pressure, but the mechanisms remain unknown. Little is known about the vulnerability of

fruit trees to embolism formation or their capability of embolism repair. If fruit trees grown in

semi-arid climates were capable of diurnal embolism repair then this could have implications for

efficient irrigation scheduling, as watering could potentially be timed to occur when it is most

beneficial for hydraulic recovery. The objective of this study was to test for diurnal embolism

formation and repair in Valencia Orange (Citrus x sinensis) grown in Southern California. Plant

water relations parameters, including leaf temperature, stomatal conductance, leaf water

potentials, stem hydraulic conductance, and percent loss of conductance (PLC) were monitored

over 24 hours periods at 4 hour intervals on nine young dwarf orange trees from fall to spring

2011/12.

Orange stems were highly embolized, with 40 to 60% loss of conductance, in fall and

winter, but there was no indication during these periods of embolism repair, even though the

trees were irrigated regularly. Nighttime leaf water potentials during these periods ranged

between -0.5 and -0.7 MPa; and no relationship was found during this time between water

potentials and the degree of xylem embolism. Findings from a warm day in spring 2012 were

substantially different. Xylem embolisms formed during the day to the same levels as previously

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observed (40 to 60% PLC) and were repaired during the night down to less than 25% PLC at leaf

water potentials of about -0.3 to -0.6 MPa, corresponding to stem water potentials of about -0.1

to -0.4 MPa. In spring, there was a significant relationship between percent loss of conductance

and water potential, with a 50% PLC estimated at -1.1 MPa. The findings suggest that orange

trees may require high nocturnal temperatures and high water potentials for embolism repair.

The finding that about half of the vessels appear to be almost permanently embolized in dwarf

Valencia Orange trees is hypothesized to be a dwarfing root stock effect.

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Introduction

The xylem is the transport system for water and minerals in plants. Trees under normal

conditions use negative pressure, which is generated by leaf transpiration, to draw water through

the xylem from the soil into the leaves to replenish water lost through transpiration. Under

drought conditions, soil water availability becomes limited, and air can be drawn into xylem

conduits through small pores connecting the conduits, which are called pit membranes. This

process is called air seeding (Hacke and Sperry 2001). The creation of air bubbles in the xylem

conduits is referred as xylem embolism formation (Hacke and Sperry 2001). Embolisms impede

the flow of water through the xylem, which can lead to leaf and branch mortality if the

embolisms are not removed. It is commonly observed that embolisms form during daytime

while the hydraulic system experiences maximum transpiration and the strongest negative

pressure (Bucci et al. 2003). Until quite recently, embolism repair was thought to take place

only in the absence of transpiration and while xylem tension is low. However, several recent

studies have indicated that embolisms can be repaired during transpiration and under negative

pressure (Clearwater and Goldstein 2005).

According to Yang and Tyree (1992), Steudle (2001), and Hacke and Sperry (2003),

embolisms can be repaired only if pressure in the xylem conduit is high enough to dissolve the

air bubbles into the xylem sap. The threshold pressure of the xylem sap required to dissolve

embolisms depends largely on the radius of the bubbles, following the Young-Laplace equation:

where pt is the threshold xylem sap pressure above which the gas bubble will collapse, P is the

gas pressure inside the bubble, T is the surface tension of water (0.00728 N m-1

, 0oC), and r is the

radius of curvature of the gas-water interface, i.e., the radius of the air bubble, which in the case

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of a completely gas-filled xylem conduit would be the radius of the conduit. If the xylem is at,

or only slightly under, atmospheric pressure then air bubbles will slowly and spontaneously

dissolve. During transpiration, xylem pressure is almost always more negative than the “-2T/r”

threshold. Embolism repair under this condition is referred as refilling under tension (Hacke and

Sperry 2003), and the mechanisms for this process are largely unknown (Zwieniecki & Holbrook

2009)

Refilling of embolized vessels under negative pressure was documented for Laurus

nobilis in Hacke and Sperry’s study (2003). The calculated minimum average -2T/r limit was a

xylem pressure of ΨPX= - 0.1 MPa. L. nobilis plants were stressed to about -2.7 MPa and were

re-watered to a target xylem pressure (Ψw) of about -0.5 MPa. The ΨPX observed during

hydraulic recovery and embolism repair was approximately -0.66 MPa, which was below the

minimum -2T/r limit of ΨPX = -0.1 MPa. The result indicated that L. nobilis embolized vessels

were refilled under negative pressure. In a study of two Brazilian savanna tree species,

Schefflera macrocarpa and Caryocar brasiliense, Bucci et al. (2003) also found that embolized

vessels were refilling under tension. During afternoon to early evening, their hydraulic specific

conductivity dramatically increased as water potential increased as well. This pattern coincided

with a partial disappearance of starch in sheath cells surrounding the vascular bundles of petioles

in the afternoon. The authors hypothesized that hydrolysis of starch creates osmosic pressure that

draws water from the adjacent cells to refill embolized conduits (Bucci et al. 2003).

Because air bubbles cannot spontaneously dissolve in xylem conduits that are under

tension, plant physiologists have proposed a number of embolism repair scenarios that could

explain embolism repair under tension. These include: 1) cell membrane osmosis, 2) pit

membranes osmosis, 3) tissue pressure, 4) membrane asymmetry (Clearwater and Goldstein

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2005), and 5) the liquid-gas membrane contactor theory (Schenk, unpublished manuscript).

Grace et al. (1993) proposed the cell membrane osmosis hypothesis, according to which solutes

are transported from the phloem into ray cells and from there through living parenchyma cells

into embolized conduits. This process could lower the osmotic pressure in the conduit, causing

water to flow from living cells into the conduit and refill it. Hacke and Sperry (2003) proposed a

variant of this hypothesis, termed the pit membrane osmosis hypothesis, to explain why high

concentrations of osmotic agents, such as salts or sugars, have not been observed in refilling

xylem. According to this hypothesis, living parenchyma cells may release solutes of high

molecular weight into conduits, which are unable to penetrate pit membranes, thereby remaining

confined to the refilling conduits, and not detectable in extracted sap.

Canny (1997) and Bucci et al. (2003) proposed a fundamentally different mechanisms,

termed the tissue pressure hypothesis. Conduit refilling according to this hypothesis is based on

the expansion of living xylem tissue. This exerts a compensating tissue pressure on cells xylem

that causes water flow into the refilling conduits. This tissue pressure and the refilling process

would be caused by increasing osmotic pressure inside living parenchyma cells and could be

caused by hydrolysis of starch. Pickard (2003) proposed a variant of the tissue pressure

hypothesis, which has been termed the membrane asymmetry hypothesis (Clearwater and

Goldstein 2005). According to this model, xylem tissue external to the xylem parenchyma

provides a source of solutes and water. During conduit refilling, solutes are loaded by membrane

transporters into xylem parenchyma, causing water uptake and increased turgor in these cells,

and this turgor pressure in turn would cause water to flow into the refilling conduit.

While all the of the proposed mechanisms discussed so far address the movement of

water into refilling conduit, the membrane contractor theory proposed by Schenk et al.

25

(unpublished manuscript) focuses on the mechanism of gas removal from conduits. According

to this theory, during nighttime transpiration, air bubbles from embolized xylem conduits diffuse

through pit membranes into the transpiration stream of functional conduits. This is possible

because xylem sap at night a cooler temperatures can dissolve large amounts of air.

Regardless of the mechanisms involved, the ability to repair embolisms may be very

important for survival, growth, and reproduction of plants during drought. Crop species,

including fruit trees, are likely to benefit from the ability to repair embolisms, as they could be

able to recover from drought after irrigation. Little information is available on embolism repair

in crops. To date, embolism repair under tension has been found in grapevine (Brodersen et al.

2010) and rice (Stiller et al. 2005).

Little is known about the drought tolerance of Citrus species. According to Spiegel-Roy

and Goldschmidt (1996) although the wild type of Citrus is of unknown origin, most citrus

commercially grown nowadays are hybrids and originated from South East Asia, indigenous to

China and India. According to Scora (1975), Citrus x. sinensis (sweet orange), the study species,

is a hybrid of C. reticulata (mandarins) and C. grandis (pomelo). Citrus became widely used in

the region as food, for its medicinal properties (used as herbal medicine), as ornamentals, for

fragrance, and as an antidote against poison. Through trade and missionaries citrus spread

through other parts of Asia and was introduced throughout the world to Europe, Africa, and not

until the early 16th

century, to America. Citrus was first introduced in Florida, then Arizona, then

California in 1769. Climatology of citrus encompasses a wide range north 40 degree (Corsica,

Japan) to south 40 degree (New Zealand). Citrus can be found near the equator in hot and humid

climates, in warm subtropical regions, and in cooler-maritime climates. The reproductive cycle

of Citrus includes blooming in spring, bearing fruit in summer, reaching maturity of fruit in fall.

26

A sufficient long and warm summer is required for citrus fruit to grow and reach maturity.

Growing citrus in Mediterranean climate with long and dry summers requires irrigation to

maintain satisfactory tree growth and fruit development.

Although Citrus did not originate from a Mediterranean climate, the plants possess a

number of apparent drought adaptations. Leaves are leathery, have thick, waxy cuticles that

prevent water loss, and have low transpiration rates (Fererest et al. 1979). The fruits are coated

with thick wax and an oily peel (Spiegel-Roy and Goldschmidt, 1996). Fereres et at. (1979)

indicated that citrus crops can withstand considerable water stress. For instance Valencia orange

tree can tolerate very low negative water pressure of – 6.6 MPa and rapidly recover within 2

hours after the irrigation started. In contrast, Poggi et al. (2007) found Citrus x clementine to be

quite vulnerable to embolism formation. My preliminary result investigating the vulnerability of

three citrus species to embolism indicated that Valencia orange, star ruby red grapefruit, and

tangerine are equally vulnerable to embolism. No study to date has investigated embolism repair

in any citrus species. The objective of this study will be to test for embolism formation and

repair in Valencia orange, one of the most widely grown varieties of sweet orange.

Stem hydraulic conductivity, leaf stomatal conductance, and branch pressure potential

will be measured over 24 hour periods. The diurnal course of stem hydraulic conductivity will

indicate whether or not embolism formation and repair occurs. The physiological conditions

under which embolisms form and/or a repaired will be documented by measurements of leaf

stomatal conductance and branch pressure potentials.

Methods

Valencia orange trees were sampled on the South side of Langsdorf Hall on the campus

of Cal State Fullerton in Fullerton, California. Tests for embolism repair were conducted during

27

three twenty-four-hour periods on November 21-22, February 25 - 26, and April 21-22,

following a protocol developed by Hessom et al. (unpublished manuscript).

On each date, nine individual Valencia orange trees were sampled in 4 hour increments

over a 24 hour period. At each time interval, each tree was measured for branch pressure

potential, stomatal conductance, and stem hydraulic conductance. To measure stomatal

conductance, a leaf porometer (Decagon Devices Inc. Leaf Porometer: Version 4.0, Pullman,

WA, USA) was used on two leaves of the same branch sampled for leaf pressure potentials and

stem hydraulic conductance. To measure leaf pressure potentials, a branch tip with leaves from

each tree was cut, and leaf pressure potentials were measured using a pressure chamber (model

1000, PMS Instruments, Eugene, OR, USA). The branches were then cut at about 50 cm long

from the tip and quickly transported to the lab.

Stem hydraulic conductance was measured on 15 cm-long stem segments using a

XYL’EM Embolism Meter (Instrutec, France. In the lab, the 50 cm branches were submerged in

a container with deionized (DI) water. Stems were cut down to 15 cm length from both ends of

the stems by cutting 1 cm at a time. The bark at the proximal end of the stems was removed.

After stem hydraulic conductivity measurement, the stem segments were submerged in water-

filled test tubes and were rehydrated under vacuum for 20 hours. After rehydration, maximum

hydraulic conductance was measured for all stem samples using the XYL’EM Embolism Meter.

Hydraulic conductivity was measured as described by Espino and Schenk (2010). A

XYL’EM embolism meter was used to measure the flow rate of water through the stems

(Bronkhorst, Montigny les Cormeilles, France). Stems were connected from their proximal ends

to a manifold, which was connected via tubing to the XYL’EM . Water used for measurements

and for hydrating stems was degassed using a membrane contactor degassing unit (Liqui-Cel

28

mini-module 1.7 x 5.5, Membrana, Charlotte, NC, USA) and passed through a 0.2 µm (model

Polycap AS 75, Whatman Inc., Piscataway, NJ). The initial flow rate of water moving into

stems was measured in the absence of a pressure differential, (Fo at 0 kPa) to account for

osmotically-driven flow. Stems were then measured under a pressure differential of 6 kPa which

was generated by raising the water supply relative to the stems. Once the flow rate was stable,

typically after one to five minutes, the flow rate was recorded as Fp. Hydraulic conductivity, k

was determined as flow rate at 6 kPa, Fp, minus flow rate at 0 kPa pressure, divided by the

pressure differential, p: k = (Fp- Fo)/ p.

Specific hydraulic conductivity (ks) of a stem with a length (L) and conducting area (A)

was determined as flow rate at 6 kPa minus flow rate at 0 kPa times length divided by the

pressure differential and conducting area: ks= (Fp – Fo) L/ p A.

All hydraulic conductivities reported in this paper include a temperature correction to

20oC to allow for changes of water viscosity with temperature. In this study, percent of loss of

conductivity was referred as percent of embolism.

Results

As hypothesized, Valencia orange was quite vulnverabe to embolism formation but was

able to repair embolisms at high water potential and low transpiration. Leaf water potential

showed a steady increase from fall 2011 to spring 2012. As the seaons changed, the temperature

increased causing an increase in stomatal conductance. Percent of embolisms were consistently

high in the Fall 2011 and early spring 2012; however, nighttime embolisms significantly dropped

in April. As leaf water potential increased, the percent of native embolism decreased.

29

In November 2011, leaf water potential during daytime was significantly lower than

during nighttime. During the day, it reached its lowest leaf water potential of -2.3 MPa while

during night, its highest leaf water potential was at -0.5 MPa (Fig. 1 A). Stomatal conductance

between daytime and nighttime also showed a great difference. The diurnal stomatal

conductance was above 150 mmol m-2

s-1

while the nocturnal stomatal conductance was very

close to 0 mmol m-2

s-1

(Fig. 1 B). Despite these great variations in leaf water potential and

stomatal conductance, percent of embolism showed minimal fluctuation over the course of the 24

hour measurement period and ranged between 40 – 60 % (Fig. 1 C).

Fig.1: Plant water relations of Citrus x sinensis over a 24 hr period on 21-22 November 2011. The

period from sunset to sunrise is marked by the shaded area. A. Leaf water potentials. B. Stomatal

conductance. C. Percent loss of conductance due to embolisms. All error bars are ± SE.

30

In February 2012, the leaf water potential increased; and there was minimal fluctuation of the

stem water potential over the 24 hr experimental period. The stem water potential reached its

lowest at -1.4 MPa which occurred at noon on February 26th

(Fig. 2 A). The nocturnal stomatal

conductance observed in February 2012 was higher than that of November 2011. The percent of

embolisms measured in February 2012 was similar to that of November 2011, ranging from 40

% to 60 % (Fig. 1 C and 2 C). On 25th

February 2012, the peak of percent loss of conductivity

occurred in the afternoon while the stem water potential was high. The percent loss of

conductance dropped from 55% to around 40% and remained at 40% from 9 a. m. to noon the

next day (Fig. 2 C).

Fig. 2: Plant water relations of Citrus x sinensis over a 24 hr period on 25- 26 February 2011.

The period from sunset to sunrise is marked by the shaded area. A. Leaf water potentials. B.

Stomatal conductance. C. Percent loss of conductance due to embolisms. All error bars are ± SE.

31

In April 2012, leaf water potential was highest compared to previous month, especially

the nighttime leaf water potential reached -0.3 MPa (Fig. 3 A). The temperature also increased

(Fig. 3 B). The stomatal conductance in April resembled that of February. The diurnal percent

of embolism still remained high; however nocturnal percent of embolism dropped to 25%, which

was denoted with the asterisks to indicate a significant drop in percent of embolisms.

Fig. 3: Plant water relations of Citrus x sinensis over a 24 hr period on 21 - 22 April 2011. The

period from sunset to sunrise is marked by the shaded area. A. Leaf water potentials. B.

Stomatal conductance. C. Percent loss of conductance due to embolisms. All error bars are ± SE.

Most importantly, the native xylem vulnerability curve showed Citrus x sinensis trees are quite

vulnerable to embolism formation. Fifty percent of embolism was at -1.57 MPa, relative high water

32

potential. There was a strong correlation between leaf water potential and percent of embolism (r2=

0.324).

Fig. 4: Native xylem vulnerability curve for Citrus x sinensis from fall 2011 to spring 2012. Native

embolism was shown in relation to leaf water potentials. The trend line is described by PLC = 100 / (1

+ (-Psi/P50)^b) with P50 = -1.57 and b = -0.482 (r2 = 0.324, p = 0.007).

Discussions

Valencia orange appears to be quite vulnerable to embolism formation. The part in my

hypothesis stating that Citrus can repair embolism during minimal transpiration and high water

potentials was partially supported because embolism repair only occurred at night in April.

Stomatal conductance during embolism repair was relatively high.

In term of regulation of stomatal conductance and the stem water potential, in November

2011, during the nighttime, the stomatal conductance in Citrus x sinensis was low at a time,

33

when the stem water potential was high. Schenk et al (2011, unpublished manuscripts) also

found the same pattern of lower stomatal conductance and higher stem water potential at night

for Encelia farinosa. Surprisingly, nocturnal stomatal conductance in February and April was

higher than in November. The differences in stomatal conductance patterns between November

2011, February and April 2012 suggest that the plants are capable of regulating their stomata,

perhaps in response to seasonal temperature changes and vapor pressure deficit.

In November 2011, the stem water potential of Citrus x sinensis showed the same pattern

as the stem water potential observed in Malosma laurina in February 2009 (Hessom, 2010).

Stem water potential was low while stomata opened and high when stomata closed. However,

the stem water potential of Citrus x sinensis in February and April 2012 remained steady

throughout the 24 hour experimental period. In November 2011, the leaf water potential was

higher at night than during the day. However the percent of embolisms remained high between

40%, despite the fact that the trees were irrigated twice a week and rained on the previous day of

the measurements.

In February 2012, percent of embolism was same as that of November even though leaf

water potential in February was higher than that in November. Approximately half of the vessels

in Citrus x sinensis appeared to be permanently non-functional, regardless of irrigation. 50%

percent loss of conductivity was approximately at -0.7 MPa to -0.8 MPa, which was quite

vulnerable compared to many other woody species (Maherali et al. 2004). In contrast, Poggi et

al. (2007) found Citrus x clementina to be much less vulnerable to embolism formation, with less

than 50% PLC at -4 MPa stem water potential. The consistently high percent of embolisms over

the two 24-hour experiments suggested that Citrus x sinensis trees were vulnerable to embolism

but did not repair during fall 2011 and early spring 2012.

34

However significant nighttime embolism repair occurred in April, which was differed

from previous dates in having higher nocturnal leaf water potentials (≥-0.5 MPa) and nocturnal

leaf temperature (≥20oC). Stomatal conductance during embolism repair was high. The findings

suggest that Citrus trees as a tropical species require high nocturnal temperature and nocturnal

water potentials to successfully repair embolism. The findings suggest that irrigation for Citrus

farms during warm nights may be more effective than during cold nights.

The knowledge about embolism formation and repair in Citrus trees could improve

irrigation scheduling. My potential graduate research project will focus on minimize water cost

while maximize Citrus fruit production. Another 24 hour measurements of plant hydraulic

conductance conducted during the hottest month in summer would be extremely beneficial

further confirm the results that Citrus repair embolisms during warmers seasons. Before

conducting these measurements, irrigation will be asked to turned off for 2 weeks to see if the

Citrus trees can still effectively repair embolisms when water supply is limited.

Acknowledgements

This research was supported by the Southern California Ecosystems Research Program, funded

by the National Science Foundation DBI – 1041203 (W. Hoese and D. Sandquist, PIs). The authors

thank Susana Espino, Miguel Macias, Kenny Duong, Matthew Laine, Sarah Taylor, Diane Tran, An Ly,

Donald Quick and Robert Wieber for helping with the measurements.

35

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