A Companion to Plant Physiology, Fifth Edition by Lincoln Taiz and Eduardo Zeiger
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Essay 18.1222

Guard Cell Photosynthesis

Tracy Lawson and James I. L. Morison, Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK

September, 2010

In the majority of species examined, guard cells contain well-developed chloroplasts (Figure 1a), unlike the other epidermal cells from which they are formed. The role of these highly conserved chloroplasts in stomatal function has long been controversial (Zeiger et al. 2002). The four primary ways that guard cell chloroplasts could contribute to stomatal opening are:

  1. Photosynthetic production of ATP and reductants
  2. Production of osmotically active sugars by photosynthetic carbon assimilation (Talbott and Zeiger, 1998)
  3. Carotenoid xanthophyll functioning as the low intensity blue-light photoreceptor
  4. They could store starch, either from carbon assimilated in the guard cell chloroplasts or imported from the mesophyll, which is then available to synthesize malate as a counter ion to K+. One of the key features of the functioning of guard cell chloroplasts is that they accumulate starch in the dark, and hydrolyze it in the light—the opposite time course to that of mesophyll chloroplasts, suggesting very different carbon assimilation mechanisms or controls.
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   (Click image to enlarge.)

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Figure 1   (a) One Commelina communis stoma, with guard cell chloroplasts showing red autofluorescence; photograph taken with an epifluorescence microscope, superimposed on a transmitted light photograph. (b) Reflected light image of a similar stoma taken using the high resolution imaging system, in visible light. (c) Corresponding image of steady state fluorescence (F′). (d) Isolation of guard cell chloroplasts from the F′ image created using the editing software developed for the imager. (e) Image of Fq′/Fm′ from guard cell chloroplasts built using only F′ and Fm′ images; color scale is from blue (highest), through green, yellow and red (lowest). (f) The effect of changing VPD on stomatal aperture (solid circles) and Fq′/Fm′ from mesophyll (solid triangles) and guard cells (solid squares) in a Commelina communis leaf at a CO2 concentration of 180 µmol mol-1. ↑ indicates the time when VPD was increased from 1.0 to 1.5 kPa and ↓ when VPD was decreased back to 1.0 kPa. (Click image to enlarge.)

This essay briefly reviews the characteristics of guard cell chloroplasts and discusses their possible photosynthetic function. In particular, we illustrate how recent developments in non-invasive methods of assessing photosynthesis using quantitative chlorophyll fluorescence are now revealing guard cell chloroplast photosynthetic function within the intact leaf (Lawson et al. 2002).

Guard Cell Chloroplasts and Photosynthetic Activity

Typically, there are only 8–15 chloroplasts per guard cell, compared to 30–70 in a palisade mesophyll cell. They are generally smaller, less well developed, and with less granal stacking than those in mesophyll cells, although these features vary across the plant families and divisions (Willmer and Fricker 1996). Although there is and less chlorophyll per cell in guard cells than in mesophyll cells (25–100 fold lower), the small volume of the guard cell (approximately 10 fold smaller) means that the chloroplasts could provide a significant energy source for these cells. The pigment composition of chlorophyll a and b and carotenoids is similar to mesophyll cells, although some studies have found differences between guard cell chloroplasts from upper and lower leaf surfaces, mimicking some of the differences found between pigment composition of sun and shade leaves. Photosynthetic electron transport, oxygen evolution, and photophosphorylation have been measured in several guard cell chloroplast studies, and the rates of photophosphorylation, on a per chlorophyll basis, can be from 70%–90% of that of mesophyll chloroplasts. Recently, it has been demonstrated that ATP produced by guard cell chloroplast photophosphorylation is used by the plasma membrane H+–ATPase and consequently is important for the ion transport that determines aperture (Tominaga et al. 2001).

Although rubisco and other Calvin cycle enzymes were found in guard cells (e.g. through biochemical assays, single-cell analyses and immunological techniques), reported amounts and activities of rubisco vary widely so the extent of Calvin cycle activity within guard cells and its role in stomatal opening is still unresolved. However, there are several lines of evidence supporting significant Calvin cycle activity: guard cell sucrose synthesis is sensitive to DCMU, an inhibitor of PSII electron transport; radiolabeled CO2 is incorporated in phosphorylated carbon compounds in illuminated guard cell protoplasts; and chlorophyll fluorescent transients in guard cell chloroplasts are similar to those of mesophyll chloroplasts. The past disagreements over the role of guard cell chloroplast photosynthetic activity have been partly due to the early difficulties in working with guard cells that form only a 5% fraction of the epidermal tissue, and because some biochemical assays have been prone to contamination from the mesophyll. The disagreements may also be a result of differences in plant material, as half a dozen species are widely used in stomatal physiological studies, from very different taxa, with different stomatal anatomy and often grown in different conditions. As Zeiger et al. (2002) recently emphasized, guard cell chloroplasts show remarkable functional plasticity, which might also account for the diversity of results. Furthermore, even when stomata are isolated in epidermal peels or as protoplasts there are difficulties in interpreting results as they are removed from the influence of the mesophyll. Guard cells in white areas of variegated plants have also been used, as they usually have chloroplasts, but these are also in unusual conditions with a non-photosynthesizing mesophyll, and hence very high internal CO2 concentrations and different light penetration.

Guard Cell Chlorophyll Fluorescence Studies

Chlorophyll fluorescence is a potent technique to elucidate photosynthetic metabolism in guard cells. Until recently, the majority of chlorophyll fluorescence measurements from guard cells were restricted to variegated leaves, epidermal peels, or guard cell protoplasts and had mainly studied fluorescence induction curves, or responses of the steady-state fluorescence signal (F′) alone. However, only limited information can be gained from steady state fluorescence due to possible changes in photochemical and non-photochemical fluorescence quenching. Recent advances have allowed the more powerful saturation light pulse methods of fluorescence quenching to be measured on single cells using microscopes, either with pulse amplitude modulation (PAM) fluorimetry (Goh et al. 1999) or using a high resolution imaging system (Lawson et al. 2002), capable of resolving details of fluorescence yield within chloroplasts (Figure 1c,d). The saturating pulse of light (several thousand µmol m-2 s-1) is applied during steady state fluorescence, this transiently shuts all PSII reaction centers and gives maximum fluorescence (Fm′), so that the quantum efficiency for PSII photochemistry (“photosynthetic efficiency”) can be calculated as Fq′/Fm′ = (Fm′-F′)/Fm′ (Figure 1e). Furthermore, the photosynthetic electron transport rate can then be estimated as the product of Fq′/Fm′ and light absorption.

Chlorophyll fluorescence is a potent technique to elucidate photosynthetic metabolism in guard cells. Until recently, the majority of chlorophyll fluorescence measurements from guard cells were restricted to variegated leaves, epidermal peels, or guard cell protoplasts and had mainly studied fluorescence induction curves, or responses of the steady-state fluorescence signal (F') alone. However, only limited information can be gained from steady state fluorescence due to possible changes in photochemical and non-photochemical fluorescence quenching. Recent advances have allowed the more powerful saturation light pulse methods of fluorescence quenching to be measured on single cells using microscopes, either with pulse amplitude modulation (PAM) fluorimetry (Goh et al. 1999) or using a high resolution imaging system (Lawson et al. 2002), capable of resolving details of fluorescence yield within chloroplasts (Figure 1c,d). The saturating pulse of light (several thousand µmol m-2 s-1) is applied during steady state fluorescence, this transiently shuts all PSII reaction centers and gives maximum fluorescence (Fm’), so that the quantum efficiency for PSII photochemistry (“photosynthetic efficiency”) can be calculated as Fq'/Fm' = (Fm'-F')/Fm' (Figure 1e). Furthermore, the photosynthetic electron transport rate can then be estimated as the product of Fq'/Fm' and light absorption.

Uniquely, the imaging system can be used on guard cells in intact, photosynthesizing leaves, where ambient lighting, CO2, O2, and humidity conditions can be altered, and where aperture changes can be measured (Lawson et al. 2002). For example (Figure 1f), when a leaf of Commelina communis is subjected to a step decrease in humidity, after a transient aperture increase due to changes in relative turgor pressure, aperture decreases sharply, which restricts the CO2 supply to the mesophyll (and to the guard cells). Therefore, Fq′/Fm′ declines in both guard and mesophyll alike, indicating a basic similarity in chloroplast function. In other experiments we measured the change of photosynthetic efficiency with light intensity (Figure 2a, and the pattern was essentially the same in both guard cell chloroplasts and mesophyll. In addition, it was clear that the major change in Fq′/Fm′ as light intensity increased was due to photochemical quenching in both cell types. Moreover, the guard cell photosynthetic efficiency was approximately 80% of that of the mesophyll cells across a wide range of light intensities (Figure 2a inset). Similar results were found in guard cell protoplasts of Vicia faba (Goh et al. 1999), confirming that photosynthetic electron transport rates in guard cell chloroplasts are substantial.

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Figure 2   (a) Response of Fq′/Fm′ of a variegated Tradescantia leaf to PPFD. Data were obtained from guard cells in white (open circles) areas of the leaf and from mesophyll (solid squares) and guard cells (solid circles). Measurements were made at a CO2 of 360 µmol mol-1. The inset in (a) shows the relationship between Fq′/Fm′ for mesophyll and guard cells in green areas over the range of PPFDs (the line is y = 0.76x; r2 = 0.92) (b) Response of Fq′/Fm′ of mesophyll (solid and open squares) and guard cells (solid and open circles) to increasing CO2 in the green areas of a Tradescantia leaf in an atmosphere containing 2% (open squares and circles) or 21% (solid squares and circles) O2. Measurements were made at a PPFD of 215 µmol m-2 s-1. (Click image to enlarge.)

The imaging system work with intact leaves also showed that low O2 concentration (2%) decreased Fq′/Fm′ in both guard and mesophyll cells of C. communis at low CO2 concentrations (<600 µmol mol-1, Figure 2b, Lawson et al. 2002). However, when CO2 supply was high, then changing the O2 concentration had little effect on Fq′/Fm′. The simplest explanation for this interaction is that when both O2 and CO2 are low, this limits the carboxylase and oxygenase activity of rubisco and reduces the sink for the products of photosynthetic electron transport, further reducing photosynthetic electron transport efficiency. Therefore, the interaction indicates that photorespiration occurs and rubisco is a major sink for ATP and NADPH produced through photosynthetic electron transport in the guard cell chloroplasts just as in the mesophyll chloroplasts. We have found similar responses of guard cell chloroplast Fq′/Fm′ to light, CO2 and O2 responses in several other species (Nicotiana tabacum, Vicia faba, Tradescantia albiflora), from a range of families, sub-classes and divisions, including a fern (Polypodium vulgare), suggesting that it is a general feature. Interestingly, the guard cell chloroplast photosynthetic efficiency of a C4 species, Amaranthus caudatus, showed this same CO2 and O2 response, while the mesophyll chloroplasts (where the primary fixation is through PEP carboxylase) did not, confirming recent immunological isolation of rubisco in guard cells in this genus.

These various chlorophyll fluorescence studies have been important in showing that guard cell chloroplasts have substantial photosynthetic activity, and point to a functioning Calvin cycle. However, they have not resolved the question of how much this contributes to the control of stomatal opening. While isolated guard cell chloroplasts can synthesize sucrose, some measurements suggest that the rate of synthesis is too low to account for the amounts of sucrose that accumulate in open stomata. Certainly, in intact photosynthesizing leaves sucrose accumulates in the guard cell apoplast (Outlaw and De Vlieghere-He 2001), and it could be that this mesophyll-derived sucrose is the source for guard cells. Such an external sucrose supply might also provide a signal that links stomatal aperture to mesophyll photosynthesis. Possibly, the high availability of sucrose could suppress carbon assimilation in guard cells (through sugar signalling and the effect this has on gene expression, which can reduce Calvin cycle enzymes). Secondly, the shape of the CO2 response of guard cell chloroplast Fq′/Fm′ evident in Figure 2b, suggests the possibility that photosynthesis is involved in CO2 sensing in guard cells. The range of CO2 concentrations, and the high sensitivity of the response are very similar to the responses of stomatal aperture or conductance to intercellular CO2, except that aperture reduces when CO2 increases. In mesophyll cells, such a CO2 response of photosynthetic efficiency (and hence photosynthetic electron transport, as these measurements were done at constant light intensity) would obviously be linked to increased CO2 fixation, leading to increased triose phosphate, and either starch or sucrose. In guard cells, if high CO2 similarly led to increased starch or sucrose, then the osmotic effect of the sucrose would increase aperture, rather than reduce it. This provides a simple reminder of the way that regulation of photosynthetic carbon assimilation in guard cells is different from that in the mesophyll. Use of antisense techniques and guard cell specific promoters to modify guard cell chloroplast carbon assimilation enzyme activities, coupled with in situ measurement of photosynthetic efficiency and stomatal function will be key ways to answer these questions.

References

Goh, C-H., Schreiber U., Hedrich R. (1999) New approach of monitoring changes in chlorophyll a fluorescence of single guard cells and protoplasts in response to physiological stimuli. Plant, Cell and Environment 22: 1057–1070.

Lawson, T., Oxborough, K., Morison, J. I. L., Baker, N. R. (2002) Responses of photosynthetic electron transport in stomatal guard cells and mesophyll cells in intact leaves to light, CO2 and humidity. Plant Physiology 128: 52–62.

Outlaw, W. H. Jr., De Vlieghere-Xe, X. (2001) Transpiration rate—an important factor controlling the sucrose content of the guard cell apoplast of Vicia faba L. Plant Physiology 126: 1716–1724.

Talbott, L. D., and Zeiger, E. (1998) The role of sucrose in guard cell osmoregulation. Journal of Experimental Botany 49: 329–337.

Tominaga, M., Kinoshita, T., and Shimazaki, K. (2001) Guard cell chloroplasts provide ATP required for H+ pumping in the plasma membrane and stomatal opening. Plant and Cell Physiology 42: 795–802.

Willmer, C., Fricker, M. (1996) Stomata, Second Edition. Chapman and Hall, London, UK.

Zeiger, E., Talbott, L. D., Frechilla, S., Srivastava, A., and Zhu, J. (2002) The guard cell chloroplast: a perspective for the twenty-first century. New Phytologist 153: 415–424.

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