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Keywords:

  • stomata;
  • plant physiology;
  • gas exchange;
  • guard cell biology;
  • evolution;
  • climate change modelling;
  • intramolecular signalling processes

Stomata are central to the physiology of land plants because environmentally induced changes in their development and movements have profound effects on the gas exchange between the atmosphere and the leaf. Recently, considerable advances have been made in the understanding of guard cell biology, and this Special Issue of New Phytologist brings together the full complement of current research approaches, from evolution and climate change modelling through to intramolecular signalling processes. But what has driven the current wave of interest?

Primary targets for molecular analysis

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References

Stomatal complexes of higher plants comprise a pair of guard cells delimiting a central pore, through which CO2 is taken up for photosynthesis at the expense of water loss. The guard cells respond to a wide variety of environmental and physiological signals such that their movements regulate the size of the pore to optimise the water use efficiency (WUE) of the plant (i.e. CO2 uptake is maximised whilst water loss is minimised (Davies et al., pp. 449–460 in this issue). In addition to regulating stomatal movements, environmental signals also alter the number and density of stomatal complexes formed during the development of the leaf. Woodward et al. (pp. 477–484) provide evidence that the regulation of stomatal development by CO2 during drought is another adaptation of stomatal biology that increases WUE. The study of stomatal biology is therefore central to understanding the physiology of land plants and guard cells have been primary targets for molecular analysis.

Such work has been accelerated by the improvement of techniques for purifying guard cells and improving the yield of guard cell RNA and proteins (Pandey et al., pp. 517–526). These developments have been coupled with advances in PCR techniques that have increased the sensitivity of detection of specific nucleic acid sequences manyfold. In parallel, stomatal physiologists have overcome the technical problems associated with working with the small guard cells of Arabidopsis (Kuchitsu et al., pp. 527–533; Pandey et al., pp. 517–526; see also Roelfsema & Prins, 1995; Pei et al., 1997; Allen et al., 1999; Webb et al., 2001). Thus, molecular techniques, forward and reverse genetics, the sequenced Arabidopsis genome and cell physiology tools have been combined in an attempt to understand the biology of this cell type.

The importance of the guard cell model

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References

The study of the cellular mechanisms of stomatal movements is important because they are central to reducing water loss and therefore to the survival of land plants (stomatal evolution is discussed by Raven on pp. 371–386). A simple experiment in which water loss from a detached leaf is quantified demonstrates the importance of abscisic acid (ABA) regulated stomatal movements in controlling evaporation from leaves (Fig. 1). Leaves from wild-type plants are able to limit the rate of water loss and do not wilt for many hours after removal, whereas leaves from plants containing a dominant mutation of the ABI1 gene are unable to regulate water loss such that 90% of the fresh weight of a leaf is lost within 40 min of excision. This unregulated water loss through stomatal pores occurs because the abi1–1 mutation renders the guard cells insensitive to ABA and the stomata fail to close. abi1–1 plants are wilty and are unable to tolerate drought, exemplifying that closure of the stomata is necessary to prevent wilting of the leaf and also demonstrating that single gene mutations can have profound effects on the physiology of stomata (see Schroeder et al. (2001) for a discussion of the effects of abi1–1 on guard cells).

image

Figure 1. Water loss from leaves detached from Arabidopsis plants. Open symbols are the Landsberg erecta background and closed symbols are abi1–1. Four rosette leaves were detached per plant and weighed at intervals during incubation at 19°C in the light. Water loss is expressed as a percentage of the fresh weight. Symbols represent the mean of three plants per genotype. The errors are ± the standard error of the mean. The genotype of the plants was confirmed using a PCR-based assay.

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ABI1 encodes a protein phosphatase 2C and is a component of the intracellular ABA signal transduction pathway in guard cells. This pathway has been extensively studied and a consensus model of the complex interactions between the increases in cytosolic free Ca2+ concentration ([Ca2+]i), other second messengers, ion channels, protein phosphatases and other components of the pathway is emerging (Assmann & Wang, 2001; Evans et al., 2001; Schroeder et al., 2001). This detailed dissection of a signal transduction pathway in a single cell type is an important contribution of stomatal research to plant biology. The focus upon a single cell type has eliminated complexities that may arise by studying ABA-signalling pathways regulating multiple responses in different cell types. This has allowed data obtained using a variety of techniques to be incorporated into a rapidly developing model.

The guard cell has a specialised physiology, but there is recent evidence that some of the fundamental steps in ABA signal transduction identified in guard cells may be common to other plant cell types. Early responses to ABA in guard cells include an influx of Ca2+ resulting in an increase in [Ca2+]i and also activation of anion efflux that depolarises the plasma membrane (Kuchitsu et al., pp. 527–533; Roelfsema & Hedrich, pp. 425–431). The same steps are required for ABA-induced RAB18 expression in Arabidopsis suspension cells (Ghelis et al., 2000a,b). In both cell types external Ca2+ is required for activation of gene expression by ABA. However, both in guard and suspension cells, elevation of the external Ca2+ concentration (and consequently [Ca2+]i), in the absence of ABA, fails to induce expression of ABA-inducible genes (Ghelis et al., 2000b; Webb et al., 2001). These data demonstrate that there are similarities between the ABA-signalling cascades regulating gene expression in guard cells and those in other cell types. Therefore, the guard cell model provides testable hypotheses for dissecting the ABA signalling networks in other plant cell types. However, signalling processes identified in the guard cell should not be assumed for other cell types: there are differences between the ABA-turgor and ABA-nuclear signalling pathways in the guard cell (Webb et al., 2001); and there are substantial differences between the guard cell and mesophyll ABA-signalling pathways (Sutton et al., 2000).

Identification of genes regulating stomatal physiology and development

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References

Early studies of the guard cell signalling pathways focused on ionic changes and the regulation of ion channel activity by pH, [Ca2+]i and phosphorylation. The adaptation of cell physiology techniques to Arabidopsis has allowed genetic resources to be used to identify genes that regulate stomatal movements and also development.

Arabidopsis mutant screens have identified a number of genes that are thought to be involved in ABA signalling in guard cells. These include genes encoding 2C protein phosphatases (ABI1, ABI2), an inositol polyphosphate 1-phosphatase (FIERY1), and a small GTP-binding protein involved in ABA-induced actin reorganisation (AtRac1) (Schroeder et al., 2001; Xiong et al., Mutant screens also have identified genes involved in the light regulation of stomatal movements (Zeiger et al., pp. 415–424; Kinoshita et al., 2001) and in the regulation of stomatal development (see Holroyd et al., pp. 433–439, for a description of the mutations affecting development).

Comparisons of guard cell physiology in wild type and mutant backgrounds have positioned genetic lesions with respect to other components of the ABA signalling cascade. For example, during ABA-induced activation of an inward Ca2+ current, the abi1–1 mutation acts upstream of the production of reactive oxygen species, whereas the abi2–1 mutation acts downstream (Murata et al., 2001). The relative position of a mutant allele in the ABA signalling pathway will often reflect the site of action of the protein encoded by the wild type gene, but this may not always be the case. First, gain-of-function mutations might implicate a gene in a signalling pathway in which the wild type gene product does not normally operate. Second, the mutation might result in compensatory alterations in gene expression, or reorganise the signalling pathways masking the wild type function of the gene. Third, pleiotropic effects of the mutation may complicate interpretation of the phenotype. Therefore, relative positions in the signalling pathway predicted by the mutant allele might require confirmation by demonstrating activity of the wild-type protein at the predicted position.

Recent work from the Schroeder lab demonstrates that the effects of a single gene mutation can be complex and that the mutation can act at distance. Hugouvieux et al. (2001) identified a recessive ABA mutant, abh1, that only affects ABA responses. Schroeder and his coworkers carried out a very detailed, multidisciplinary study to determine how the abh1 mutation alters the sensitivity of stomata to ABA. They demonstrated that abh1 exhibits hypersensitive ABA-induced stomatal closure, reduced wilting during drought and hypersensitive [Ca2+]i increases. These are consistent with an effect early in the ABA signalling pathway. ABH1 encodes an mRNA cap binding protein which may function in the transcriptional alteration of ABA signalling elements. The abh1 mutation causes the down regulation of expression of a number of genes thought to be involved in Ca2+-based signalling pathways. One of these is AtPP2C, which encodes a protein phosphatase 2C. Members of this class of proteins are negative regulators of ABA signalling and the phenotypes of abh1 are consistent with the down regulation of the expression of PP2Cs. Therefore, ABH1 is a potent modulator of ABA signalling and the abh1 mutation most likely renders stomata hypersensitive to ABA by reducing the expression of a PP2C, a negative regulator in the ABA signalling pathway. It is not known whether ABH1 also acts as a signalling element.

Not all genes can be identified by classical genetics. These include genes that are functionally redundant – a mutation is unlikely to occur simultaneously in two or more redundant genes, therefore a phenotype may not be detectable (e.g. KAT1 might not have been identified by classical genetics because it is partially redundant) (Szyroki et al., 2001; Kwak et al., 2001). Classical genetics also fails to identify genes in which a mutation results in a lethal phenotype. For example, mutant screens have not identified positive regulators of stomatal development, possibly because a loss of function allele of such a gene would result in a lethal lesion in stomatal development. For these reasons a number of complementary approaches have been used to identify genes important in guard cell function. These approaches include promoter trapping (HIC1, Holroyd et al., pp. 433–439), enhancer trapping (Fig. 2; A. J. Baker & A. A. R. Webb, unpublished), reverse genetics (GPA1, Wang et al., 2001; AtMRP5, Gaedeke et al. 2001), complementation of yeast mutations (KAT1, Anderson et al., 1992), microinjection of guard cell cDNA into Xenopus oocytes (Nt-SYR1, Blatt, pp. 405–413) and identification on the basis of expression profile (carbon metabolism genes, Kopka et al., 1997; AAPK1, Li et al., 2000). Subsequent knockout using reverse genetic approaches has assigned functions to many of these genes.

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Figure 2. Confocal laser scanning photomicrographs of the GAL4-green fluorescent protein (GFP) enhancer trap line Q2480. GFP expression is driven by transactivation by GAL4, which in turn is expressed under the control of an endogenous enhancer. In this line, GFP is expressed exclusively in the guard cells in the shoot (a) but also in epidermal and cortical cells of the root (b). GFP expression is shown in green and the cell wall stain propidium iodide in red. Bars, 20 µm.

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Future prospects

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References

The goal for stomatal physiology is a description of the molecular interactions that occur during stomatal movements. To achieve this goal will require identification of all of the genes expressed in the guard cell. The development of microarray technology, in which fluorescent dye-labelled guard cell RNA (or cDNA) is probed with the genome or subsets of the genome bound to a substrate, will result in an explosion of information concerning the guard cell transcriptome. The challenge will be to assign function to all of the genes expressed in the guard cell. This will represent a formidable task. The efficient assignment of guard cell gene function might require consortia of laboratories using both high throughput genetic manipulation techniques and high throughput assays for stomatal phenotypes. These high throughput assays will benefit from guard cell targeting of gene expression using transactivation in enhancer-trap lines (Fig. 2) or guard cell-specific promoters (Plesch et al., 2001), remote sensing of stomatal behaviour using techniques such as thermal imaging and imaging of noninvasive reporters of cellular activity such as aequorin, cameleon-green fluorescent protein (GFP) and luciferase.

One target for functional genomic studies is the identification of the ABA-receptor(s). It is disappointing that the start of the ABA signalling cascade remains unknown. Assuming that the receptor is a protein, it should be described in the genomic DNA, RNA or protein sequences predicted by the guard cell transcriptome. However, the identity of the ABA-receptor may not be immediately apparent from the sequence data and it is likely that a combination of microarray, functional genomics and reverse genetics will be required to identify and characterise the receptor(s).

Other targets for functional genomic analysis of guard cells are the ‘calcium sensor’ proteins. Oscillations in [Ca2+]i are central to the control of stomatal movements and information is encoded in the oscillatory pattern resulting in a ‘calcium signature’ (McAinsh et al., 1995; Allen et al., 2001). However, the mechanisms by which the oscillations are decoded are obscure. Reverse genetic approaches including gene knock-out and cell-specific misexpression of candidate ‘calcium-sensor’ proteins coupled with physiological and biochemical approaches will be required to identify the ‘calcium-sensor’ protein(s) and determine how they are regulated by oscillations of [Ca2+]i (Evans et al., 2001).

Powerful genomic tools will place the emphasis of future research upon genetically encoded signalling elements. However, signalling pathways are much more than a series of interacting proteins. Lipid and carbon metabolism, ionic changes, protein phosphorylation/dephosphorylation and other post-translational modifications are all components of ABA signalling. The pathways by which other signals, such as CO2, light and the circadian clock regulate stomatal movements also require further investigation, not least because in well-watered plants these can represent the major regulators of stomatal movements. Similarly, the pathways by which CO2 and light regulate the patterning of stomata during leaf development are beginning to emerge and are likely to be the subject of intense investigation (Holroyd et al., pp. 433–439).

Genetic manipulation of stomata for crop improvement

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References

The signalling pathways regulating stomatal movements and changes in stomatal density provide molecular candidates for genetic manipulations aimed at improving crop WUE with the goals of increasing survival during drought and reducing fresh water use. Recent advances in understanding the role of the chloroplast in light-regulated stomatal movements and carbon metabolism suggest that guard cell plastid function also is a candidate target for genetic manipulation (Zeiger et al., pp. 415–424; but see Kinoshita et al., 2001). Genetic improvements might be complemented by advances in measuring cell physiological parameters in planta using both invasive (Roelfsema & Hedrich, pp. 425–431) and noninvasive techniques such as thermal imaging and low light imaging of cameleon-GFP, aequorin and luciferase. It is possible to envision the physiology of the guard cell acting as a visible indicator of crop stress in the field. Noninvasive recombinant reporters of changes in guard cell physiology might, in the future, alert growers to the onset of stress. This will allow the growers to take remedial action to prevent visible crop damage and reductions in yield.

This Special Issue demonstrates the breadth and depth of the knowledge concerning the control of stomatal behaviour. Much of this knowledge is already finding application in agriculture and horticulture. For example, Davies et al., (pp. 449–460) describe how the chemical regulation of stomatal movements by ABA is being used to decrease the water used by wine grape growers in Australia. Because the next decade will see substantial advances in understanding the biology of stomata, the outlook for decreasing crop fresh water usage and improving WUE, particularly in the wine industry, is looking increasingly ‘rosé’.

Acknowledgements

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References

Inspiration for papers in this Special Issue came from the 7th New Phytologist Symposium, ‘Stomata 2001’, held in Birmingham in July 2001 (see Drake, 2001). AJB is funded by a BBSRC UK Research Committee Studentship. AARW is grateful to the Royal Society for the award of a University Research Fellowship.

References

  1. Top of page
  2. Primary targets for molecular analysis
  3. The importance of the guard cell model
  4. Identification of genes regulating stomatal physiology and development
  5. Future prospects
  6. Genetic manipulation of stomata for crop improvement
  7. Acknowledgements
  8. References
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