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29th New Phytologist Symposium: Stomata 2012, in Manchester, UK, July 2012
The transition of plants from sea to terrestrial environments is undoubtedly one of the key steps during evolution. It required formation of a gas-impermeable cuticle and guard cells, which form stomatal pores that act as valves for gas-exchange between plants and surrounding environment. Closed stomata shield above-ground plant tissues from harmful factors, such as drought, air pollution and pathogenic microorganisms. Opening of these microscopic pores allows plants to assimilate vast amounts of the greenhouse gas CO2 and return more than half of the terrestrial rainfall to the atmosphere. Because of these large gas fluxes, stomata are not only of crucial importance for plant growth and agriculture, but also have a major impact on our climate (Fig. 1). No wonder that the function, development and evolution of stomata have attracted the attention of numerous scientists worldwide. The New Phytologist Symposium ‘Stomata 2012’ provided a platform for them to discuss recent progress as well as unsolved issues.
‘… it is now possible to reconstitute the ABA-signaling pathway of guard cells in a heterologous expression system, or even in vitro. However, compared to the knowledge about ABA signaling, still little information is available for guard cell responses to environmental factors, such as CO2, ozone, or light.’
Global scales – climate models and improved crop production
Due to the enormous fluxes of water through stomata, the conductance of these pores has a significant influence on our climate. In most global climate models, the conductance of stomata is calculated with equations developed by Timothy Ball, Joseph Berry and co-workers in the late 1980s (Collatz et al., 1991). Their models relate the leaf conductance to photosynthesis, air humidity and the CO2 concentration at the leaf surface. However, these models do not take into account that the distribution and shape of stomata differs considerably between species. Due to these morphological differences, the maximum conductance can vary considerably. This limitation can be overcome by defining the typical conductance properties of plants within certain global vegetation zones and use these data to improve climate models, as pointed out by Ian Woodward (University of Sheffield, UK).
Water availability is limiting crop production in many areas of the world. Thus, programs to improve the efficiency of water usage in agriculture are required to ensure food for future generations. A very successful project in China was presented by Jianhua Zhang (Hong Kong Baptist University, China); a crop researcher with astonishing achievements for improving agricultural water use (Marris, 2008). Using a technique called ‘partial root zone drying’, the water usage of many crop plants could be optimized (Hu et al., 2011a). In northwest China, crops are nowadays grown with only half of the irrigation water as compared to a decade ago.
In addition to improved irrigation protocols, agriculture also benefits from breeding programs for crop plants with a lower demand for water. In field conditions, crop plants are often exposed to rapid changes in environmental conditions, to which they have to adapt their stomatal conductance. During such transition periods plants exhibit a sub-optimal conductance, leading to unnecessary loss of water or low photosynthetic rates. Tracy Lawson (University of Essex, UK) brought up that changes in stomatal conductance tend to be an order of magnitude slower than those of photosynthesis. Selecting genetic traits that speed up stomatal responsiveness therefore will help to optimize the water usage and carbon gain. A combined technique enabling imaging of transpiration and chlorophyll fluorescence is developed by Tracy Lawson and co-workers to screen plants with optimal stomatal conductance responses.
C4-photosynthesis is probably the most important adaptation of plants to low atmospheric CO2 levels and dry environment, since it enables plants to maintain high carbon assimilation rates, despite of a limited stomatal conductance. Colin Osborne (University of Sheffield, UK) pointed out that C4-plants tend to have small stomata, just as the plants that evolved during periods of low atmospheric CO2 concentrations. Due to the smaller stomata C4-plants have an improved ability to keep up their water balance and sustain photosynthesis at periods of limited water supply (Taylor et al., 2012).
Stomatal development and evolution
Plants with appropriate stomatal movements are not only favored in agriculture, but also have selective advantages in natural environments. During evolution, stomatal properties changed with changes in atmospheric CO2 level. During the periods of low CO2, the average size of stomata tended to decrease, whereas their density on the leaf surface increased. Peter Franks put forward that the small sizes of guard cells correlate with a small genome size (Franks et al., 2012). This suggests that genome sizes may have been subject to changes too, during periods of high and low atmospheric CO2 levels in evolution.
Plants can use two alternative strategies to alter their stomatal conductance, in the short-term stomata can be opened or closed, whereas a reduction in the stomatal number and size has a long-term effect. According to Jennifer McElwain (University College Dublin, Ireland) plants use either of the strategies to adapt to low atmospheric CO2 concentrations. Species with rapid CO2-induced stomatal closure response do not alter their stomatal density under elevated CO2 and vice versa (Haworth et al., 2011). However, no strong phylogenetic relation can be observed for these traits. During evolution plants thus may have switched between fast and slow adaptive responses in stomatal conductance.
Several lycophyte and fern species, which were studied by Tim Brodribb (University of Tasmania, Australia) and co-workers, did not show fast stomatal responses to ABA. Nevertheless, their stomata closed in response to excision and at low air humidity, suggesting an ABA-independent drought response in these plant groups (Brodribb & McAdam, 2011; McAdam & Brodribb, 2012). Based on these data, it is likely that ABA responses of stomata only developed in seed plants. However, this hypothesis does not match the data from the groups of Julie Gray (University of Sheffield, UK) and Alistair Hetherington (University of Bristol, UK), who carried out studies with the moss Physcomitrella patens and the lycophyte Selaginella (Chater et al., 2011; Ruszala et al., 2011). In contrast to Brodribb and co-workers, the latter research groups found that guard cells of these divisions resemble those of Arabidopsis, since they respond to ABA and CO2. Even the molecular mechanisms that provoke ABA-induced stomatal closure appear to be conserved between Arabidopsis and Physcomitrella. The ost1 mutant of Arabidopsis could be rescued with the homologous PpOST1 protein kinase of P. patens. Future studies may clarify these very exciting, but at the moment apparently contradictory, findings.
A link between short- and long-term stomatal conductance responses was elegantly shown by Cawas Engineer (University of California, San Diego, USA). He and his co-workers used carbonic anhydrase mutants of Arabidopsis, which were previously shown to have an impaired stomatal closure in response to elevated CO2 concentrations (Hu et al., 2011b). These mutants also showed altered CO2-induced stomatal density responses and could be used to identify an apoplastic signal peptide that is related to CO2 signaling. The role of similar signal peptides regulating stomatal development has been established for Epidermal Patterning Factors (EPFs) (Hara et al., 2007; Hunt & Gray, 2009; Abrash & Bergmann, 2010). Caspar Chater (University of Sheffield, UK) showed that these genes are also present in P. patens, which develop 10–15 stomata at the bottom of their spore capsules. Because of the high homology between the genes found in Arabidopsis and Physcomitrella it is likely that stomata have evolved only once in evolution.
Guard cell development
In Arabidopsis, the EPF1 and 2 peptides are involved in early and late stages of stomatal development, respectively, as explained by Julie Gray (University of Sheffield, UK). In the enthusiastic talk given by Keiko Torii (University of Washington, WA, USA) it was shown that these EPF-peptides are recognized in the apoplast by the ERECTA-family of receptor kinases, which act as negative regulators of stomatal development (Lee et al., 2012). The development of stomata is apparently controlled by leaf cells that are releasing EPF peptides, which in turn are sensed by cells capable of developing into guard cells. The ERECTA-like receptor kinases act in concert with the Too Many Mouth (TMM) receptor and regulate the activity of a set of transcription factors. Dominique Bergmann (Stanford University, CA, USA) explained that certain transcription factors like SPEECHLESS, MUTE or FAMA can be assigned to specific stages of guard cell development, whereas the ICE/SCREAM transcription factor is involved in all stages (MacAlister & Bergmann, 2011). The SPEECHLESS transcription factor binds the promotor of TMM, which thus may act as a feedback loop in the development of stomata.
Stomata develop from meristemoid cells in the epidermis, which can transform into guard mother cells and eventually divide into two guard cells. The transition of the undifferentiated meristemoid cells to the highly specialized guard cells is an intriguing process, which is still not well described at the molecular level. However, gene expression arrays probed with cDNA of cells within the stomatal lineage point to an important role for POLAR (Pillitteri et al., 2011). The asymmetrical localization of POLAR in meristomoid cells depends on the presence of BASL, which was shown to be involved in Breaking of Asymmetry in the Stomatal Lineage (Dong et al., 2009).
Guard cell signaling and stomatal movement
Mature guard cells can be regarded as top specialists in ion uptake and release. Experiments in the 1960s revealed an important role for K+-ions, which accumulate in the guard cells during stomatal opening. The uptake and release of K+ is facilitated by K+ selective ion channels that have been characterized in detail (for details see Roelfsema & Hedrich (2005) and references cited therein). Recent studies, however, tend to focus on the roles of plasma membrane anion channels, as well as H+-ATPases, which appear to be key players in the regulation of stomatal movements (Kollist et al., 2011; Roelfsema et al., 2012).
Plasma membrane H+-ATPases
Stomatal opening is fuelled by H+-ATPases in the guard cell plasma membrane, that establish a H+-gradient as well as plasma membrane, inside negative, potential. Thus changes in H+-ATPase activity can provoke stomatal movements as was visualized by a modeling approach of the laboratory of Michael Blatt (University of Glasgow, UK; Hills et al., 2012). H+ pumps require constant ATP supply; but the sources of ATP remain largely unresolved. Alistair Hetherington and co-workers presented data showing that rapid light-induced stomatal opening depends on the catabolism of stored triacylglycerols. The poster of Deirdre McLachlan (University of Bristol, UK) dedicated to this topic was chosen as best poster presentation. The importance of H+-ATPases was also underlined by Nathalie Leonhardt (CEA–Cadarache, France) and co-workers, who draw attention to three differentially regulated isoforms of plasma membrane H+-ATPases, AHA1, AHA2 and AHA5. Dominant point mutations in the plasma membrane ATPase AHA1/OST2 that lead to constitutive activation of the H+-pump also cause severely more open stomata and insensitivity to ABA (Merlot et al., 2007).
In wild type plants, plasma membrane H+ pumps are stimulated by light. Blue light is perceived by phototropins and causes phosphorylation and activation of H+-ATPases (Kinoshita & Shimazaki, 1999; Kinoshita et al., 2001). However, phototropins do not seem to interact directly with the H+-ATPase, but instead act through type 1 protein phosphatases and the protein kinase BLUS1, recently identified by Ken-ichiro Shimazaki (Kyushu University, Tokyo, Japan) and co-workers. It is likely that the emerging signaling pathway through BLUS1 is also involved in the blue light-induced inhibition of guard cell anion channels (Marten et al., 2007).
Whereas H+-ATPases can be regarded as the motor for stomatal opening, anion channels represent the primary valves that trigger ion release during stomatal closure. Jaakko Kangasjärvi (University of Helsinki, Finland) explained that the loss of the guard cell plasma membrane slow anion channel SLAC1 leads to severely impaired stomatal closure responses (Negi et al., 2008; Vahisalu et al., 2008). In Kangasjärvi's group, SLAC1 was identified during a screen for ozone sensitive mutants. He highlighted the virtue of these mutants in deciphering the new regulators of ROS-induced stomatal closure. The guard cell plasma membrane not only harbors SLAC1, but also SLAH3, a second slow-type anion channel, as well as the quickly activating anion channel QUAC1 (AtALMT12) (Meyer et al., 2010). In contrast to SLAC1, SLAH3 is a nitrate-dependent slow-type anion channel. Extracellular nitrate enhances SLAH3 activation by shifting its activation potential towards guard cell resting membrane potential (Geiger et al., 2011).
The data from Rainer Hedrich's (Würzburg University, Germany), Sheng Luan's (University of California, Berkeley, USA) and Hannes Kollist's (University of Tartu, Estonia) groups showed that SLAC1 is phosphorylated by the guard cell specific protein kinase OST1 (Geiger et al., 2009; Lee et al., 2009; Vahisalu et al. 2010). OST1 not only activates SLAC1, but recent experiments from the Hedrich laboratory indicate that it also stimulates the activity of QUAC1. In turn, OST1 is controlled by ABA, its PYR/PYL/RCAR receptors and a group of type 2 C protein phosphatases (PP2Cs). During drought, ABA binds to its receptors, causing inhibition of PP2Cs and activation of OST1. OST1 phosphorylates anion channels and causes stomatal closure, which will shield above plant tissues for loss of water.
In addition to OST1, also calcium dependent protein kinases (CPKs) can activate SLAC1 as well as SLAH3 (Geiger et al., 2010; Brandt et al., 2012). Just as for OST1, PP2Cs are controlling the activity of CPKs in an ABA-dependent manner. Thus, CPKs represent a Ca2+-dependent ABA signaling pathway, whereas signaling through OST1 is Ca2+-independent. Julian Schroeder (University of California, San Diego, USA) showed that the whole guard cell ABA signaling pathway involving CPK6 can be reconstituted in Xenopus oocytes (Brandt et al., 2012).
Studies based on Arabidopsis indicate that OST1-dependent activation of SLAC1 is crucial to trigger rapid stomatal closure in response to all tested stomatal closure-inducing stimuli. Obviously, a future challenge is to address whether this holds true also in other species. Unexpected results presented by Sanna Ehonen (University of Helsinki, Finland) showed that in some Populus species the SLAC1 gene is incomplete and apparently these plants also lack rapid stomatal response to elevated CO2 and ozone.
Stimulus-induced stomatal closure
The protein kinase OST1 is not only important for guard cell responses to ABA, but also for stimuli such as CO2 and Microbe Associated Molecular Patterns (MAMPs) (Melotto et al., 2006; Schulze-Lefert & Robatzek, 2006). The induction of stomatal closure by MAMPs was discussed by Silke Robatzek (Sainsbury Laboratory, Norwich, UK). She showed that the bacterial effector HopM1 inhibits MAMP-triggered stomatal closure via the brassinosteroid signaling pathway. In addition, an imaging system was presented that enables high throughput studies of MAMP and other stimuli-induced stomatal closure responses.
Changes in the guard cell volume during stomatal movements are accompanied by rearrangements of their actin filaments. Alistair Hetherington (University of Bristol, UK) showed that the hrs3 mutation encoding a protein of the Actin Related Protein2/3 complex impaired stimulus-induced stomatal movements (Jiang et al., 2012). In wild type, actin filaments were disrupted during ABA-induced stomatal closure whereas in hrs3 guard cells they remained largely bundled.
The presentations at Stomata 2012 made clear that research on these pores is advancing at a fast pace. Much of the progress has been made with the aid of genome and transcriptome data, which are available for Arabidopsis. It is likely that the guard cell metabolome, currently deciphered by Sarah Assmann's (Penn State University, PA, USA) team, soon can be added to these tools. The genome and transcriptome databases certainly have had a major impact in unraveling several steps in guard cell development and functioning. As a result, it is now possible to reconstitute the ABA-signaling pathway of guard cells in a heterologous expression system, or even in vitro (Geiger et al., 2011; Brandt et al., 2012). However, compared to the knowledge about ABA signaling, still little information is available for guard cell responses to environmental factors, such as CO2, ozone, or light. Identification of molecular components in the signaling pathways of the latter stimuli will certainly be a major task for future studies.
Even though Arabidopsis has provided excellent tools for research, stomata in this weed are unlikely to resemble those of crop plants in every respect. The comparison of genome- and transcriptome data of selected crop plants with Arabidopsis may help to uncover species-specific properties of guard cells. Information about guard cell genes in crop plants may in turn help to assist breeding programs for plants with improved stomatal properties. Genome data comparisons already have been used to study stomatal properties of various plant divisions. These studies revealed that the ABA signaling pathway is relatively conserved (Chater et al., 2011; Ruszala et al., 2011). At the moment these data seem contradictory to those obtained by the group of Tim Brodribb (University of Tasmania, Australia), who found that stomata of ferns and lycophytes are ABA-insensitive. Further studies, including more species, may help to understand these contradictory results and point out how the ability of guard cells, to sense and respond to a large number of hormones and environmental stimuli, has evolved.
Stomata are not only of major importance for efficient water use in agriculture, but also play an important role in global water fluxes. The models used to describe these water fluxes will benefit from extended knowledge about factors influencing the stomatal conductance. For instance, it is now possible to manipulate the stomatal density with single gene alterations. This opportunity was used in two unrelated studies of Graham Dow (Stanford University, USA) and Julie Gray (University of Sheffield, UK) to explore the role of stomatal density on the leaf conductance in a virtually unchanged genetic background. The results of these studies, as well as those on guard cell signaling mechanisms, provide excellent means for improving the prediction of stomatal conductance changes in climate models. In conjunction with improved crop plants, advanced climate models can help society to prepare for the predicted global warming, in the decades to come.