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How Do Stomata Sense Reductions in Atmospheric Relative Humidity?

2013; Elsevier BV; Volume: 6; Issue: 5 Linguagem: Inglês

10.1093/mp/sst055

1674-2052

Hubert Bauer, Peter Ache, Florian Wohlfart, Khaled A. S. Al‐Rasheid, Sophia Sonnewald, Uwe Sonnewald, Susanne Kneitz, Alistair M. Hetherington, Rainer Hedrich,

Plant Physiology and Cultivation Studies

Dear Editor, It has been known since the work of Francis Darwin that, in response to a reduction in atmospheric relative humidity (rh), stomatal aperture decreases. Screening for Arabidopsis mutants compromised in stomatal responses to reduced rh resulted in the identification of two genes, OST1 and ABA2, that are involved in stomatal response to low rh conditions. Interestingly both encode proteins previously known to be involved in ABA signaling (Xie et al., 2006Xie X. Wang Y. Williamson L. Holroyd G.H. Tagliavia C. Murchie E. Theobald J. Knight M.R. Davies W.J. Leyser H.M et al.The identification of genes involved in the stomatal response to reduced atmospheric relative humidity.Curr. Biol. 2006; 16: 882-887Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, and references therein). These findings strongly suggested that, at least in part, the stomatal response to low rh is mediated by ABA and the intracellular ABA signaling pathway. Our most recent data show that low rh-induced stomatal closure can proceed by guard cell autonomous ABA synthesis (Bauer et al., 2013Bauer H. Ache P. Lautner S. Fromm J. Hartung W. Al-Rasheid K.A. Sonnewald U. Kneitz S. Lachmann N et al.The stomatal response to reduced relative humidity requires guard cell autonomous ABA synthesis.Curr. Biol. 2013; 23: 53-57Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), suggesting that there is significant overlap between the guard cell low air humidity-induced signaling pathway and the guard cell ABA signaling pathway. Such convergence has been noted before (Xie et al., 2006Xie X. Wang Y. Williamson L. Holroyd G.H. Tagliavia C. Murchie E. Theobald J. Knight M.R. Davies W.J. Leyser H.M et al.The identification of genes involved in the stomatal response to reduced atmospheric relative humidity.Curr. Biol. 2006; 16: 882-887Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, and references therein) and the example of Open STomata 1 (OST1/SRK2E) that encodes a serine–threonine protein kinase and the SLow Anion Channel SLAC1 may represent a common mechanism for most other closure-inducing signals, too. This can be explained by the fact that OST1 is known to regulate SLAC1. When the rh drops, stomata located in the leaf boundary to the atmosphere are the first to face dry air. Upon perception of the signal, guard cells produce their 'own' ABA (Bauer et al., 2013Bauer H. Ache P. Lautner S. Fromm J. Hartung W. Al-Rasheid K.A. Sonnewald U. Kneitz S. Lachmann N et al.The stomatal response to reduced relative humidity requires guard cell autonomous ABA synthesis.Curr. Biol. 2013; 23: 53-57Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). The endogenous stress hormone is then recognized by a cytosolic ABA receptor. The underlying fast ABA signaling pathway is initiated when the cytosolic threshold of the stress hormone is passed and binding of ABA to the cytosolic receptor facilitated. This in turn results in the activation of guard cell anion channels in a process involving OST1. Specifically, OST1 phosphorylates and thereby activates SLAC1 (Geiger et al., 2009Geiger D. Scherzer G. Mumm P. Stange A. Marten I. Bauer H. Ache P. Matschi P. Stange S. Liese A. Al-Rasheid K.A et al.Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase–phosphatase pair.Proc. Natl Acad. Sci. U S A. 2009; 106: 21425-21430Crossref PubMed Scopus (642) Google Scholar; Lee et al., 2009Lee S.C. Lan W. Buchanan B.B. Luan S A protein kinase–phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells.Proc. Natl Acad. Sci. U S A. 2009; 106: 21419-21424Crossref PubMed Scopus (452) Google Scholar). Opening of anion channels depolarizes the plasma membrane and the anions and K+ released are accompanied by the loss of water from guard cells. In turn, turgor drops and stomata close (for review, see Joshi-Saha et al., 2011Joshi-Saha A. Valon C. Leung J Abscisic acid signal off the STARting block.Mol. Plant. 2011; 4: 562-580Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Although we are making progress in the dissection of the intracellular signaling pathway that underlies rapid rh-induced closure, we know much less about either how rh induces ABA synthesis or the molecular basis of longer-term (adaptive) stomatal responses to reduced rh. To begin to address these questions, we adopted a transcriptomic approach. We used the two well-characterized genes known to be involved in guard cell ABA and rh signaling: OST1 (Xie et al., 2006Xie X. Wang Y. Williamson L. Holroyd G.H. Tagliavia C. Murchie E. Theobald J. Knight M.R. Davies W.J. Leyser H.M et al.The identification of genes involved in the stomatal response to reduced atmospheric relative humidity.Curr. Biol. 2006; 16: 882-887Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar; Geiger et al., 2009Geiger D. Scherzer G. Mumm P. Stange A. Marten I. Bauer H. Ache P. Matschi P. Stange S. Liese A. Al-Rasheid K.A et al.Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase–phosphatase pair.Proc. Natl Acad. Sci. U S A. 2009; 106: 21425-21430Crossref PubMed Scopus (642) Google Scholar; Lee et al., 2009Lee S.C. Lan W. Buchanan B.B. Luan S A protein kinase–phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells.Proc. Natl Acad. Sci. U S A. 2009; 106: 21419-21424Crossref PubMed Scopus (452) Google Scholar) and SLAC1, which represents a prime target of OST1 (Joshi-Saha et al., 2011Joshi-Saha A. Valon C. Leung J Abscisic acid signal off the STARting block.Mol. Plant. 2011; 4: 562-580Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar; Laanemets et al., 2013Laanemets K. Wang Y.F. Lindgren O. Wu J. Nishimura N. Lee S. Caddell D. Merilo E. Brosche M. Kilk K et al.Mutations in the SLAC1 anion channel slow stomatal opening and severely reduce K+ uptake channel activity via enhanced cytosolic [Ca2+] and increased Ca2+ sensitivity of K+ uptake channels.New Phytol. 2013; 197: 88-98Crossref PubMed Scopus (44) Google Scholar). We reasoned that, by comparing the transcriptomes of ost1-2 and slac1-5 mutants with wild-type (wt) and guard cell transcriptome responses to ABA and low rh, we might reveal key elements common to the overall control of these responses. In order to obtain data sets that were not biased by genes expressed in response to protoplasting, such as the presence of fungal elicitors and the loss of turgor, we isolated intact guard cells by the blender method and then performed expression-profiling using Agilent V4 gene expression microarrays (44k), as described in Bauer et al., 2013Bauer H. Ache P. Lautner S. Fromm J. Hartung W. Al-Rasheid K.A. Sonnewald U. Kneitz S. Lachmann N et al.The stomatal response to reduced relative humidity requires guard cell autonomous ABA synthesis.Curr. Biol. 2013; 23: 53-57Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar. We compared samples ost1-2 and slac1-5 guard cells with their wt. The resulting genes were then filtered for (1) guard cell specificity, (2) ABA regulation (4 h after spraying with ABA), and (3) regulation following a drop in atmospheric relative air humidity from 80% to 20% rh, as described in Bauer et al., 2013Bauer H. Ache P. Lautner S. Fromm J. Hartung W. Al-Rasheid K.A. Sonnewald U. Kneitz S. Lachmann N et al.The stomatal response to reduced relative humidity requires guard cell autonomous ABA synthesis.Curr. Biol. 2013; 23: 53-57Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar. We compared the guard cells' expression pattern of ost1-2 mutants with that of wt and identified 96 genes affected by the loss of OST1 (Supplemental Table 1). With slac1-5 guard cells, a set of 99 genes appeared differentially regulated relative to wt (Supplemental Table 2). From the pool of 35 ost1-2-affected preferentially guard cell-expressed genes, we identified 10 which also responded to low humidity. Among these, eight genes responded to ABA as well. In the slac1-5 mutant, there were 72 genes preferentially guard cell-expressed and, of these, nine also appeared sensitive to dry air, with five of them additionally regulated by ABA (Figure 1A). Overall, the lack of OST1 and SLAC1 activity addresses a common set of four guard cell-enriched genes controlled by low humidity and ABA (Figure 1B). These are: the transcription factor MYB74, the dehydrin and widely used stress marker RAB18, the sucrose synthase SUS3, and the AAA+-type ATPase DAA1, involved in protein unfolding and degradation. Besides its function in the activation of the anion channel SLAC1 (Geiger et al., 2009Geiger D. Scherzer G. Mumm P. Stange A. Marten I. Bauer H. Ache P. Matschi P. Stange S. Liese A. Al-Rasheid K.A et al.Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase–phosphatase pair.Proc. Natl Acad. Sci. U S A. 2009; 106: 21425-21430Crossref PubMed Scopus (642) Google Scholar), OST1 is known to affect transcription in the nucleus (Fujita et al., 2011Fujita Y. Fujita M. Shinozaki K. Yamaguchi-Shinozaki K ABA-mediated transcriptional regulation in response to osmotic stress in plants.J. Plant Res. 2011; 124: 509-525Crossref PubMed Scopus (668) Google Scholar). The set of genes affected by the loss of both OST1 or SLAC1 that are identified here might thus function as core genes in ABA-mediated low-humidity adaptation of guard cells. The transcripts of these four genes were validated in respective RNA samples by qPCR and the data obtained matched with the array observations (Supplemental Table 3). Interestingly, an Aramemnon search (http://aramemnon.botanik.uni-koeln.de ) predicted co-expression of SUS3, DAA1, and RAB18 (Supplemental Figure 1). When searching for loss-of-function mutants with the four rh/ABA core genes, confirmed knockouts for SUS3 and DAA1 were identified, but not for MYB74 and RAB18. To gain insights into guard cell SUS3 and DAA1 function, we compared low-humidity signaling with Arabidopsis mutants and wt. The sucrose synthase is involved in guard cells' control of cytoplasmic sugar level and the SWEETs, a new class of sugar exporters with 17 members, in sugar transport (Chen et al., 2010Chen L.Q. Hou B.H. Lalonde S. Takanaga H. Hartung M.L. Qu X.Q. Guo W.J. Kim J.G. Underwood W. Chaudhuri B et al.Sugar transporters for intercellular exchange and nutrition of pathogens.Nature. 2010; 468: 527-532Crossref PubMed Scopus (934) Google Scholar). Guard cells express SWEET 1, 4, and 5 (Supplemental Table 4). Upon a drop in rh, expression of these SWEET genes was increased two- to sixfold in the wt, while this induction of these same genes was suppressed in sus3 and even more so in daa1 (Figure 1C). In daa1 low-humidity-induced rise in SUS3, RAB18 mRNA as well as that of ABA signaling 2C protein phosphatase HAI1 and OST1 were also strongly down-regulated. Interestingly, the low-humidity response (Bauer et al., 2013Bauer H. Ache P. Lautner S. Fromm J. Hartung W. Al-Rasheid K.A. Sonnewald U. Kneitz S. Lachmann N et al.The stomatal response to reduced relative humidity requires guard cell autonomous ABA synthesis.Curr. Biol. 2013; 23: 53-57Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) of daa1 and sus3 mutant did not differ from control. To test whether or not the mutants are impaired in ABA signaling, we used the non-invasive pressure probe to monitor ABA-induced stomatal closure (cf. Ache et al., 2010Ache P. Bauer H. Kollist H. Al-Rasheid K.A. Lautner S. Hartung W. Hedrich R Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non-invasive pressure probe measurements.Plant J. 2010; 62: 1072-1082PubMed Google Scholar). Thereby, we observed an incomplete ABA response with stomata from daa1 and sus3 mutants compared with wt (Figure 1D). Stomata in darkness and under ambient CO2 are closed, but not completely. ABA treatment following prolonged incubation usually leads to full closure, which is reflected by the wt curves (Figure 1D). Both mutants seem to be unable to close their stomata to the same extent as the wt. It is thus tempting to speculate that this behavior might be the result of impaired sugar metabolism and transport, when the respective ABA-/rh-responsive genes are affected (see Figure 1C). Together, these findings indicated that, among the four rh/ABA core genes identified, (1) DAA1 seems to trigger an early step in dry air signaling and that (2) the low rh sensing/response appears to involve guard cell sugar metabolism and transport. How, then, can humidity changes be sensed in guard cells? It has been recorded that sugars in addition to potassium salts ions can drive stomatal movement (Talbott and Zeiger, 1996Talbott L.D. Zeiger E Central roles for potassium and sucrose in guard-cell osmoregulation.Plant Physiol. 1996; 111: 1051-1057PubMed Google Scholar). Interestingly, guard cell sugar metabolism and sugar transport have not been studied in relation to rh signaling but our results strongly suggest that they are involved. There are at least two non-exclusive possibilities. It is possible that the sugars are directly involved and exported from guard cells to facilitate turgor loss. Alternatively or additionally, as the guard cell wall is loaded with sugars (Lu et al., 1997Lu P. Outlaw W.H. Smith B.G. Freed G.A A new mechanism for the regulation of stomatal aperture size in intact leaves: accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba.Plant Physiol. 1997; 114: 109-118PubMed Google Scholar) and sugars are hygroscopic, it is possible that changes in rh could influence the osmotic potential of the cell wall. This in turn could trigger ABA biosynthesis in guard cells. In this context, the sucrose synthase SUS3 is specifically expressed in guard cells (eFP browser: http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi ). The role of SUS3 and SWEET transporters would be consistent with either direct movement of sugars as osmotica or in sugar loading of the cell wall occurring as part of a rh sensing mechanism. In conclusion, our transcriptomic-based study highlighted the commonality of the guard cell response to ABA and reduced rh. By comparing wt, ost1-2, and slac1-5 mutants, we identified a set of genes that are likely to form the core of the signaling pathway that is used in the guard cell response to both reduced rh and ABA. Among these genes, the guard cell SUS3 could provide a hygroscopic sugar moiety to sense changes in ambient humidity and/or could be involved in sugar transport supporting turgor-induced changes in stomatal aperture. Mutants with SUS3, as well as the other three rh core genes, will be thoroughly characterized and particularly tested through exposure to dry air for alterations in guard cell rh/ABA gene expression and will thus serve to unravel longer-term stomatal adaptive responses to drought episodes. Supplementary Data are available at Molecular Plant Online. This work was supported by grants of the Bayerische Forschungsallianz (BayFOR), FORPLANTA to U.S. and R.H.; R.H. and K.A.S.A.-R. were supported by grants from the Strategic Technologies Program—National Science, Technology and Innovation Plan, Saudi Arabia Project No. 10-ENV1181–02. No conflict of interest declared.