Regulation of Guard-Cell Function by the Regulatory Apoplastic Photosynthate Pool Yun Kang

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THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES

REGULATION OF GUARD-CELL FUNCTION BY THE

REGULATORY APOPLASTIC PHOTOSYNTHATE POOL

YUN KANG

A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Fall Semester, 2005

The members of the committee approve the dissertation of Yun Kang defended on November 4, 2005.

______William H. Outlaw Jr. Professor Directing Dissertation

______Michael Blaber Outside Committee Member

______Hank W. Bass Committee Member

______George W. Bates Committee Member

______Ross W. Ellington Committee Member

______Laura R. Keller Committee Member

Approved: ______Timothy S. Moerland, Chair, Department of Biological Science

The Office of Graduate Studies has verified and approved the above named committee members.

AKNOWLEDGEMENTS

I would like to thank my major advisor, Dr. William H. Outlaw Jr., for his guidance, support and assistance. Special thanks are extended to Drs. Hank W. Bass, George W. Bates, Michael Blaber, and Laura R. Keller for helpful advice. I would like to thank Dr. Karthik Aghoram, Fanxia Meng, Danielle M. Sherdan and Tianran Jiang for assistance in experiments as well as writing. Kara L. Chamberlain and Guorong Zhang are thanked for help in revision of the dissertation. I thank Bruce N. Smith and Giordano B. Fiore for conducting the sugar measurements with HPLC, and Kim A. Riddle and Xixi Jia for microscopy. I also thank Paul Burress and Jason A. Johnson for making the liquid scintillation measurements. Finally, I would like to thank my family for their unconditional love and support. This work was partially supported by a grant from NASA awarded to Dr. William H. Outlaw Jr.

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TABLE OF CONTENTS

LIST OF FIGURES ...... vi

LIST OF ABBREVIATIONS...... viii

ABSTRACT...... ix

INTRODUCTION ...... 1

GUARD-CELL APOPLASTIC PHOTOSYNTHATE ACCUMULATION IS LIMITED OR ABSENT IN THE SYMPLASTIC PHLOEM LOADING PLANT OCIMUM BASILICUM...... 7

Introduction ...... 7 Materials and Methods...... 10 Plant Material ...... 10 Chemicals ...... 10 Phloem Exudation ...... 10 Leaf Conductance...... 11 Semiquantitative Histochemical Estimates of Guard-cell Starch Contents and of Guard-cell Potassium Contents ...... 11 Quantitative Histochemical Assay of Guard-cell Sucrose Content...... 11 Bulk-leaf Apoplastic Sap Collection...... 12 Bulk-leaf Apoplastic Water Content Measurement ...... 12 Transmission Electron Microscopy (TEM) and Dwarf Basil Guard-cell-wall Volume and Guard-cell Volume Calculation...... 13 Movement of Extra-foliar Xylem-source Mannitol through the Leaf...... 13 Guard-cell-sugar Extraction for HPLC ...... 14 HPLC Analysis of Sugars in Phloem Exudates, Bulk-leaf Apoplastic Sap and Guard-cell Extracts...... 14 Results ...... 15 Sugar Analysis of Phloem Exudates Confirmed that Dwarf Basil is a Symplastic Phloem Loader...... 15 Diurnal Kinetics of Dwarf-basil Leaf Conductance, Stomatal Aperture Size, and Guard-cell Starch and Potassium Contents Was Typical...... 16 Dwarf-basil Bulk-leaf Apoplastic Sugar and Galactinol Concentrations were in the Micromolar Range...... 18

Mannitol Fed via the Petiole Accumulated in the Guard-cell Apoplast, Indicating the Continuity of the Bulk-leaf Apoplast in Dwarf Basil...... 19 Guard-cell Apoplastic Photosynthate Accumulation was Limited or Absent in Dwarf-basil Plants ...... 23 Discussion ...... 25 Dwarf Basil, a Model Symplastic Phloem Loading Species for Guard-cell Studies ...26 Relevance of Sugars in the Bulk-leaf apoplast of Symplastic Phloem loaders to Photosynthesis-dependent Transpiration-linked Sugar Accumulation in the Guard-cell Apoplast...... 27 Absence of Guard-cell Apoplastic Photosynthate Accumulation in Symplastic Phloem Loading Plant Dwarf Basil...... 28 Physiological and Ecological Implications of Stomatal Control in Symplastic Phloem Loaders and Apoplastic Phloem Loaders...... 29 Summary...... 30

GUARD-CELL APOPLASTIC SUCROSE CONCENTRATION―A LINK BETWEEN LEAF PHOTOSYNTHESIS AND STOMATAL APERTURE SIZE ...... 31

Introduction ...... 31 Materials and Methods...... 32 Plant Material ...... 32 Photosynthesis Rate Measurement...... 33 Plant Shading and Growth-cabinet CO2-Concentration Manipulation ...... 33 Quantitative Histochemical Assay of Guard-cell Sucrose Content...... 33 Bulk-leaf Apoplastic Sap Collection...... 34 Guard-cell-sugar Extraction for HPLC ...... 34 HPLC Analysis of Sugars in Bulk-leaf Apoplastic Sap and Guard-cell Extracts ...... 35 Results ...... 35 Lowered Photosynthesis Rate without Altering Transpiration Rate Was Effected by Shading Plus Decreased Ambient CO2 Concentration...... 35 Bulk-leaf Apoplastic Sucrose, Glucose and Fructose Concentrations were Lower in Shaded than in Control Plants ...... 37 Lower Leaf Photosynthesis Rate Resulted in Lower Guard-cell Apoplastic Sucrose Content ...... 38 Discussion ...... 41

OVERALL SUMMARY ...... 46

REFERENCES ...... 48

BIOGRAPHICAL SKETCH ...... 58

LIST OF FIGURES

Fig. 1. Leaf cross of apoplastic phloem loaders (A) and symplastic phloem loaders (B), showing the accumulation of sucrose around the guard cell under active transpiration...... 5

Fig. 2. Two phloem-loading pathways in source leaves...... 6

Fig. 3. The sugar composition (mole percent, mean±SE, n=4 plants) of dwarf-basil phloem exudates...... 16

Fig. 4. Patterns of leaf conductance, stomatal aperture size, guard-cell potassium content and guard-cell starch content of dwarf basil over the course of a day...... 18

Fig. 5. Diel pattern of sugar concentrations (mean±SE) in the dwarf-basil bulk-leaf apoplast...... 20

Fig. 6. Representative pressure-volume curve of dwarf-basil leaf...... 22

Fig. 7. Time course of dwarf-basil leaf transpiration rate (mean±SE) when excised leaves were supplied with water (n=6 plants, two growth lots).or 5 mM mannitol (n=7 plants, two growth lots) through the petiole...... 23

Fig. 8. 14C-mannitol accumulation (mean±SE) in the bulk-leaf apoplast and in the guard-cell apoplast of dwarf-basil ...... 23

Fig. 9. Sucrose content (mean±SE) in the dwarf-basil guard-cell symplast and guard-cell apoplast at maximum leaf conductance (1100 h)...... 25

Fig. 10. Stomatal aperture sizes (mean±SE, n=180 stomata from six plants, two growth lots) of control and shaded/low-CO2 broad-bean plants ...... 37

Fig. 11. Photosynthesis rate (mean±SE, n=6 plants, two growth lots) of control and shaded/low-CO2 broad-bean plants...... 38

Fig. 12. Bulk-leaf apoplastic sucrose, glucose, and fructose concentrations (mean±SE, n=6 plants, two growth lots) of control and shaded/low-CO2 broad-bean plants...... 39

Fig. 13. Guard-cell symplastic sugar and guard-cell apoplastic sugar content of control or shaded/low-CO2 broad-bean plants with either HPLC (mean±SE, n=3 measurements, each

contained 180 guard-cell pairs) or histochemical method (mean±SE, n=43 guard-cell pairs from six plants)...... 41

Fig. 14. Relationship between leaf apoplastic sucrose concentrations (Fig. 12) and guard-cell apoplastic sucrose contents (histochemical method, Fig. 13)...... 41

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LIST OF ABBREVIATIONS

HPLC: High Performance Liquid Chromatography PAR: Photosynthetically Active Radiation PFD: Photon Flux Density pd: Plasmodesmata RH: Relative Humidity RSOs: Raffinose Series Oligosaccharides SE-CCC: Sieve Element-Companion Cell Complexes TEM: Transmission Electron Microscope VPD: Vapor Pressure Difference

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ABSTRACT

Stomata, each delimited by a pair of guard cells, are crucial for gas exchange in terrestrial plants. Guard-cell apoplastic sucrose concentration was proposed to be a signal that integrates the information from bulk-leaf apoplastic sucrose concentration and leaf transpiration rate. In apoplastic phloem loaders, the bulk-leaf apoplastic sucrose concentration is ~2 mM during the photoperiod, but is concentrated to >150 mM in the guard-cell apoplast by transpiration, which could diminish aperture size by up to 3 µm. However, guard-cell apoplastic sucrose accumulation is greatly reduced by decreased leaf transpiration rate. Here, two approaches were used to study the relationship between the bulk-leaf apoplastic photosynthate concentration and the guard-cell apoplastic photosynthate concentration. Firstly, a symplastic phloem loading plant, dwarf basil (Ocimum basilicum cv Minimum), was used because it has a naturally low bulk-leaf apoplastic photosynthate concentration. As typical for symplastic phloem loaders, dwarf basil predominately transported Raffinose Series Oligosaccharides (RSOs) in the phloem instead of sucrose. 14C-mannitol fed via the leaf petiole accumulated around guard cells, indicating an open leaf apoplast. Fluctuations in guard-cell contents of K+ and starch, and the leaf conductance were typical, establishing this as the first symplastic phloem loading model species for guard-cell research. The sum of sugar (RSOs + sucrose + glucose + fructose) concentrations in the bulk-leaf apoplast was <0.3 mM. The upper limit of RSOs (stachyose + raffinose) in the guard-cell apoplast was 10 mM, and sucrose, glucose, and fructose in the guard-cell apoplast were not detectable (p>0.2 compared with concentration zero). Therefore, symplastic phloem loaders lack stomatal osmotic regulation by bulk-leaf apoplastic photosynthate. Secondly, with the apoplastic phloem loader broad bean, leaf photosynthesis rate was lowered by shading and leaf transpiration remained constant by lowering ambient CO2 concentration. With shading/low-CO2 treatment, bulk-leaf apoplastic sucrose concentration decreased to 0.4 mM, one third of the control value, whereas the guard-cell apoplastic sucrose concentration decreased to ~40 mM, less than

one-fourth of the control value. This phenomenon indicates that in apoplastic phloem loaders, sucrose accumulation in the guard-cell apoplast is a direct function of the bulk-leaf apoplastic sucrose concentration and, thus, the rate of photosynthesis.

INTRODUCTION

The evolution of stomata was an important adaptation for plants to survive in a terrestrial environment. Stomata are pores distributed primarily in the leaf and stem surfaces, and each is flanked by a pair of specialized epidermal cells, guard cells. Stomata allow CO2 to enter leaves for photosynthesis and for water to leave leaves through transpiration. Through the swelling and shrinking of guard cells, stomatal aperture size is adjusted to optimize the ratio of CO2 gain to water loss. Stomatal movements are influenced by a variety of exogenous and endogenous signals (recently reviewed in Outlaw, 2003; Fan et al., 2004), e.g. light, humidity, environmental CO2 concentration, and phytohormones. Stomatal movements also follow a circadian rhythm (e.g. Gorton et al., 1989; Meidner and Willmer, 1993). Stomatal movements are achieved by guard-cell water uptake or loss via accumulation or dissipation of osmotica. As osmotica accumulate in guard cells upon stimulation, water enters guard cells and guard cells swell. Because guard-cell walls are thicker in the area surrounding the central stomatal pore, the pair of guard cells bows away from each other and thus opens the stoma between them. Stomatal closure is essentially the opposite of opening: osmotica leave guard cells or are metabolized, water leaves, and stomata close as a consequence. Potassium is the major fluctuating osmoticum in guard cells (reviewed in Outlaw, 1983; Zeiger, 1983). The K+ level fluctuations associated with stomatal movements are accompanied by changes in counterions of K+, such as malate and Cl-. However, potassium salt fluctuations can not provide adequate osmotic pressure required for stomatal opening under all conditions (reviewed in Lu et al., 1995); in addition to K+ salts, sucrose is also a major fluctuating osmoticum involved in osmoregulation of guard cells (Tallman and Zeiger, 1988; Poffenroth et al., 1992; Talbott and Zeiger, 1993; Lu et al., 1995; Amodeo et al., 1996; Talbott and Zeiger, 1996, 1998). The sucrose that accumulates in guard cells potentially comes from three sources: starch degradation, guard-cell

photosynthesis and apoplastic sucrose (sucrose from the space outside of the cell membrane). However, starch degradation can only provide a limited amount of sucrose, ~250 fmol pair-1 (~30 mM, calculated from Outlaw and Manchester, 1979). Under what conditions and to what extent guard-cell photosynthesis provides sucrose is still under debate. In this context, apoplastic sucrose is the most important source for the sucrose that accumulates in guard cells during stomatal opening. In addition, sugar transporters have been detected in guard cells of a number of plants such as potato (Kopka et al., 1997) and Arabidopsis (Stadler et al., 2003). Ultimately, bulk-leaf apoplastic sucrose is from photosynthesis in mesophyll cells (Lu et al., 1997). In addition to serving as a source for guard-cell symplastic sucrose, guard-cell apoplastic sucrose has an osmotic role in regulating stomatal aperture size (Lu et al., 1995, 1997). As illustrated in Fig. 1A (Outlaw, 2003), in some plants such as broad bean (an apoplastic phloem loader), sucrose in the mesophyll cells, produced from leaf photosynthesis, is released to the bulk-leaf apoplast in the vicinity of the phloem complex. As the transpiration stream arrives at the bulk-leaf apoplast, it carries the bulk-leaf apoplastic sucrose and travels towards the stomatal subcavity, causing significant sucrose accumulation at the guard-cell apoplast (as modeled with the accumulation of petiolar-fed plasma membrane impermeable 14C-mannitol at the guard-cell apoplast in broad-bean leaves (Ewert et al., 2000)). High concentrations of sucrose (>150 mM, Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001, calculations according to Ewert et al., 2000)) at the guard-cell apoplast diminish stomatal aperture size, and may serve as a signal in the sensing of transpiration rate, leaf photosynthesis rate and phloem translocation rate (Lu et al., 1997). In broad bean (Outlaw and De Vlieghere-He, 2001), when the leaf transpiration rate was lowered through increasing environmental RH from 60% to 90%, guard-cell apoplastic sucrose concentration decreased from nominally 170 mM to 50 mM, thus proving that transpiration is a positive factor in affecting sucrose in the guard-cell apoplast. As mentioned above, the osmotic role of guard-cell apoplastic sucrose has been tested in broad bean, an apoplastic phloem loader. However, another major group of vascular plants, i.e. symplastic phloem loaders, has not been studied on this subject. Therefore, how transpiration stream causes leaf apoplastic photosynthates to accumulate in

the guard-cell apoplast of symplastic phloem loaders is unknown (Fig. 1B). Apoplastic phloem loaders and symplastic phloem loaders are different because they have different phloem loading strategies (Fig. 2, recently reviewed in Lalonde et al., 2004). In apoplastic phloem loading, the sucrose synthesized by mesophyll cells is transported to the phloem parenchyma cell or bundle sheath cell symplastically, and then is released to the apoplast. From the apoplast, sucrose is loaded into SE-CCC by sucrose/H+ symporters in the plasma membrane of companion cells and sieve elements (e.g. Gottwald et al., 2000; Williams et al., 2000). Companion cells have virtually no or very few plasmodesmata (pd) connected to surrounding phloem parenchyma cells or bundle sheath cells, whereas companion cells and enucleated sieve elements are connected through enlarged and abundant pd in all plants. In the phloem, sucrose is the predominant sugar transported. In contrast, in symplastic phloem loading, sucrose is presumably transported from mesophyll cells into the SE-CCC symplastically, and then is used to synthesize RSOs in the companion cells. Companion cells are connected to phloem parenchyma cells or bundle sheath cells through numerous pd. In most of the plants with symplastic loading pathways, RSOs, instead of sucrose, are the predominant sugars transported in the phloem (Turgeon, 1995; Flora and Madore, 1996; Turgeon, 1996; Fiehn, 2003). Because of the presence or absence of an apoplastic step in loading of sucrose into the phloem complex, apoplastic phloem loaders and symplastic phloem loaders have different leaf apoplastic sucrose concentrations, i.e. 2-6 mM in typical apoplastic phloem loaders (Voitsekhovskaja et al., 2000; Knop et al., 2001; Lohaus et al., 2001) vs. less than 0.5 mM in typical symplastic phloem loaders (Voitsekhovskaja et al., 2000). As indicated, vascular plants can be categorized into two groups in a broad sense, apoplastic phloem loaders and symplastic phloem loaders (Stitt, 1996). However, in recent years, many studies indicate that, although some plants are relatively strict symplastic (e.g. cucumber, coleus, basil) or apoplastic phloem loaders (e.g. broad bean, pea), some plants probably use both loading pathways, and both loading pathways may possibly coexist in the same minor vein (van Bel et al., 1988; van Bel and Gamalei, 1992; Grusak et al., 1996; Goggin et al., 2001; Knop et al., 2001). Some plants even have no apparent phloem loading (Turgeon and Medville, 1998). A third group of plants with a mixed phloem

loading configuration has been brought up by some researchers (van Bel and Gamalei, 1992). Thus, the leaf apoplastic sucrose concentrations may vary accordingly. In the present study, two hypotheses were tested to investigate the relationship between the bulk-leaf apoplastic sucrose concentration and the guard-cell apoplastic sucrose concentration. The first hypothesis is, In a plant with typical symplastic phloem loading, bulk-leaf apoplastic photosynthates do not accumulate to osmotically significant levels around guard cells during times of active transpiration because the low bulk-leaf apoplastic photosynthate concentrations limit the potential of their accumulation at the guard-cell apoplast. This will allow comparison with the well-characterized apoplastic phloem loader plant broad bean. The second hypothesis is, In the apoplastic phloem loader broad bean, the accumulation of guard-cell apoplastic sucrose to physiologically significant levels (>150 mM) is associated with high leaf photosynthesis rate. This hypothesis is based on the observation that mesophyll photosynthesis is the direct source of bulk-leaf apoplastic sucrose (Lu et al., 1997). If the leaf photosynthetic rate is lowered while keeping the transpiration rate unchanged, the guard-cell apoplastic sucrose may not accumulate to osmotically significant concentrations. My final results supported these two hypotheses. In summary, in the first chapter, in the typical symplastic phloem loader I selected, dwarf basil, the upper limit of RSO (stachyose plus raffinose) concentration in the guard-cell apoplast was 10 mM, and other sugars (glucose, fructose and sucrose) in the guard-cell apoplast were not detectable (p>0.2, compared with concentration zero). Thus, the guard-cell apoplastic photosynthate concentration was significantly lower than that in apoplastic phloem loaders and exerted no significant osmotic effect on guard cells. In the second chapter, with the apoplastic phloem loader broad bean, after shading of the leaf while keeping constant leaf transpiration, bulk-leaf apoplastic sucrose concentration decreased to one third of the control value, whereas the guard-cell apoplastic sucrose concentration decreased to ~40 mM, less than one-fourth of the control value. This phenomenon indicates that for apoplastic phloem loaders, when the leaf-apoplast-sucrose concentration is reduced, guard-cell apoplastic sucrose may not exert physiologically significant osmotic effects on guard cells.

(A) Apoplastic phloem loaders (B) Symplastic phloem loaders

Fig. 1. Leaf cross of apoplastic phloem loaders (A) and symplastic phloem loaders (B), showing the accumulation of sucrose and/or RSOs around the guard cell under active transpiration. (A) Apoplastic phloem loaders. Sucrose (1) (red line or dots) that is synthesized in the mesophyll cell is transported symplastically to the phloem parenchyma cell (2) and is released into the apoplast (3). From the apoplast, sucrose is loaded into the sieve tube element-companion cell (4) complex and is transported out of the leaf through the translocation stream (5) (blue line) in the phloem. Water that comes from the root through the xylem arrives at the bulk-leaf apoplast and goes to the stomatal sub-cavity along with the transpiration stream (6). Sucrose in the apoplast travels in the transpiration stream and accumulates at the guard-cell apoplast (7) when water evaporates (8) through the stoma. (B) Symplastic phloem loaders. Sucrose that is synthesized in the mesophyll cell is transported symplastically to the sieve tube element-companion cell (4) complex and is used to synthesize RSOs (in purple line or dots) in the companion cell (4). How the transpiration stream (5) causes leaf apoplastic sucrose or RSOs to accumulate in the guard-cell apoplast (7) is unknown. (Modified from Outlaw, 2003)

Fig. 2. Two phloem-loading pathways in source leaves. (A) Apoplastic phloem loading. Sucrose is produced in mesophyll cells and transported symplastically to the bundle sheath cell/phloem parenchyma cell, then it is released to the apoplast. Next, the sucrose is loaded into the sieve tube element-companion cell complex through sucrose/H+ symporters. Sucrose is the predominate solute transported in the phloem. The possibility that sucrose is released from all the mesophyll cells is not excluded. (B) Symplastic phloem loading. Sucrose moves symplastically from mesophyll cells to companion cells. RSOs are probably produced in the companion cell and are the predominant sugars transported in the phloem. (Modified from Raven et al. (2005))

CHAPTER 1

GUARD-CELL APOPLASTIC PHOTOSYNTHATE ACCUMULATION IS LIMITED OR ABSENT IN THE SYMPLASTIC PHLOEM LOADING PLANT OCIMUM BASILICUM

Introduction

The evolution of adjustable stomata was an important adaptation for survival in a terrestrial environment. Aperture size changes are effected by the osmotic gain or loss of water by the guard-cell symplast, which is isolated from other cells. Potassium salts are the usual major fluctuating osmotica inside guard cells (Outlaw, 1983; Zeiger, 1983), but potassium-salt fluctuations are inadequate under some conditions (e.g. MacRobbie and Lettau, 1980). In addition to those by potassium salts, sucrose-concentration fluctuations osmoregulate guard cells (Tallman and Zeiger, 1988; Poffenroth et al., 1992; Lu et al., 1995; Amodeo et al., 1996; Talbott and Zeiger, 1996, 1998). The sucrose that accumulates in guard cells potentially comes from three sources: starch degradation, guard-cell photosynthesis (e.g. Poffenroth et al., 1992; Talbott and Zeiger, 1993) and apoplastic sucrose. However, starch degradation (~250 fmol sucrose pair-1 ~30 mM, calculated from Outlaw and Manchester, 1979) and guard-cell photosynthesis (e.g. Reckmann et al., 1990) can only provide a limited amount of sucrose (reviewed in Outlaw, 2003). Therefore, by default, bulk-leaf apoplastic sucrose is the most important source for the sucrose that accumulates in guard cells during stomatal opening. In addition, in planta kinetic studies (Lu et al., 1997) of sucrose movement to guard cells, sucrose uptake by isolated guard cells (Reddy and Rama Das, 1986; Outlaw, 1995), documentation of sugar transporters in guard cells (Kopka et al., 1997; Stadler et al., 2003), and guard-cell enzyme patterns

typical of a sucrose sink (Hite et al., 1993) are supportive. Ultimately, bulk-leaf apoplastic sucrose is from photosynthesis of leaf mesophyll cells (Lu et al., 1997), as discussed later. In addition to serving as a source for guard-cell symplastic sucrose, guard-cell apoplastic sucrose itself plays an osmotic role in regulating stomatal aperture size (Lu et al., 1995). Our model for apoplastic phloem loaders (Outlaw, 2003) posits that photosynthetically produced sucrose is transported symplastically from the mesophyll to the phloem, where it is released to the bulk-leaf apoplast. From there, sucrose is loaded into companion cells and sieve tubes for translocation. The transpiration stream arriving at the bulk-leaf apoplast mingles with this bulk-leaf apoplastic sucrose transport pool and carries a portion of it to the guard-cell apoplast. Evaporation of the transpiration stream causes sucrose accumulation in the guard-cell apoplast, which diminishes stomatal aperture size, and thus serves as a physiological signal in the sensing of the rates of transpiration, photosynthesis and translocation. In addition to the cited evidence for this model, the effect of apoplastic sucrose has been mimicked by petiolar-fed 14C-mannitol accumulation in the guard-cell apoplast (Ewert et al., 2000). Bulk-leaf apoplastic sucrose concentrations range widely among plants and correlate with different phloem-loading patterns. In typical apoplastic phloem loaders, the bulk-leaf apoplastic sucrose concentration is 2–6 mM (Delrot et al., 1983; Lohaus et al., 2000; López-Millán et al., 2000; Voitsekhovskaja et al., 2000; Lohaus et al., 2001), whereas in typical symplastic phloem loaders, bulk-leaf apoplastic sucrose concentration is <0.5 mM (Voitsekhovskaja et al., 2000). This difference results from the absence of an apoplastic intermediate transport pool in symplastic phloem loaders, in which sucrose moves symplastically via plasmodesmata into the companion-cell/sieve-element complex. As a means of maintaining the sucrose gradient for passive transport into the complex, sucrose there is removed and used to synthesize RSOs (Raffinose Series Oligosaccharides), which are the predominant export species (e.g. Flora and Madore, 1996; Turgeon, 1996; Fiehn, 2003). The absence of an apoplastic step in photosynthate movement obviously limits transpiration-rate-dependent regulatory alteration of the guard-cell apoplastic photosynthates by leaf photosynthesis. Model plants for guard-cell studies are either typical apoplastic phloem loaders, e.g. Arabidopsis thaliana (Truernit and Sauer, 1995; Gottwald et al., 2000; Haritatos et al.,

2000; Weise et al., 2000) and Vicia faba (Delrot and Bonnemain, 1981; Bourquin et al., 1990; Gamalei, 1991; Flora and Madore, 1996), or use both symplastic and apoplastic phloem loading pathways, such as Commelina communis (Eschrich and Fromm, 1994). These three model plants have been used extensively in guard-cell studies (e.g. Hanstein and Felle, 2002; Ritte and Raschke, 2003; Stadler et al., 2003; Leonhardt et al., 2004). However, strict symplastic phloem loaders have not been studied perhaps because they are primarily trees and shrubs (Gamalei, 1991; van Bel and Gamalei, 1992), which are less amenable to laboratory manipulation. However, use of these plants has two advantages. First, they are a large segment of the biome (of the 515 species characterized by Zimmermann and Ziegler, 1975, 65% transport detectable amount of RSOs and 20% transport more RSOs than sucrose) and, as discussed, stomatal regulation is hypothesized to lack key integrative components found in apoplastic phloem loaders, which, if verified, would be a notable phenomenon (see Hanstein and Felle, 2002; Roelfsema and Hedrich, 2002). Second, the low photosynthate concentration in the bulk-leaf apoplast resembles that of important crop plants under conditions of low photosynthesis. The overall aim of this study was to determine whether bulk-leaf apoplastic sugars accumulate in the guard-cell apoplast and exert osmotic and other effects on guard cells in symplastic phloem loaders during the photoperiod. As a necessary step toward this goal, a model symplastic phloem loader, dwarf basil (Ocimum basilicum cv Minimum), was identified, selected and characterized with regard to stomatal physiology (diurnal stomatal aperture changes, guard-cell starch and potassium content changes), transport physiology (sugars in the bulk-leaf apoplast and sugar derivatives in phloem exudates) and anatomy (continuity of the bulk-leaf apoplast). The key finding confirmed our hypothesis: guard-cell apoplastic photosynthate concentration was much lower than that in apoplastic phloem loaders and probably exerted no biologically significant osmotic effect on guard cells.

Materials and Methods

Plant Material

Dwarf-basil (Ocimum basilicum cv Minimum) seeds were purchased from Harris Seeds Inc. (Rochester, NY, USA). The seeds were germinated in Fafard No. 2 soil-less potting medium (Conrad Fafard, Inc., Agawam, MA, USA) in plastic market packs (~100 ml per unit) under a fluorescent-light bank (~200 µmol m-2 s-1). When the plants were two weeks old, they were individually transplanted to 1-liter pots and further culture was in a growth cabinet (for details, see Ewert et al., 2000). In brief, the cabinet was programmed for a 16-h day (25/20 ºC day/night temperature; PAR, 500 µmol m-2 s-1) at a constant 60% RH. The first youngest fully expanded pairs of simple opposite leaves of 24- to 28-day-old plants were used in all experiments.

Chemicals

Enzymes used for guard-cell sucrose histochemical assays were purchased from Boehringer Mannheim Corp. (Mannheim, Germany). Malic dehydrogenase used for the detection of cell-content contamination in bulk-leaf apoplastic sap was purchased from F. Hoffmann-La Roche Ltd. (Indianapolis, IN, USA). Mannitol (Sigma-Aldrich Co., St. Louis, MO, USA), used for petiolar feeding, was pre-washed with methanol to remove possible ABA contamination as described in Ewert et al. (2000). 14C-mannitol, 2.1 GBq mmol-1, was from PerkinElmer, Inc. (Shelton, CT, USA). HPLC standards for sucrose (Fisher Chemicals, Fairlawn, NJ, USA), and glucose, fructose, stachyose, raffinose and galactinol (Sigma-Aldrich Co., St. Louis, MO, USA) were obtained commercially.

Phloem Exudation

Phloem exudation collection was according to Büchi et al. (1998). Four hours into the light period, leaves were excised by cutting through the petiole, which was submerged in water; then, the petiole was re-cut under phloem-exudation buffer

(5 mM KH2PO4/K2HPO4, pH 7.5, containing 5 mM EDTA (disodium salt)). Incubation

was at 100% RH and darkness (to avoid transpiration) and room temperature with the petiole submerged in phloem-exudation buffer (2 leaves per 0.8-ml-centrifuge tube containing 0.6 ml phloem exudation buffer). Exudates collected during the initial 2 h were discarded. Exudates collected during three successive 3-h periods were pooled for analysis by HPLC.

Leaf Conductance

Leaf conductance was measured with a LI-1600 steady-state porometer (LI-COR Inc., Lincoln, NE, USA).

Semiquantitative Histochemical Estimates of Guard-cell Starch Contents and of Guard-cell Potassium Contents

Guard-cell starch was stained with iodine-phenol-potassium iodide (I2KI) reagent as in Heath (1949) and scored zero to five semiquantitatively.

Guard-cell potassium was stained with the hexanitrocobaltate (Na3Co(NO2)6) reagent (Sigma-Aldrich Co., St. Louis, MO, USA) and scored zero to five semiquantitatively according to Green et al. (1990).

Quantitative Histochemical Assay of Guard-cell Sucrose Content.

Histochemical procedures for single-cell sucrose analysis were according to Lu et al. (1997). (For a general description of these methods, see Outlaw and Zhang, 2001). In brief, tissue was frozen in liquid-nitrogen slurry and stored at –80 ºC until freeze-drying at –35 ºC and <10 µm Hg. Then, guard-cell pairs were individually dissected in a climate-controlled environment and the sucrose content of each was measured with oil-well (initial volume=1 µl) and enzymatic-cycling techniques (0.1-1 pmol). Guard cells that were dissected from whole-leaf fragments contained both symplastic and apoplastic sucrose. Guard cells that were dissected from epidermis that had been washed before freezing contained only symplastic sucrose. Apoplastic contents were

calculated by subtraction; SEs were calculated by an algorithm that provided a maximum variance in the apoplastic pool (see Outlaw and De Vlieghere-He, 2001).

Bulk-leaf Apoplastic Sap Collection

Petioles of excised leaves were quickly wrapped with Parafilm and then inserted immediately through the sealing grommet of a pressure chamber (Model 1000, PMS Instrument Co., Albany, OR, USA). The first droplet to be extruded, ~2 µl, was removed by blotting with tissue paper and discarded. The next aliquot, 8–10 µl, was collected with a restriction pipette and stored at –80 ºC until analysis. The maximum pressure exerted was 1.1 MPa. Malic dehydrogenase activity (according to López-Millán et al., 2000) in the bulk-leaf apoplastic sap was <1.5% of that in the leaf homogenate (mass basis), a determination made to ensure that cells were not ruptured at high pressures.

Bulk-leaf Apoplastic Water Content Measurement

The pressure chamber was also used to determine the leaf aqueous apoplastic volume, expressed as a percentage of the total leaf aqueous volume (Ewert et al., 2000). The tops of well-watered plants were cut off and discarded. Then the shoots were cut off under water near the crown, and then transferred to individual flasks with the bases submerged in water to assure hydration. After a 4-h incubation at 100% RH in darkness, one leaf of each pair of simple opposite leaves was used for fresh weight, area, and dry weight. The other leaf (with the intact petiole) was fixed into the pressure chamber as described above. Pressure was increased in a stepwise manner (0.1-0.2 MPa/step) and extruded sap was collected at each step. Pressure-volume curves were constructed, permitting extrapolation to aqueous bulk-leaf apoplastic volume (for details, see Turner, 1988).

Transmission Electron Microscopy (TEM) and Dwarf Basil Guard-cell-wall Volume and Guard-cell Volume Calculation

TEM procedure was according to Ewert et al. (2000). The middle section images of the guard cell were used to obtain the guard-cell dimension and the guard-cell wall thickness. The guard-cell-wall volume calculation was based on the geometric model of a cylinder (h, 36.3 µm, from light microscope images (n=10); r (outer dimension), 4.6 µm, from TEM images (n=4)). The average thickness (0.8 µm) of the guard-cell wall was calculated assuming equal wall thickness from TEM images (n=4). The final calculation was corrected for a 15% shrinkage rate in each dimension (Ewert et al., 2000). As before (Ewert et al., 2000), the aqueous guard-cell-wall volume was assumed to be half of the total wall volume. Calculated thus, the aqueous space in the wall of one dwarf basil guard-cell pair was 1.2×10-15 m3, and the guard-cell volume was 4.5×10-15 m3.

Movement of Extra-foliar Xylem-source Mannitol through the Leaf.

As a means of determining whether the bulk-leaf apoplast of dwarf basil is uninterrupted, two types of experiments based on feeding the plasma-membrane impermeant solute mannitol to excised leaves were conducted (see Ewert et al., 2000). In both types of experiments, leaves were excised and then re-cut under water. Then, the leaf petioles were transferred to plastic tubes (2.7×0.5 cm) containing either 5 mM mannitol, 5 mM 14C-labeled mannitol or water and were kept in the growth cabinet. Solutions were replenished every 20 min for up to 3 h. In the first type of experiment, the accumulation of mannitol in the guard-cell apoplast was inferred by the diminution of transpiration rate. In the second type of experiment, the mannitol content of the guard-cell apoplast was calculated directly from the 14C content of guard cells that were dissected from freeze-dried 14C-mannitol-fed leaves (as for sucrose analysis above). Contents were converted to concentration using the average guard-cell aqueous apoplastic volume of this species (determined above).

Guard-cell-sugar Extraction for HPLC

Fragments of dwarf-basil leaf or epidermis were sampled at 1100 h and freeze-dried at -35 ºC according to Lu et al. (1997), then were stored under vacuum at –20 ºC until dissection of guard cells. Guard cells were dissected from washed epidermis (containing symplastic sugars) or from leaf fragments (containing both symplastic and apoplastic sugars) as for histochemical analysis. Guard cells were pooled (up to 1000 pairs for RSOs, and up to 120 pairs for sucrose and reducing sugars) for extraction in 30 µl of ice-cold water, which was immediately elevated to 95 ºC, where it remained for 30 min. The extraction solution was then stored at –20 ºC before being used for HPLC analysis of sugars.

HPLC Analysis of Sugars in Phloem Exudates, Bulk-leaf Apoplastic Sap and Guard-cell Extracts

Sugars and galactinol were analyzed with a Waters 2695 Alliance Separation Module with temperature-controlled column chamber and autosampler (Waters Co., Milford, MA). The column used was 250×4.1 mm Hamilton RCX-10 anion exchange HPLC column (Hamilton Co., Reno, Nevada). Samples were injected in a volume of 20 µl and the mobile phase was 150 mM NaOH running at a speed of 1 ml min-1. The detector was ESA Coulochem II electrochemical detector with gold electrode (ESA Biosciences Inc., Chelmsford, MA). The peaks of sugars were identified by comparing their retention times with those of standard sugars with Millenium32 data-analysis software from Waters Co. (Milford, MA). Internal standards to insure recovery (>90%) were added to guard-cell extracts before injection. The detection limit (p <0.001) of sugars with this method was 4 pmol (2×10-7 M, 20 µl), i.e. if a total of 1000 pairs of guard cells were pooled in each 30-µl extraction solution, the detection limit of each sugar was 0.006 pmol pair-1.

Results

Sugar Analysis of Phloem Exudates Confirmed that Dwarf Basil is a Symplastic Phloem Loader

Apoplastic phloem loading and symplastic phloem loading are the prototypical extremes of phloem-loading patterns in plants. However, prototypical symplastic phloem-loading species have not been used as model plants for guard-cell studies. As a means of studying the unique guard-cell environment in symplastic phloem loaders, dwarf basil was selected as a model after evaluation of a taxonomically diverse range of candidates. Dwarf basil is in the Lamiaceae, a family generally recognized as symplastic phloem loaders (Gamalei, 1991). However, dwarf basil itself had not been documented as a symplastic phloem loader. Thus, the carbohydrate profile of phloem exudates of dwarf basil was determined to assure its symplastic phloem loading pattern (Turgeon and Medville, 2004). Stachyose was, by far, the predominate sugar of the five detected in phloem exudates (Fig. 3). Alone, stachyose accounted for 68±3% (molar basis; 84%, carbon basis) of the five sugars. Together, RSOs accounted for ~76% (molar basis; 91%, carbon basis) of total sugars. Sucrose was the least abundant sugar (4±0.5% on molar basis; 3%, carbon basis). Glucose and fructose were also detected in dwarf basil phloem exudate (14±2% and 5±2% on molar basis, and 4% and 2% on molar basis, respectively). This carbohydrate profile is a distinguishing characteristic of symplastic phloem loaders (e.g. Bachmann et al., 1994; Flora and Madore, 1996; Fiehn, 2003) and contrasts sharply with that of an apoplastic phloem loader (e.g. in broad bean, sucrose accounted for 90% (molar basis; 97% on carbon basis) of total sugars, and RSOs were undetected (data not shown)).

80 )

20 Sugar concentration (mole% concentration Sugar 0 Glucose Fructose Sucrose Raffinose Stachyose

Fig. 3. The sugar composition (mole percent, mean±SE, n=4 plants) of dwarf-basil phloem exudates. Sugars were analyzed with HPLC.

Diurnal Kinetics of Dwarf-basil Leaf Conductance, Stomatal Aperture Size, and Guard-cell Starch and Potassium Contents Was Typical

Because the value of a model species depends on its conformity to the normal (cf. Outlaw et al., 1982), basic stomatal physiological parameters of dwarf basil were examined and compared with those of broad bean, an apoplastic phloem loading plant that is a model for guard-cell studies. In dwarf basil, leaf conductance (Fig. 4A) and stomatal aperture size (Fig. 4B) increased in the morning, reached a steady-state maximum near noon and decreased before the end of the photoperiod. The maximum stomatal aperture size was ~7 µm, which is ~2 µm smaller than the correlate value of broad bean (Lu et al., 1995; Outlaw and De Vlieghere-He, 2001) and is consistent with the somewhat smaller guard cells in dwarf basil (36×11 µm vs. 46×11 µm (Willmer and Fricker, 1996)). However, the maximum leaf conductance of dwarf basil (~0.5 mol m-2 s-1) was 3-4-fold higher than that of broad bean (~0.14 mol m-2 s-1, Lu et al., 1995; Ewert et al., 2000), which probably reflects the ~1.5-fold higher stomatal density of dwarf basil (11,000 cm-2 vs. ~6000 cm-2 on the lower epidermis). The increase in guard-cell potassium content and the decrease in guard-cell starch content of dwarf basil correlated strongly with the increase in stomatal aperture size

following the onset of illumination. The extremes of these parameters coincided temporally, at 1100 h (Fig. 4B). However, after 1100 h, guard-cell potassium content appeared to decline faster than stomatal aperture size. Between 1100 h and 1400 h, stomatal aperture size only decreased moderately, from 6.7±0.3 µm to 5.9±0.2µm, but guard-cell potassium content decreased from a score of 3.7±0.1 to 2±0.1, a decrease of 46% of the original value. The decline in potassium content without a commensurate decrease in stomatal aperture size is reminiscent of observations in onion and broad bean (Amodeo et al., 1996; Talbott and Zeiger, 1996). Guard-cell starch content appeared to be inversely correlated to stomatal aperture size throughout the photoperiod, which is often observed, but with many exceptions (reviewed in Heath, 1949).

Fig. 4. Patterns of leaf conductance, stomatal aperture size, guard-cell potassium content and guard-cell starch content of dwarf basil over the course of a day. (A) Leaf conductance (mean±SE, n=7 plants). (B) Stomatal aperture size, guard-cell potassium content and guard-cell starch content. Data are means (±SEs) of 180 guard-cell pairs from plants that were different from that used for leaf conductance measurement (from three independent experiments).

Dwarf-basil Bulk-leaf Apoplastic Sugar and Galactinol Concentrations were in the Micromolar Range Mature guard cells are symplastically isolated from surrounding cells (e.g. Wille and Lucas, 1984) and guard cells have no or limited capacity for photosynthetic carbon reduction (Outlaw, 2003). Consequently, the bulk-leaf apoplast supplies guard cells with carbohydrates, which may accumulate in the guard-cell apoplast and osmotically diminish stomatal aperture size (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001). In addition to sugars themselves, other components, e.g ABA (Zhang and Outlaw, 2001c) and malate (Hedrich et al., 1994) of the bulk-leaf apoplastic sap are signaling substances that target guard cells. Therefore, at least preliminary characterization of the bulk-leaf apoplastic sap is essential in establishing a model species for guard-cell studies. Stachyose and glucose were the most abundant of the five sugars detected at each time assayed (Fig. 5). The diurnal concentration kinetics of these two sugars increased nominally 2-3-fold from the onset of illumination to reach a maximum of nominally 90 µM at 1100 h. As apparently did the other less abundant sugars, stachyose and glucose (p=0.01) decreased after 1100 h. Note that the stachyose and glucose were also the most abundant sugars in the phloem exudates at 1100 h (Fig. 3), but differed in relative abundance and absolute concentrations in the apoplastic sap (Fig. 5). Galactinol was present in the bulk-leaf apoplastic sap, but at only about 20 µM at 1100 h (not shown). In summary, the combined maximum (total sugar + galactinol) concentration was nominally 300 µM at its peak, 1100 h (when the average stomatal aperture size was also the highest, Fig. 4B). By way of comparison, RSOs and galactinol are not detectible in the broad-bean bulk-leaf apoplastic sap, in which sucrose, at ~2 mM, is the most abundant and glucose and fructose are each <1 mM (Voitsekhovskaja et al., 2000; Lohaus et al., 2001 and our unpublished data).

Fig. 5. Diel pattern of sugar concentrations (mean±SE) in the dwarf-basil bulk-leaf apoplast. Bulk-leaf apoplastic sap was collected with a pressure chamber. Sugar concentrations were measured with HPLC. n=5 plants at 600 h; n=7 plants at 1100 h; for other times, n=3 plants.

Mannitol Fed via the Petiole Accumulated in the Guard-cell Apoplast, Indicating the Continuity of the Bulk-leaf Apoplast in Dwarf Basil

Membrane-impermeant solutes have been introduced into the transpiration stream as a marker to determine its fate. The results have been interpreted by Canny (1995) and others as evidence for barriers within the bulk-leaf apoplast that prevent the transpiration stream from reaching stomata. However, 14C-mannitol fed to excised broad-bean leaflets via the petiole accumulated in the guard-cell apoplast and diminished transpiration rate (Ewert et al., 2000), proving continuity of the apoplast, at least in that species. Corroboratively, ABA fed via the intact petiole (Zhang and Outlaw, 2001a) or via elevation of xylem ABA by water stress (Zhang and Outlaw, 2001b, 2001c) accumulated around broad-bean guard cells. Finally, and again with broad bean, sucrose produced by photosynthesis accumulated in the guard-cell apoplast (Lu et al., 1997) and this accumulation depended upon transpiration (Outlaw and De Vlieghere-He, 2001). Altogether, these results with broad bean are consistent with the absence of apoplastic

barriers. Here, similar experiments were conducted with dwarf basil (Fig. 6, Fig. 7 and Fig. 8) to test the continuity of the bulk-leaf apoplast in that species. As preliminary to interpretation of the results of mannitol-feeding experiments, the volume of the bulk-leaf apoplast and of the guard-cell apoplast of dwarf basil was determined in order to estimate mannitol concentrations in the bulk-leaf apoplast and the guard-cell apoplast. The bulk-leaf apoplastic aqueous volume was 39±3% (n=9) (average 102 µl per leaf) of the total leaflet water, as determined by extrapolation of the pressure-volume curve (see Fig. 6 for a representative plot (Turner, 1988)). By comparison, that of the broad bean is ca. 29% (Ewert et al., 2000). The aqueous guard-cell-wall volume of dwarf basil was ca. 1.2×10-15 m3 pair-1 calculated based on the geometric model of a cylinder. As support for this estimate, the aqueous space of wall volume of a broad bean guard-cell pair was calculated similarly and the value obtained was 3.9×10-15 m3, which is essentially identical to that (~4.2×10-15 m3 ) obtained by serial sectioning and 3-D computational analysis (Ewert et al., 2000). The transpiration rate of excised leaves of dwarf basil increased (p=0.02) over a 3-h period if the petioles were submerged in water (Fig. 7). (A common observation, this increase results from loss of resistance to water flow due to removal of the roots and shoot.) The typical turnover rate of bulk-leaf apoplastic water through transpiration -2 -1 (assuming 3 mmol H2O m s ) was two times per hour. When the leaves were fed 5 mM mannitol instead of water, the average of the transpiration rates was relatively stable during the first hour then declined steadily over the next 2 h (Fig. 7). The difference in transpiration rate between mannitol- and water-fed leaves was manifested by 120 minutes (p=0.02) and was highly significant by the end of the time course (p<0.001), when the transpiration rate of mannitol-fed leaves was only 64% of controls. These results provide indirect, but only equivocal, evidence for an open apoplast. That is, the simplest interpretation is that mannitol accumulated around guard cells and diminished aperture size (Fig. 7), but plausible and more complex interpretations such as mannitol-evoked ABA increase are not excluded. As a means of directly testing mannitol movement from the petiole to stomata, 5 mM 14C-mannitol was fed to excised leaves via the petiole for 2 h and then 14C-mannitol in guard cells was assayed (Fig. 8). Transpirational water loss during the 2 h of petiolar

feeding concentrated the 14C-mannitol in the bulk-leaf apoplast to an average of 20 mM. However, after 2 h of feeding 14C-mannitol, the average guard-cell apoplastic 14C-mannitol concentration was ca. 300 mM, implying not only continuity of the apoplast, but also the potential for transpiration-linked accumulation of bulk-leaf apoplastic solutes (Fig. 8). Altogether, these results proved that the bulk-leaf apoplast of dwarf basil is continuous, just as it is in broad bean (Ewert et al., 2000).

) 3.0 -1

2.5

2.0 y = -0.0042x + 1.37 1.5 R2 = 0.99 1.0

0.5

Inverse balance pressure (MPa pressure balance Inverse 0.0 0 100 200 300 400 Cumulative volume removed (µl)

Fig. 6. Representative pressure-volume curve of dwarf-basil leaf. Dwarf-basil bulk-leaf apoplastic solution was collected in consecutive drops as the pressure inside the pressure chamber increased in a stepwise manner. The interception of the linear plot with the x-axis indicates the volume of the total leaf symplastic water. The total leaf aqueous volume was calculated by subtracting the dry weight from the fresh weight.

Fig. 7. Time course of dwarf-basil leaf transpiration rate (mean±SE) when excised leaves were supplied with water (n=6 plants, two growth lots) or 5 mM mannitol (n=7 plants, two growth lots) through the petiole. The experiments were conducted inside an illuminated growth chamber and began at 1100 h. The transpiration rate was measured directly by replenishing the solution supplied to the petiole.

Fig. 8. 14C-mannitol accumulation (mean±SE) in the bulk-leaf apoplast and in the guard-cell apoplast of dwarf-basil plants after 5 mM 14C-mannitol was fed to the leaf through the petiole for 2 h. The experiment was conducted inside an illuminated growth chamber and began at 1100 h. 14C-mannitol of the guard cells (n=15 pairs) was assayed with liquid scintillation counting. Calculation was based on the bulk-leaf apoplastic aqueous volume was 39% of the total leaflet water and the aqueous guard-cell wall volume was 1.2×10-15 m3. Other details are given in Fig. 7.

Guard-cell Apoplastic Photosynthate Accumulation was Limited or Absent in Dwarf-basil Plants

As the essence of this study, the photosynthate concentrations in the guard-cell apoplast were determined during mid-day, when transpiration was at a maximum. Complementarily, photosynthates in the guard-cell symplast were also assayed. The source of transpiration-linked photosynthate accumulation in the guard-cell apoplast is the bulk-leaf apoplast (see kinetics analysis of Lu et al., 1997). Thus, the initial focus was to measure RSOs, the most abundant sugars in the bulk-leaf apoplast, in the guard-cell apoplast. Neither stachyose nor raffinose was detectible in guard cells dissected from freeze-dried whole leaf or rinsed epidermis (HPLC method, maximum of 1000 pairs of guard cells pooled for each measurement). These results place the upper limit of these compounds at 0.006 pmol per whole guard-cell pair and 2 mM in the guard-cell symplast (based on the guard-cell volume is 4.5×10-15 m3). It is implausible that sugars would accumulate in the guard-cell apoplast, but be absent in the symplast (where they would be metabolized). However, in this extreme scenario—if RSOs were at the detection limit and were restricted to only the aqueous volume of the guard-cell wall—the maximum concentration in the guard-cell wall would be 10 mM. Two methods were used for assay of the sucrose content of the guard-cell apoplast and of the guard-cell symplast of dwarf basil during transpiration. First, using quantitative histochemistry, the sucrose content of the guard-cell apoplast was 0.018±0.02 pmol guard-cell pair-1 (n=47) and that of the symplast was 0.36±0.03 pmol guard-cell pair-1 (n=47). These values were almost identical to values obtained with HPLC from extracts of pooled guard cells (0.004±0.1 and 0.45±0.13 pmol guard-cell pair-1 for the apoplastic and symplastic compartments, respectively.) The reducing sugars were at lower concentration (Fig. 9; glucose, 0.05±0.03 and 0.16±0.05 pmol guard-cell pair-1 and fructose, 0.01±0.03 and 0.07±0.03 pmol guard-cell pair-1 for the apoplastic and symplastic compartments, respectively.) than sucrose. In summary, the sucrose concentration in the guard-cell symplast in dwarf basil (~80 mM, calculated based on the guard-cell volume was 4.5×10-15 m3) is similar to that in broad bean (~110 mM, Lu et al., 1997; Outlaw and De Vlieghere-He, 2001). However, the dwarf basil guard-cell apoplastic sucrose

concentration was not detectable (viz., 15±17 mM (histochemical method) and 3±80 mM (HPLC), calculated based on the guard-cell volume was 1.2×10-15 m3). Overall, the sugar concentration in the dwarf basil guard-cell apoplast was low, i.e. the upper limit of RSO concentration was 10 mM, and neither sucrose, glucose nor fructose was detectable (p>0.2, compared with concentration zero). In contrast, the broad bean guard-cell apoplastic sucrose concentration can be easily more than 150 mM (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001, calculated according to Ewert et al. (2000)) at higher rates of transpiration. Thus, guard-cell apoplastic photosynthate concentration was significantly lower in typical symplastic phloem loader dwarf basil than that in apoplastic phloem loaders and exerted no biologically significant osmotic effect on guard cells.

Fig. 9. Sugar content (mean±SE) in the dwarf-basil guard-cell symplast and guard-cell apoplast at maximum leaf conductance (1100 h). Sucrose was measured with both HPLC (n=5 plants, two growth lots) and histochemistry (n=47 pairs of guard cells from six plants, two growth lots); glucose and fructose were measured with HPLC method (n=5 leaves from five plants, two growth lots) only.

Discussion

The bulk-leaf apoplast plays important roles in plant-signal transduction (e.g. Maier-Maercker, 1999; Chikov and Bakirova, 2004; Gao et al., 2004), transport of water and nutrients (e.g. Canny, 1995; Sattelmacher, 2001), and plant-pathogen interactions (e.g. Gau et al., 2004). As a special case, the guard-cell apoplast is particularly important because guard cells are symplastically isolated at maturity (Wille and Lucas, 1984; Palevitz and Hepler, 1985), the bulk-leaf apoplast is continuous (e.g. Ewert et al., 2000), and the guard-cell apoplast is the terminal point in the evaporative pathway (Maier-maercker, 1983; Fricke, 2004). These facts have several implications. For example, xylem-source ABA accumulates in the guard-cell apoplast in broad bean during transpiration and the accumulated ABA results in stomatal closure (Zhang and Outlaw, 2001a, 2001b). More directly relevant to this investigation, apoplastic sucrose, a product of recent photosynthesis in the mesophyll, accumulates to high concentration in the guard-cell apoplast (>150 mM, Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001, calculated according to Ewert et al. (2000)) in this species, which is a strict apoplastic phloem loader. This sucrose accumulation is dependent upon transpiration (Outlaw and De Vlieghere-He, 2001) and the bulk-leaf apoplastic sucrose concentration (Y. Kang, W. H. Outlaw and P. C. Anderson, unpublished), which itself is positively related to the rate of photosynthesis (Y. Kang, W. H. Outlaw Jr. and P. C. Anderson, unpublished). Sucrose accumulation in the guard-cell apoplast osmotically diminishes stomatal aperture size (Lu et al., 1995, 1997) and thus is a mechanism by which transpiration rate is sensed (see Mott and Parkhurst, 1991; Monteith, 1995). Therefore, the basic elements of the hypothesis (Lu et al., 1997) that the concentration of photosynthate in the guard-cell apoplast is a signal that integrates the rates of transpiration, of photosynthesis, and of translocation in apoplastic phloem loaders have been established. As discussed above, the theory describing the integrative role of the guard-cell apoplastic photosynthate pool was based on the physiology of a strict apoplastic phloem loader, broad bean. In such plants, sucrose is the usual transport species, and it initially moves symplastically toward the phloem parenchyma, but is released to the apoplast

before reaching the SE-CCC. Sucrose is loaded from the apoplast into the SE-CCC by H+/sucrose symporters (e.g. Gottwald et al., 2000; Williams et al., 2000). In contrast, the phloem loading strategy used by symplastic phloem loaders (reviewed by Lalonde et al., 2004) limits the photosynthate source pool for the guard-cell apoplast. In these species, sucrose is transported from mesophyll cells into the SE-CCC symplastically, and then is used to synthesize RSOs in the companion cells (e.g. Turgeon and Gowan, 1992; Sprenger and Keller, 2000; Fiehn, 2003). Therefore, RSOs, not sucrose, are translocated in strict symplastic phloem loaders. In essence, the presence of an apoplastic step in apoplastic phloem loaders accounts for the relatively high sucrose concentration in the bulk-leaf apoplast of apoplastic phloem loaders (2–6 mM, Voitsekhovskaja et al., 2000; Knop et al., 2001; Lohaus et al., 2001) and the absence of an apoplastic step in symplastic phloem loaders limits the total bulk bulk-leaf apoplastic photosynthate concentration in symplastic phloem loaders (<0.5 mM, Voitsekhovskaja et al., 2000). On the basis of these differences, it was hypothesized that photosynthesis-dependent transpiration-linked accumulation of bulk-leaf apoplastic photosynthate in the guard-cell apoplast of symplastic phloem loaders does not occur. The results reported here support this hypothesis. In dwarf basil, a model species characterized for this study, RSOs were not detected in the guard-cell apoplast (total <10 mM, cf. >150 mM sucrose in broad bean, Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001, calculated according to Ewert et al. (2000)). Sucrose, fructose or glucose were not detected (p>0.2) in the guard-cell apoplast either. Thus, guard-cell apoplastic photosynthates do not exert a biologically significant osmotic effect on guard cells of dwarf basil, implying that the integrative theory developed by study of broad bean does not apply to symplastic phloem loaders.

Dwarf Basil, a Model Symplastic Phloem Loading Species for Guard-cell Studies

Symplastic phloem loaders are common. Of the 515 species characterized by Zimmermann and Ziegler (1975), 65% transport RSOs and 20% transport predominantly RSOs. Yet, no strict symplastic phloem loader has been used for guard-cell studies (see Introduction). The bias against symplastic phloem loaders probably stems from their being mostly trees and shrubs (Gamalei, 1991), which are not amenable to laboratory culture.

Exceptionally, the Lamiaceae generally are herbaceous symplastic phloem loaders (Gamalei, 1991), and have been studied for this reason (e.g. Flora and Madore, 1996; van Bel et al., 1996; Büchi et al., 1998). Following comparisons of taxonomically diverse symplastic phloem loaders (not shown), a member of this family, dwarf basil, emerged as the choice for the present studies. Previously uncharacterized, dwarf basil primarily transports RSOs (Fig. 3) indicating that this species is a symplastic phloem loader (cf. sweet basil (Büchi et al., 1998)). In general, dwarf basil is a good physiological model for guard-cell studies: (i) a compact erect plant, it produces several mature leaves within one month from seed; (ii) flat, homobaric, and opposite pairs of leaves facilitate porometry, peeling of epidermis, and assure uniform illumination for control and treatments; (iii) long petioles (~2.5 cm) simplify use of a pressure chamber; (iv) large guard cells (36×11 µm, about twice those of Arabidopsis (Willmer and Fricker, 1996) improve the relative precision of aperture measurements and are especially valuable for dissection that is required for quantitative histochemistry; and (v) diel changes in leaf conductance, stomatal aperture size, guard-cell starch content and potassium content are typical (Fig. 4B).

Relevance of Sugars in the Bulk-leaf apoplast of Symplastic Phloem loaders to Photosynthesis-dependent Transpiration-linked Sugar Accumulation in the Guard-cell Apoplast.

The few studies (Madore and Webb, 1981; Voitsekhovskaja et al., 2000) of bulk-leaf apoplastic sugars of symplastic phloem loaders indicate only micromolar concentrations of sucrose, hexoses, and RSOs (as well as galactinol, an RSO precursor, Madore and Webb, 1981). Similar results obtained for dwarf basil, in which the sum of sugars was less than 300 µM (Fig. 5). The most abundant sugars in the bulk-leaf apoplast of dwarf basil were stachyose and glucose (Fig. 5). Only these two sugars increased from the onset of illumination to midday (Fig. 5) and therefore only they are candidates for photosynthesis-dependent sugar accumulation in the guard-cell apoplast in this species. In particular, stachyose is important because it at least partially originates from the leakage of the phloem in the leaf (Ayre et al., 2003) or elsewhere (van Bel, 1993; Lalonde et al., 2003; Chikov and Bakirova, 2004). However, the potential for a role of stachyose

accumulation in the guard-cell apoplast, analogous to that of sucrose in apoplastic phloem loaders, is small because of its low absolute concentration in the bulk-leaf apoplast (100 µM (Fig. 5) vs. 2-6 mM sucrose in apoplastic phloem loaders (Voitsekhovskaja et al., 2000; Knop et al., 2001; Lohaus et al., 2001)). The potential for a role of glucose is also small because the absolute concentration in the dwarf basil apoplast is 4× less than that in broad bean (Lohaus et al., 2001; Y. Kang, W. H. Outlaw Jr. and P. C. Andersen, unpublished), in which glucose content of the guard-cell apoplast is small and statistically independent of the rate of photosynthesis (Y. Kang, W. H. Outlaw Jr. and P. C. Anderson, unpublished). Whereas the preceding indirectly precludes an osmotically important photosynthesis-dependent solute accumulation in the guard-cell apoplast, a transpiration-linked accumulation merits consideration independently. The potential for this simpler mechanism is also limited. In broad bean, only sucrose accumulates to an osmotically important level in the guard-cell apoplast, where it is maximally ~100× that of the bulk-leaf apoplast (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001). In the unlikely scenario that all the solutes in the bulk-leaf apoplast of dwarf basil accumulated to this maximum extent, the sum change in osmolality would be minor, <40 mM, compared with the value in broad bean.

Absence of Guard-cell Apoplastic Photosynthate Accumulation in Symplastic Phloem Loading Plant Dwarf Basil

Despite the negative evaluation of the hypothesis in the preceding section, two compelling facts implied the value of a direct test. First, the bulk-leaf apoplast of dwarf basil (Fig. 8) is continuous and thus provides a structural pathway for solute movement to the guard-cell apoplast. Second, transpirational flux, if calculated to a guard-cell basis, is possibly substantial (0.34 pmol (total solutes) stoma-1 hr-1, in the extreme case in which the entire unaltered transpiration stream reaches guard cells), but still is only 0.25× that of potential sucrose delivery to guard cells in broad bean (Outlaw and De Vlieghere-He, 2001). The direct test comprised analyses of the RSO, sucrose and hexose contents of individually dissected guard cells harvested from leaves that were under conditions of high

transpiration and photosynthesis. RSOs in the guard-cell apoplast of dwarf basil were not detected, indicating that the maximum concentration could not have exceeded 10 mM (p<0.001). Neither sucrose, fructose, nor glucose was detected (p>0.2) in the guard-cell apoplast of dwarf basil, but biological variability in these sugars in the guard-cell symplast unavoidably compromised the precision of these assays (Fig. 9). However, with almost exactly the same limitation (80 mM sucrose in the guard-cell symplast of dwarf basil, and ~110 mM in broad bean (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001), the high sucrose content of the guard-cell apoplast of broad bean was easily measured (e.g. SE/x, ~0.2; Outlaw and De Vlieghere-He, 2001). Altogether, these facts indicate the absence of transpiration-linked, photosynthesis-dependent photosynthate accumulation in the guard-cell apoplast of dwarf basil.

Physiological and Ecological Implications of Stomatal Control in Symplastic Phloem Loaders and Apoplastic Phloem Loaders

No single attribute is entirely responsible for fitness of a plant to a particular environment. Certainly, that is the case for regulation of the loss of water, generally the limiting resource for a terrestrial plant. Indeed, many independent biochemical adaptations, such as CAM (Crassulacean Acid Metabolism), and morphological adaptations, such as stomatal position, act synergistically. Here, another feature is reported: the absence of transpiration-linked photosynthate accumulation in the guard-cell apoplast of symplastic phloem loaders provides stomata with less feedback control. Complementary empirical evidence for this conjecture is lacking, however, because there are no surveys of “humidity” responses and absolute transpiration rates of morphologically similar plants that only differ in phloem loading strategies. It is striking, though, that conductance of dwarf basil, 0.5 mol m-2 s-1, is higher than that of the average herbaceous plant, ~0.26 mol m-2 s-1 (Willmer and Fricker, 1996). In broad bean, apoplastic sucrose is not only an external osmolyte, but it is taken up by guard cells, increasing the internal sucrose pool size (Lu et al., 1997) and providing carbon for replenishing starch reserves (Y. Kang, W. H. Outlaw Jr. and P. C. Andersen, unpublished). Contrary to this situation, the small guard-cell apoplastic sugar pool in

dwarf basil limits its potential as a source for guard-cell symplastic sugars. Nevertheless, as stated, the guard-cell symplast of dwarf basil and of broad bean contain similar concentrations of sucrose, respectively, 80 mM vs ~110 mM (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001). The question is whether the flux of sucrose into guard cells of symplastic phloem loaders is sufficient to allow its use as a fluctuating internal guard-cell osmolyte in symplastic phloem loaders as is the case with apoplastic phloem loaders (Tallman and Zeiger, 1988; Amodeo et al., 1996; Talbott and Zeiger, 1996, 1998).

Summary

In summary, dwarf basil was established as a model for guard-cell studies of symplastic phloem loaders. Use of this plant demonstrated that the phloem loading strategy has implications for stomatal control. In dwarf basil, guard-cell apoplastic photosynthate accumulation, if present, was insignificant compared with that in apoplastic phloem loaders. Therefore, the theory that transpiration rate is sensed by photosynthate accumulation around guard cells holds only for apoplastic phloem loaders. Similarly, the diminution of conductance by excess leaf photosynthate also holds only for apoplastic phloem loaders.

CHAPTER 2

GUARD-CELL APOPLASTIC SUCROSE CONCENTRATION―A LINK BETWEEN LEAF PHOTOSYNTHESIS AND STOMATAL APERTURE SIZE

Introduction

Sucrose plays several roles in stomatal movements. In the guard-cell symplast, sucrose is an important osmoticum that supplements or replaces potassium and is thus involved in sustaining stomatal opening (Tallman and Zeiger, 1988; Poffenroth et al., 1992; Lu et al., 1995; Amodeo et al., 1996; Talbott and Zeiger, 1996, 1998). For example, in broad bean (an apoplastic phloem loader), guard cells accumulate up to 110 mM sucrose (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001) and the concentration oscillates diurnally, corresponding to stomatal movements and the fluctuations in guard-cell-potassium content (Lu et al., 1995; Talbott and Zeiger, 1996). Under photosynthetic conditions, transpiration also drives sucrose accumulation in the guard-cell apoplast (>150 mM, Lu et al., 1995; Outlaw and De Vlieghere-He, 2001). This concentration is osmotically sufficient to diminish stomatal aperture size by 3 µm (Lu et al., 1995). Aside from its external osmotic effects, guard-cell apoplastic sucrose is also the source for guard-cell symplastic sucrose: guard cells have insufficient carbon-reduction capacity to replenish carbohydrate stores (Outlaw, 2003) and guard cells have an enzyme complement typical of a sink (Hite et al., 1993). Corroboratively, isolated guard cells take up exogenous sucrose (Reddy and Rama Das, 1986; Outlaw, 1995; Ritte et al., 1999) probably via sugar transporters in the guard-cell plasma membrane (Kopka et al., 1997; Stadler et al., 2003) and when the guard-cell apoplastic sucrose concentration is lowered

experimentally, guard cells fail to replenish starch (W. H. Outlaw Jr., M. Pearson and T. Jiang, unpublished), as they normally do at the end of the photoperiod. Finally, sucrose potentially regulates guard-cell gene expression as it is reported to do in many other tissues (Koch, 1996; Coruzzi and Bush, 2001; Rolland et al., 2002; Baier et al., 2004). As implied above, an increase in the guard-cell apoplastic sucrose concentration is a signal that effectively measures transpiration rate, the relevant parameter when stomata close in response to increased vapor-pressure difference (Monteith, 1995). As guard-cell apoplastic sucrose is a recent product of photosynthesis in mesophyll cells (Lu et al., 1997), its concentration is hypothesized to correlate with the rate of photosynthesis as well as transpiration. We (Outlaw and De Vlieghere-He, 2001) have thus proposed that guard-cell apoplastic sucrose concentration is an integrative signal that inhibits further opening of stomata when photosynthesis and transpiration are high, a modulation that would optimize CO2 gain and water loss. In this study, we have addressed the hypothesized effects of leaf photosynthesis rate on guard-cell apoplastic sucrose accumulation using broad bean, a plant often used for stomatal studies (e.g. Talbott and Zeiger, 1998; Outlaw and De Vlieghere-He, 2001; Taylor and Assmann, 2001; Ritte and Raschke, 2003). As hypothesized, lowering photosynthesis rate without altering transpiration decreased bulk-leaf apoplastic sucrose concentration >2-fold and decreased guard-cell apoplastic sucrose concentration >4-fold, from more than 170 mM to <40 mM. Additional investigations indicated that reducing sugars in the guard-cell apoplast had only a negligible osmotic effect under all conditions.

Materials and Methods

Plant Material

Broad-bean (Vicia faba L. cv Longpod) seeds were purchased from Harris Seeds (Rochester, NY, USA). Broad-bean plants were grown in Fafard No. 2 soil-less potting medium (Conrad Fafard, Inc., Agawam, MA, USA) in a growth cabinet (for details, see Ewert et al., 2000). In brief, the cabinet was programmed for a 16-h day (25/20 ºC

day/night temperature; PAR, 500 µmol m-2 s-1) at a constant 60% RH. The first youngest fully expanded pair of leaflets of ~3-week-old plants was used in all experiments.

Photosynthesis Rate Measurement

Leaf photosynthesis rate was measured with a CIRAS-1 Portable Photosynthesis System (PP systems Co., Amesbury, MA, USA).

Plant Shading and Growth-cabinet CO2-Concentration Manipulation

Broad-bean leaves were shaded with a custom-fabricated shading device. Lattices of transparent plastic monofilament lines (spacing=1 cm) were mounted in two plastic frames (20×10 cm2) that sandwiched the leaf, holding it horizontally. In treatments, neutral-density film (26 cm2) was mounted between the lights and leaves. Light intensity of control leaves was 450–500 µmol m-2 s-1 and that of shaded plants, 120-150 µmol m-2 s-1.

Growth cabinet CO2 concentration was lowered manually by either adding bottled

CO2-free air (80/20, nitrogen/oxygen) or by pumping internal air through 1M NaOH solution inside the growth cabinet. Growth cabinet RH was kept at 60% during CO2 concentration manipulation. CO2 concentration was monitored with an infra-red gas analyzer (Model 225 MK3, the Analytical Development Co., Hoddesdon, United

Kingdom). Growth cabinet CO2 concentration under the low CO2 condition was 350-360 µmol mol-1, 100 µmol mol-1 lower than that of control conditions.

Quantitative Histochemical Assay of Guard-cell Sucrose Content.

measured with oil-well (initial volume=1 µl) and enzymatic-cycling techniques (0.5-2.5 pmol). Guard cells that were dissected from whole-leaf fragments contained both symplastic and apoplastic sucrose. Guard cells that were dissected from epidermis that had been washed before freezing contained only symplastic sucrose. Apoplastic contents were calculated by subtraction; SEs were calculated by an algorithm that provided a maximum variance in the apoplastic pool (see Outlaw and De Vlieghere-He, 2001).

Bulk-leaf Apoplastic Sap Collection

Petioles of excised leaves were inserted immediately through the sealing grommet of a pressure chamber (Model 1000, PMS Instrument Co., Albany, OR, USA). The first droplet to be extruded, ~3 µl, was removed by blotting with tissue paper and discarded. The next aliquot, 8–10 µl, was collected with a restriction pipette and stored at –80 ºC until analysis. The maximum pressure exerted was 1.1 MPa. Malic dehydrogenase activity (according to López-Millán et al., 2000) in the bulk-leaf apoplastic sap was <0.5% of that in the leaf homogenate (mass basis), a determination made to ensure that cells were not ruptured at high pressures.

Guard-cell-sugar Extraction for HPLC

Fragments of broad-bean leaf or epidermis were sampled at 1100 h and freeze-dried according to Lu et al. (1997). In brief, tissue was frozen in liquid-nitrogen slurry and stored at –80 ºC until freeze-drying at –35 ºC and <10 µm Hg. Then, tissues were stored under vacuum at –20 ºC until dissection of guard cells. Guard cells were dissected from washed epidermis (containing symplastic sugars) or from leaf fragments (containing both symplastic and apoplastic sugars) for histochemical analysis. Individually dissected guard cell pairs were pooled (180 pairs) for extraction in 30 µl of ice-cold water, which was immediately elevated to 95 ºC, where it remained for 30 min. The extraction solution was then stored at –20 ºC before being used for HPLC analysis of sugars.

HPLC Analysis of Sugars in Bulk-leaf Apoplastic Sap and Guard-cell Extracts

Sugars were analyzed with a Waters 2695 Alliance Separation Module with temperature-controlled column chamber and autosampler (Waters Co., Milford, MA, USA). The column used was 250×4.1 mm Hamilton RCX-10 anion exchange HPLC column (Hamilton Co., Reno, Nevada, USA). Samples were injected in a volume of 20 µl and the mobile phase was 150 mM NaOH running at a speed of 1 ml min-1. The detector was ESA Coulochem II electrochemical detector with gold electrode (ESA Biosciences Inc., Chelmsford, MA, USA). The peaks of sugars were identified by comparing their retention times with those of standard sugars with Millenium32 data-analysis software from Waters Co. (Milford, MA, USA). Internal standards to insure recovery (>90%) were added to guard-cell extracts before injection. The detection limit (p<0.001) of sugars with this method was 4 pmol (2×10-7 M, 20-µl injection). Standard sucrose (Fisher Chemicals, Fairlawn, NJ, USA), and glucose, fructose, stachyose, raffinose and galactinol (Sigma-Aldrich Co., St. Louis, MO, USA) were obtained commercially.

Results

Lowered Photosynthesis Rate without Altering Transpiration Rate Was Effected by

Shading Plus Decreased Ambient CO2 Concentration

Bulk-leaf apoplastic sucrose is the intermediate pool between sucrose produced by recent photosynthesis in the mesophyll and that in the guard-cell apoplast in broad bean, a typical apoplastic phloem loader (Lu et al., 1997). We (Lu et al., 1997) hypothesized that a diminished bulk-leaf apoplastic sucrose pool by lowered photosynthesis would limit the size of the guard-cell apoplastic sucrose pool. Here, shading was used to decrease leaf photosynthesis, which did lower the bulk-leaf apoplastic sucrose pool, as expected (e.g. Walsh et al., 1998). In addition, to interpret the results in the context of our overall integrative hypothesis (see Introduction), it was necessary to maintain stomatal aperture size in order to distinguish the effects of lowering the leaf photosynthesis rate from

lowering the leaf transpiration rate. Thus, after 5 h of shading (PFD=40% of control) leaves on intact plants, the stomatal aperture size decreased from 10.2±0.1 µm to

7.8±0.1 µm (p<0.001) (column 2, Fig. 10). Compensatorily, decreased CO2 concentration eliminated this effect (column 3, Fig. 10): with shading plus lowered ambient CO2 concentration, stomatal aperture size was essentially the same as that of the control (p=0.49, Fig. 10), whereas leaf photosynthesis rate decreased (p<0.001) more than five -2 -1 -2 -1 fold (Fig. 11) from 9.8±0.6 µmol CO2 m s to 1.5±0.2 µmol CO2 m s .

Fig. 10. Stomatal aperture size (mean±SE, n=180 stomata from six plants, two growth lots) of control, shaded and shaded/low-CO2 broad-bean plants at 1100 h. Non-control plants were shaded from the onset of light period at 0600 h. PAR at experimental leaves of control plants was 450–500 µmol m-2 s-1 and of shaded plants, 120–150 µmol m-2 s-1. -1 Growth cabinet CO2 concentration under low CO2 condition was 350–360 µmol mol (100 µmol mol-1 lower than that of controls).

Fig. 11. Photosynthesis rate (mean±SE, n=6 plants, two growth lots) of control and shaded/low-CO2 broad-bean plants at 1100 h. For other details, see Fig. 10.

Bulk-leaf Apoplastic Sucrose, Glucose and Fructose Concentrations were Lower in Shaded than in Control Plants

The effect of diminished leaf photosynthesis rate under steady-state conductance on bulk-leaf apoplastic sugar concentration was assessed as a prerequisite to testing the relationship between photosynthesis rate and sugar accumulation at the guard-cell apoplast. Glucose and fructose were characterized in addition to sucrose because broad-bean bulk-leaf apoplastic sap contains glucose and fructose in addition to the dominant sugar sucrose (Delrot et al., 1983; Lohaus et al., 2001). As shown in Fig. 12, the average bulk-leaf apoplastic sucrose concentration was

~1.3 mM in control plants and ~0.4 mM in shaded/low-CO2 plants (p=0.002). Glucose and fructose concentrations in the bulk-leaf apoplast were less than half (p=0.009 for glucose and p=0.02 for fructose) that of sucrose in control plants, and shading/low-CO2 caused these sugars to decrease in concentration also (p=0.03 for glucose and p=0.02 for fructose, Fig. 12).

Fig. 12. Bulk-leaf apoplastic sucrose, glucose, and fructose concentrations (mean±SE, n=6 plants, two growth lots) of control and shaded/low-CO2 broad-bean plants at 1100 h, 5 h from the onset of light period at 0600 h. For other details, see Fig. 10.

Lower Leaf Photosynthesis Rate Resulted in Lower Guard-cell Apoplastic Sucrose Content

As shown above (Fig. 12), shading caused a significant decrease in bulk-leaf apoplastic sugar concentrations. However, leaf transpiration rate was not altered because the stomatal aperture size (Fig. 10) and vapor pressure difference were constant. Here, guard-cell apoplastic sugar contents of control and shaded plants were measured to assess the effects of lowered photosynthesis rate on sugar accumulation at the guard-cell apoplast. Guard-cell apoplastic and guard-cell symplastic sucrose, glucose, and fructose of control broad-bean plants were assayed by HPLC (Fig. 13). Guard-cell apoplastic glucose (0.11±0.15 pmol pair-1, equivalent to ~26 mM) and fructose (0.08±0.17 pmol pair-1, equivalent to ~20 mM) contents were much lower (p=0.009 for glucose and p=0.015 for fructose) than that of sucrose (0.63±0.16 pmol pair-1) (Fig. 13, HPLC method) of control plants. In summary, the concentrations of hexoses were low and unimportant osmotically in control plants. Therefore, guard-cell apoplastic glucose and fructose contents were not

determined in shaded/low-CO2 plants where their concentrations would be expected to be even lower. Guard-cell apoplastic and guard-cell symplastic sucrose contents of both control and shaded/low-CO2 plants were assayed with quantitative histochemical methods. After shading, guard-cell apoplastic sucrose content (Fig. 13) decreased almost 4-fold, from 0.75±0.1 to 0.16±0.08 pmol pair-1 (p<0.001), which correlates with the decrease from 1.3 mM to 0.4 mM (P=0.002 Fig. 12) in the bulk-leaf apoplastic sucrose concentration. In contrast, guard-cell symplastic sucrose content was the same in control and shaded/low-CO2 plants (p=0.16) (histochemical method, Fig. 13). Results from quantitative histochemistry and HPLC agreed. With histochemistry, the sucrose content of the guard-cell symplast was 1.09±0.07 pmol guard-cell pair-1 (n=43) and that of the apoplast was 0.75±0.1 pmol guard-cell pair-1 (n=47) of control plants. These values are almost identical to values obtained with HPLC from extracts of pooled guard cells (1.26±0.2 and 0.63±0.2 pmol guard-cell pair-1 for the symplastic and apoplastic compartments, respectively.). A linear relationship (Fig. 14, R2=0.80) between bulk-leaf apoplastic sucrose concentrations and corresponding guard-cell apoplastic sucrose contents exists. The highest bulk-leaf apoplastic sucrose concentration was ~2 mM, similar to that of Outlaw De Vlieghere-He (2001) and Lu et al. (1997). The highest guard-cell apoplastic sucrose content (control plant) was ~0.9 pmol guard-cell pair-1, which is also in the range of previous reports (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001). The guard-cell apoplastic sucrose contents of shaded/low-CO2 plants (0.07-0.25 pmol guard-cell pair-1) are similar to those of broad-bean plants with closed stomata (0.22 pmol guard-cell pair-1, Lu et al., 1995), or with low leaf transpiration rate (0.2 pmol guard-cell pair-1, Outlaw and De Vlieghere-He, 2001).

Fig. 13. Guard-cell symplastic and guard-cell apoplastic sugar content of control or shaded/low-CO2 broad-bean plants with either HPLC (mean±SE, n=3 measurements, each contained 180 guard-cell pairs) or histochemical method (mean±SE, n=43 guard-cell pairs from six plants) at 1100 h. 5 h from the onset of light period at 0600 h. For other details, see Fig. 10.

Fig. 14. The relationship between bulk-leaf apoplastic sucrose concentration (Fig. 12) and guard-cell apoplastic sucrose content (histochemical method, Fig. 13). Samples were from 11 plants; five were control plants, and six were shaded/low-CO2 plants. There was no statistical difference (p=0.49) in stomatal aperture size between control and shaded/low-CO2 plants. For other details, see Fig. 13.

Discussion

In the guard-cell symplast, sucrose is an important osmoticum that supplements or replaces potassium salts and is thus involved in sustaining stomatal opening (e.g. Tallman and Zeiger, 1988; Lu et al., 1995; Talbott and Zeiger, 1998). In the guard-cell apoplast, enough sucrose may accumulate (Lu et al., 1995) during the photoperiod to diminish stomatal aperture size osmotically by ~3 µm (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001). The hypothetical mechanism for guard-cell apoplastic sucrose accumulation in apoplastic phloem loaders (Outlaw, 2003) is outlined in the following. Photoassimilated sucrose is released to the bulk-leaf apoplast in the vicinity of the phloem. The transpiration stream sweeps some of this sucrose towards stomata. Water evaporation from the guard-cell apoplast deposits the sucrose there, a mechanism modeled with petiolar-fed plasma membrane impermeant 14C-mannitol (Ewert et al., 2000). High concentration of sucrose in the guard-cell apoplast was hypothesized to be a signal that integrates transpiration rate, leaf photosynthesis rate, and phloem translocation rate (Lu et al., 1997) in the fine regulation of gas exchange. Of these three hypothetical factors, the role of transpiration rate was proven earlier (Outlaw and De Vlieghere-He, 2001). Here, we report a direct robust correlation (Fig. 14) between the concentration of sucrose in the bulk-leaf apoplast and the concentration of sucrose in the guard-cell apoplast at constant physiological transpiration rates. Lowering the photosynthesis rate lowered the bulk-leaf apoplastic sucrose concentration (Fig. 12) and diminishing translocation increases the bulk-leaf apoplastic sucrose concentration (e.g. Krapp et al., 1991; Gottwald et al., 2000). Therefore, the importance of all three elements of the integrative hypothesis has now been established empirically. Isolation of the independent relationship of bulk-leaf apoplastic sucrose concentration to guard-cell apoplastic sucrose concentration required experimentation at constant transpiration and under physiological conditions. Transpirational flux is the product of the driving force for water effusion and conductance. For comparisons among equivalent leaves positioned similarly in a closed cabinet, the relative driving forces simplify to VPDs and the monitored parameter was ambient RH at constant temperature. -2 -1 Under control conditions, the photosynthesis rate was 9.8±0.6 µmol CO2 m s (Fig. 11),

which is typical for broad-bean plants (e.g. Hariadi and Shabala, 2004). Simply lowering the light intensity as a means of lowering the photosynthesis rate (and hence bulk-leaf apoplastic sucrose concentration) had the predicted effect on conductance (stomatal aperture size) (Fig. 10, e.g. Stitt et al., 1991; Whitehead and Teskey, 1995; Peek et al., 2004). In preliminary experiments (not shown), we empirically established the means to restore conductance to the control value (Fig. 10) by a small modulation of CO2 concentration, a well-known stomatal effector (Willmer, 1988; Assmann, 1999). Therefore, the bulk-leaf apoplastic sucrose concentration was lowered (from an average of

1.3±0.2 mM to 0.4±0.01 mM, Fig. 12) by a combination of shading and lowered CO2 concentration at a constant, typical transpiration rate for this species (Ewert et al., 2000) permitting an effect on sucrose accumulation around guard cells independent of transpiration to be assessed. The effect was striking: in control plants, the average guard-cell apoplastic sucrose content was 0.75±0.1 pmol guard-cell pair-1 (similar to our earlier work (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001)) whereas the -1 correlate value in shaded/low-CO2-treated plants was 0.16±0.08 pmol guard-cell pair (similar to that at the onset of illumination (Lu et al., 1995) and that of low-transpiration plants (Outlaw and De Vlieghere-He, 2001)). The experimental design (collection of bulk-leaf apoplastic sap from one leaflet and guard cells from the other leaflet of the pair) permitted pairwise comparisons for an even stronger conclusion. Thus, over a ~4-fold range of leaf apoplastic sucrose concentrations, there was a corresponding (R2=0.80) ~3.6-fold range in guard-cell apoplastic sucrose content (Fig. 14). As a means of providing perspective, here and elsewhere (Outlaw and De Vlieghere-He, 2001), sucrose concentrations have been calculated from the sucrose contents and a conversion factor, the aqueous volume of the guard-cell apoplast. Note that these calculations provide only imprecise estimates. Although the guard-cell wall volume of embedded tissue of broad bean has been meticulously quantified (Ewert et al., 2000), how much of the wall volume is available to bulk solution and how much is occupied by structural elements is uncertain. Our conversion factor apportions equal volumes to these two elements (see Fry, 1988), implying that the calculated concentrations are potentially overestimated by up to 2-fold or underestimated. Neither of these potentials for error

undermines the overall conclusions (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001) The minimum average calculated concentration of sucrose in the guard-cell apoplast (Fig. 13) was 38±19 mM, or 100-fold that of the bulk-leaf apoplast during active transpiration. This minimum guard-cell apoplastic sucrose concentration holds for plants at the end of the normal dark period (Lu et al., 1995) and for plants under minimum transpiration conditions (Outlaw and De Vlieghere-He, 2001). Even more confounding is the sucrose content—0.47±0.1 pmol guard-cell pair-1 of the apoplast of guard cells of excised leaves maintained at 100% RH and zero CO2 concentration (Y. Kang, T. Jiang, and W. H. Outlaw Jr., unpublished). Moreover, in none of these cases can the high minimum sucrose concentration be explained away by sampling, assay or biological variation. Because the bulk-leaf apoplast turns over about every 20 min (Ewert et al., 2000) during normal transpiration and the half-time for the guard-cell apoplastic sucrose pool is less than 40 min (Lu et al., 1997), the minimum high sucrose concentration in the guard-cell apoplast apparently cannot be explained for all these different conditions by simple diffusional limitations either. A second unexplained phenomenon is the large increase in the guard-cell symplastic sucrose concentration that occurs in response to an abrupt decrease in transpiration (Outlaw and De Vlieghere-He, 2001). A third phenomenon that likewise defies explanation is the osmotically relevant increase in guard-cell symplastic sucrose concentration toward the end of the photoperiod (Talbott and Zeiger, 1996, 1998). Kinetic (Reddy and Rama Das, 1986; Outlaw, 1995; Lu et al., 1997) and molecular bases (Kopka et al., 1997) for guard-cell sucrose transport are known, and the existence and potential importance for regulation illustrated long ago (Outlaw, 1995), but signaling and regulation have only begun to be studied (Kopka et al., 1997; Stadler et al., 2003; Liang et al., 2005). In conclusion, the physical model that we proposed (Lu et al., 1997) apparently overrides other underlying mechanisms, which undoubtedly serve other functions, such as homeostasis in sucrose-regulated gene expression (reviewed in Smeekens, 2000; Rolland et al., 2002), and merit study. Apoplastic phloem loading is the advanced condition (Gamalei, 1991), and the integrative theory described in this paper provides evolutionary pressure for its selection because gas exchange is finely regulated by current physiological parameters. That is,

when transpiration rate is too high, sucrose accumulates in the guard-cell apoplast and modulates aperture size. Similarly, when the bulk-leaf apoplastic sucrose concentration is too high—indicating that the release of sucrose into the apoplast exceeds retrieval— stomatal aperture size diminishes, conserving water as the demand for CO2 is lessened. However, this mechanism might dampen stomatal responses, too, which benefits a plant by maintaining sufficient conductance in a fluctuating light environment. Thus, when a leaf is temporarily shaded, the absence of light and the rise of Ci tend to decrease conductance, but the loss of sucrose from the guard-cell wall tends to increase conductance. Kinetic studies on the rate of sucrose dissipation in an ecological context will be needed to assess this dampening hypothesis. Albeit at lower concentrations than that of sucrose, glucose and fructose are still major solutes in the bulk-leaf apoplast of broad bean (Fig. 12, Delrot et al., 1983; Lohaus et al., 2001) and both decreased there, though proportionately somewhat less than sucrose, in response to the shading/low-CO2 treatment (Fig. 12). However, glucose and fructose were not detected in the guard-cell apoplast and were not major osmolytes (each <30 mM) there under control conditions. Therefore, no attempt was made to measure these hexoses under the lowered photosynthesis rate. The low hexose concentration in the guard-cell apoplast is consistent with the absence of invertase there (Hite et al., 1993). Whereas sucrose in the guard-cell apoplast is directly a product of recent photosynthesis in the mesophyll (Lu et al., 1997), the data are consistent with the hexoses in the bulk-leaf apoplast, particularly fructose, having a different origin (cf. Fig. 12, Fig. 13) such as the transpiration stream (Minchin and McNaughton, 1987; Chikov and Bakirova, 2004; Chikov et al., 2005), perhaps as a result of phloem leakage (Ayre et al., 2003). However, sound conclusions require further study, which is merited because hexoses, like sucrose, regulate gene expression (reviewed in Koch, 1996; Pego et al., 2000). A non-osmotic role for hexoses in the guard-cell apoplast cannot be excluded because of the imprecision of quantifying a low-concentration guard-cell apoplastic solute when it is present in relatively large and variable amounts in the guard-cell symplast as discussed (Y. Kang, W. H. Outlaw Jr. and G. B. Fiore, unpublished). Specifically, our conservative means used to express guard-cell apoplastic solute contents exaggerates the

error as it incorporates the error associated with corresponding assays of that solute in the guard-cell symplast.

OVERALL SUMMARY

In the guard-cell symplast, sucrose is an important osmoticum that supplements or replaces potassium and is thus involved in sustaining stomatal opening (e.g. Tallman and Zeiger, 1988; Lu et al., 1995; Talbott and Zeiger, 1998). In the guard-cell apoplast, sucrose plays an osmotic role in diminishing stomatal aperture size during high transpiration (Lu et al., 1995, 1997; Outlaw and De Vlieghere-He, 2001). Guard-cell apoplastic sucrose has been hypothesized (Lu et al., 1997) to be a signal that integrates the rates of transpiration, of photosynthesis, and of translocation in apoplastic phloem loaders. Presumably, this process is also one of the mechanisms for apoplastic phloem loaders to respond to high transpiration rate and avoid excessive water loss. Of the three factors that affect guard-cell apoplastic sucrose accumulation, leaf transpiration rate has been proven to be a positive factor (Outlaw and De Vlieghere-He, 2001), which is in consistent with the above hypothesis. In the present research, we first explored the influence of altering phloem translocation on the guard-cell apoplastic photosynthates accumulation. As an extreme example, we selected a typical symplastic phloem loader plant, dwarf basil, which lacks an apoplastic step in phloem loading, thus it has much lower bulk-leaf apoplastic photosynthate concentrations compared with typical apoplastic phloem loaders (Tetlow and Farrar, 1993; López-Millán et al., 2000; Lohaus et al., 2001). Dwarf basil belongs to the Lamiaceae family; plants in this family are believed to be symplastic phloem loaders (Gamalei, 1991) and have been used widely in previous studies (e.g. Flora and Madore, 1996; van Bel et al., 1996; Büchi et al., 1998). The analysis of the dwarf basil phloem exudates confirmed its symplastic phloem loading pattern with 76% (molar basis) of RSOs in the phloem exudates. The diurnal leaf conductance, stomatal aperture, guard-cell starch content, and guard-cell potassium content changes were typical and assured dwarf basil’s conformity to the normal plants. Stachyose, raffinose and galactinol were found in the dwarf basil leaf apoplast in addition to sucrose, glucose and fructose. Overall, dwarf basil

bulk-leaf apoplastic photosynthates concentration was low, total <0.3 mM and sucrose is the lowest at <0.03 mM. Finally, the essential parts of results, the guard-cell apoplastic photosynthates were characterized. RSOs (stachyose plus raffinose), the dominant sugars in the bulk-leaf apoplast, in the guard-cell apoplast were not detectable, i.e. <10 mM (HPLC of guard-cell extracts). The guard-cell apoplastic sucrose, glucose, or fructose was also not detectable (p>0.2). Thus, guard-cell apoplastic photosynthate concentration was significantly lower than that in apoplastic phloem loaders and exerted no biologically significant osmotic effect on guard cells. Therefore, typical symplastic phloem loaders with low bulk-leaf apoplastic photosynthate concentration lack or have diminished stomatal osmotic regulation by bulk-leaf apoplastic photosynthate. In the second part of research, we tested the effect of leaf photosynthesis rate on guard-cell apoplastic sugar accumulation in apoplastic phloem loader broad bean. After shading of the leaf while maintaining constant leaf transpiration through lowering ambient

CO2 concentration, bulk-leaf apoplastic sucrose concentration decreased to one third of the control value, whereas the guard-cell apoplastic sucrose concentration decreased to 38 mM, less than one-fourth of the control value. In addition, shading also caused about two-fold concentration decrease of glucose and fructose in the bulk-leaf apoplast. Glucose and fructose concentration in the guard-cell apoplast were low under normal conditions, i.e. <30 mM, thus their contents in the guard-cell apoplast under shaded conditions were not quantified. These results indicate that for apoplastic phloem loaders, when the bulk-leaf apoplastic sucrose concentration is reduced, guard-cell apoplastic sucrose may not exert physiologically significant osmotic effects on guard cells. In summary, the data presented here support the overall hypothesis that guard-cell apoplastic sucrose is a signal that integrates the information from the transpiration rate, leaf photosynthesis rate and phloem translocation rate (Lu et al., 1997). Previous work (Outlaw and De Vlieghere-He, 2001) proved the role of leaf transpiration in positively affecting guard-cell apoplastic sucrose accumulation. Here, leaf photosynthesis was also shown to positively affect guard-cell apoplastic sucrose accumulation in broad bean. In addition, for dwarf basil, a typical symplastic phloem loading species with low bulk-leaf apoplastic photosynthate concentration, guard-cell apoplastic photosynthate accumulation is much lower than that in broad bean, which is again consistent with the hypothesis.

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BIOGRAPHICAL SKETCH

DATE AND PLACE OF BIRTH December 12, 1975 Qufu, Shandong Province, P.R. China

EDUCATION Ph.D. Candidate (Biological Science) 1999 – present Department of Biological Science, Florida State University (FSU), Tallahassee Major Advisor: Dr. William H. Outlaw Jr.

M.S. (Plant Physiology) 1996 – 1999 College of Biological Science, China Agricultural University (CAU), Beijing, China Major Advisor: Dr. Xuechen Wang

B.S. (Crop Science) 1992 – 1996 Department of Agronomy, Laiyang Agricultural College (LAC), Laiyang, China

PUBLICATIONS Kang Y, Outlaw WH Jr Guard-cell apoplastic sucrose concentration―a link between photosynthesis and stomatal aperture size. In preparation. Kang Y, Outlaw WH Jr Guard-cell apoplastic photosynthate accumulation is limited or absent in a symplastic loading plant Ocimum basilium. In preparation. Outlaw WH Jr, Jiang T, Pearson M, Kang Y Apoplastic sucrose―necessary for typical diurnal fluctuation of guard-cell starch content. Research completed, manuscript in preparation. Huang RF, Zhu MJ, Kang Y, Chen J, Wang XC (2002) Identification of plasma membrane aquaporin in guard cells of Vicia faba and its role in stomatal movement. Acta Botanica Sinica 44: 42-48 Zhu MJ, Kang Y, Chen J, Wang XC (1999) Plant aquaporins and their regulation.

Chinese Bulletin of Botany 16: 44-50 Wang XC, Zhu MJ, Kang Y, Chen J (1999) Antibody preparation of the plasma membrane aquaporin and its localization in the guard cells of Vicia faba L. Academic Periodical Abstracts of China 6: 1234-1235

CONFERENCE PRESENTATIONS Kang Y, Outlaw WH Jr, Fiore D (2005) Guard-cell apoplastic photosynthate accumulation is limited or absent in a model symplastic phloem loading species. University of Florida Plant Molecular and Cellular Biology workshop, May 2005 Kang Y, Jiang T, Pearson M, Outlaw WH Jr (2004) Leaf transpiration rate affects guard-cell carbon metabolism. ASPB (Southern Section) annual meeting, March 2004

TEACHING EXPERIENCE 1999 – 2004 Teaching Assistant, Department of Biological Science, FSU • General Biology Lab for Majors (BSC 2010L), four semesters • General Biology Lab for Majors (BSC 2010L), Senior Teaching Assistant, three semesters • Botany Lab (BOT 3015L), three semesters • Experimental Biology Lab (BSC 3402L), one semester • General Biology Lab for non-majors (BSC 1005L), two semesters 1997 Teaching Assistant, Department of Biological Science, CAU • Plant Physiology Lab, one semester

HONORS • “Notable Mentions” presentation award, University of Florida Plant Molecular and Cellular Biology workshop, 2005, Florida • University Scholarship for four consecutive years, 1992 – 1996, LAC • University Three-Good Student for four consecutive years (equivalent to Honors Student), 1992 – 1996, LAC • Selected “Model Student", 1994, LAC

• First Prize in the LAC 4th English Competition, 1993, LAC

AFFILATION Member of the American Society of Plant Biologists (ASPB)