Current Biology 16, 882–887, May 9, 2006 ª2006 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2006.03.028
Report The Identification of Genes Involved in the Stomatal Response to Reduced Atmospheric Relative Humidity Xiaodong Xie,1 Yibing Wang,1 Lisa Williamson,2 Geoff H. Holroyd,1 Cecilia Tagliavia,1 Erik Murchie,3 Julian Theobald,1 Marc R. Knight,4 William J. Davies,1 H.M. Ottoline Leyser,2 and Alistair M. Hetherington1,* 1 Department of Biological Sciences University of Lancaster Lancaster LA1 4YQ United Kingdom 2 Department of Biology University of York York YO10 5YW United Kingdom 3 Division of Agricultural and Environmental Sciences University of Nottingham Sutton Bonington Campus Loughborough LE12 5RD United Kingdom 4 School of Biological and Biomedical Sciences Durham University South Road Durham DH1 3LE United Kingdom
Summary Stomatal pores of higher plants close in response to decreases in atmospheric relative humidity (RH). This is believed to be a mechanism that prevents the plant from losing excess water when exposed to a dry atmosphere and as such is likely to have been of evolutionary significance during the colonization of terrestrial environments by the embryophytes. We have conducted a genetic screen, based on infrared thermal imaging, to identify Arabidopsis genes involved in the stomatal response to reduced RH. Here we report the characterization of two genes, identified during this screen, which are involved in the guard cell reduced RH signaling pathway. Both genes encode proteins known to be involved in guard cell ABA signaling. OST1 encodes a protein kinase involved in ABA-mediated stomatal closure while ABA2 encodes an enzyme involved in ABA biosynthesis. These results suggest, in contrast to previously published work, that ABA plays a role in the signal transduction pathway connecting decreases in RH to reductions in stomatal aperture. The identification of OST1 as a component required in stomatal RH and ABA signal transduction supports the proposition that guard cell signaling is organized as a network in which some intracellular signaling proteins are shared among different stimuli.
*Correspondence:
[email protected]
Results and Discussion Stomata are pores on the surface of leaves that control the uptake of CO2 from the atmosphere and the loss of water vapor from the plant. It is believed that the acquisition of functional stomata together with the development of a relatively impermeable cuticle and a vascular system were key events in the evolution of higher plants [1]. Stomatal aperture is regulated by the turgor of the two guard cells that surround the pore relative to the turgor of the epidermal cells. Stomata can be regarded as structures that tailor plant gas exchange to suit the prevailing environmental conditions, and in line with this role, guard cell turgor is regulated by various environmental cues and endogenous plant hormones [2]. One of the environmental variables that controls stomatal aperture, as observed by Francis Darwin, is the relative humidity of the atmosphere (RH) [3]. In general, stomata close in response to a reduction in RH. This is believed to help prevent the plant from losing excessive water to a dry atmosphere [4]. It is perhaps surprising, given the potential importance of the stomatal humidity response to the establishment, spread, and evolution of the embryophytic flora, that we know rather little about the molecular and physiological events that underlie the response of stomata to this environmental variable. Current evidence supports the suggestion that stomata do not detect alterations in RH as such, but rather it is the change in transpiration rate that is perceived [5–7]. We know less about the intracellular signaling pathway responsible for coupling the perception of alterations in RH to changes in guard cell turgor. The current view, resulting from work carried out in Arabidopsis, is that stomatal RH signaling is ABA independent [8]. However, what is clear is that the guard cell RH response is important as, in addition to contributing to the ability of the plant to conserve water in dry atmospheres, it acts to condition the stomatal response to other environmental signals. For example, in Vicia faba, Talbott and colleagues [9] showed that RH controls short-term stomatal sensitivity to CO2 while Pospisilova [10] demonstrated that a functional stomatal apparatus only developed after Phaseolus vulgaris seedlings were transferred from a high- to a low-RH environment. In order to identify genes required for the stomatal RH response, we screened M2 plants from an (ethyl methanesulphonate) EMS-mutagenized population by thermal imaging. Figure 1 shows the images produced from two of the mutant lines, named 7C and 30A, identified in this screen in comparison to the parental wild-type (wt) Columbia (Col-2). When exposed to a sudden drop in atmospheric RH, both lines display leaf thermal profiles that are lower than wt. Genetic analysis demonstrated that these phenotypes were caused by single, recessive, nonallelic Mendelian mutations. These data suggest that the genes mutant in 7C and 30A are required to reduce stomatal aperture in response to a drop in atmospheric humidity. At 6 weeks, 7C is morphologically
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Figure 1. Infrared Images of 5-Week-Old Wild-Type Col-2 and the 30A and 7C Mutants 40 Minutes after They Experienced a Drop in Relative Humidity from 65% to 25% Methods are as follows. 20,000 seeds from an Arabidopsis thaliana (ecotype Col-2) ethyl methanesulphonate (EMS) M2 population representing 40 independent pools (each pool corresponding to approximately 1000 M1 plants) [24] were germinated and grown in a 3:1 mix of peat-based compost (SHL, Multi purpose, William Sinclaire Horticulture, UK) and washed horticultural silver sand (Gem Horticulture, UK) in acrylic horticultural propagators (William Sankey Products, UK). The propagators were placed in a growth room (10.5 hr photoperiod, PPFD 150 mmol m22 s21; air temperature 23ºC 6 2ºC; RH 25% 6 5%), and the plants were grown until they were 3 weeks old. The relative humidity inside the propagators was 65%. 40 min prior to infrared thermography, the acrylic lid was removed from the propagator. This caused the plants to experience a drop in relative humidity from 65% to 25%. Plants displaying aberrant leaf temperatures were detected by infrared thermography conducted with an Inframetrics ThermaCam SC1000 focal plane array (256 3 256 pixel platinum silicide) imaging radiometer (3.4–5 mm) fitted with a 16º lens (Flir Systems Inc), and the data were analyzed with ThermaGRAM 95 Pro image analysis software (Thermoteknix Systems Ltd., UK). Mutants exhibiting altered leaf surface temperature compared to wild-type were selected, and self-pollinated and seed (M3) was collected for further investigation. Backcross seed (F1s) were obtained by using the mutant lines as female and Columbia as male, and the F2 was used for segregation analysis. Mutants segregating in F2 were backcrossed to wt Columbia for another two generations before being used for fine mapping and phenotypic analysis.
similar to wt whereas 30A is noticeably smaller and in comparison to wt displays a wilty appearance (Figure 2). We next used gas exchange techniques to investigate further the response of the mutants to a reduction in RH. Figure 3 shows that in comparison to wt, the 7C and 30A mutants displayed a lower rate of reduction in stomatal conductance after transfer from 75% RH to 5% RH.
These data are consistent with the IR thermal imaging results (Figure 1) and suggest that 7C and 30A carry lesions that impair the stomatal response to reduced RH. However, it is important to note that both mutants retained a capacity to respond to a reduction in RH. However, this was markedly reduced compared to wt. We also checked whether there was any alteration in stomatal index in either of the mutants. Relative to wt, there was no statistically significant alteration in stomatal index, stomatal density, or epidermal cell density in the 7C mutant. In 30A there was a statistically significant increase in stomatal density and epidermal cell density but no alteration in stomatal index (a measure of the proportion of epidermal cell that are stomata). These data suggest that in this mutant the epidermal cells and stomata are smaller than wild-type but that the rule governing the ratio of stomata to epidermal cells is not violated (data not shown). To determine the identity of the genes responsible for the phenotype of the 7C and 30A lines, we adopted a map-based cloning strategy. By means of a range of simple sequence length polymorphism (SSLP) and cleaved amplified polymorphic sequence (CAPS) markers chosen from the The Arabidopsis Information Resource (TAIR) (http://www.Arabidopsis.org/), we mapped 7C to a region on chromosome 4 between the markers AtMYB3R and Nga1139 and 30A to a region on chromosome 1 between CWI1 and SGCSNP69. Each of these regions includes a gene involved in ABA action. The 7C region includes the OST1 gene [11, 12] also known as SRK2e [13, 14]. This gene encodes a protein kinase known to be involved in guard cell ABA signaling [11, 12, 15]. The 30A region includes the ABA2 gene. This gene encodes a short chain alcohol dehydrogenase, which acts in the ABA biosynthetic pathway, converting xanthoxin to abscisic aldehyde [16–19]. Since ABA is known to regulate stomatal aperture, we considered these genes to be good candidates for the genes responsible for the 7C and 30A phenotypes. To test this idea, we looked for mutations in OST1 and ABA2 in the 7C and 30A backgrounds, respectively, and we assessed F1 complementation between 7C, 30A, and known ost1 and aba2 mutant alleles. In the 7C line, the OST1 gene was found to contain a G178Q missense mutation in a highly conserved region of the protein, and in the F1 of crosses between 7C and ost1-2 [12], no complementation was observed. When the ABA2 gene was sequenced from the 30A line, it was found to include an insertion of an A, leading to a frameshift at amino acid 30, which is predicted to lead to the
Figure 2. The Morphology of 6-Week-Old Wild-Type, 7C, and 30A Mutants The backcrossed 7C and 30A lines were outcrossed to Landsberg-erecta (Ler) plants, and the resulting progeny were self-fertilized. Mutant individuals were selected from the F2 and DNA was prepared from leaf samples with Dneasy 96 plant kits according to the manufacturer’s instructions (Qiagen, Germany). The DNA samples were used to genotype the F2s with respect to known CAPS and SSLP markers (http://www.Arabidopsis.org/), allowing cosegregation and relative position to be assessed for each marker.
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Figure 3. Typical Responses of Stomatal Conductance after a Drop in Atmospheric Humidity for Wild-Type Col-2 and the 7C and 30A Mutants To measure stomatal conductance, plants were grown in propagators as described above to 6 weeks old. Gas exchange measurements were made with a Licor 6400 (Licor, NE) photosynthesis system with a 6400-15 Arabidopsis leaf chamber attachment. Illumination was provided to the adaxial side by a fiberoptic attached to a Schott lamp and was 90 mmol m22s21 at the leaf surface. Air temperature within the leaf chamber was maintained at 22ºC throughout. Plants were taken directly from the growth chamber and placed in the leaf chamber, where the humidity was set at 75% (RH) 6 10%. Stomatal conductance was monitored graphically on a computer, and data were collected automatically every 5 s. Stabilization of stomatal conductance typically took 25–35 min. After stabilization, humidity was reduced to 5% RH over a period of 7 min, and measurements resumed immediately after this period, continuing until g was stable once more at a lower value. Analysis of the stomatal conductance response curves took place with Sigmaplot 9.0 (Point Richmond, CA) and showed that the data fitted an equation for exponential decay. Converting these data to a logarithmic scale gave straight lines (example shown here). Experiments were repeated four times for each line: the slope of the linearized response was statistically significantly greater for wild-type than for both 7C and 30A (0.0043 6 0.0007, 0.0017 6 0.0002, 0.003 6 0.0007, respectively). Before the decline in humidity, the initial steady-state values for stomatal conductance in 7C and 30A were 0.56 6 0.02 mol m22s21and 0.58 6 0.12 mol m22s21, respectively, more than twice that of wild-type (0.23 6 0.05 mol m22s21). After the decline in humidity, however, the final values as a percentage of the initial were 53%, 55%, and 32% for wild-type, ost1-4, and aba2-13, respectively.
insertion of three additional amino acids and then a premature stop. In the F1 of crosses between the 30A line and three independent aba2 mutant alleles, no complementation of the stomatal closure phenotype was observed. These data indicate that the 7C phenotype is caused by a mutation allelic to OST1, and thus we have renamed it ost1-4; the data also indicate that 30A phenotype is caused by a mutation allelic to ABA2 and thus we have renamed it aba2-13. Both of these genes are required for wt response of stomatal aperture to RH. As these results suggest that ABA is involved in stomatal humidity signal transduction, we confirmed that ABA biosynthesis was disrupted in the aba2-13 mutant. Figure 4A shows the result of an experiment in which
ABA levels were quantified in plants from which water had been withheld for 6 days. As expected, at the end of the experiment, we observed a significant increase in ABA levels in wt and the ost1-4 mutant relative to well-watered control plants. In contrast, but also as expected, there was only a slight increase in ABA in aba2-13, this despite the fact that water status of these plants with an impaired stomatal response to reduced RH must be more unfavorable than the water relations of the wild-type. These water relations would be expected to stimulate ABA biosynthesis. We also confirmed that the ost1-4 stomata displayed reduced sensitivity to applied ABA in the inhibition of stomatal opening (Figure 4B) and promotion of stomatal closure (see Figure S1 in the Supplemental Data available with this article online) bioassays. Figure 4B and Figure S1 also show that aba2-13 and wt plants exhibited wild-type responses to applied ABA in the inhibition of stomatal opening and promotion of stomatal closure bioassays. We also confirmed that both genotypes exhibited wildtype behavior in the inhibition of germination by ABA (data not shown). The primary focus of these experiments has been the identification of genes involved in the stomatal response to reductions in atmospheric RH. Previous work based solely on gas exchange provided evidence that trienoic fatty acids were involved in the process. However, the effect on guard cell signaling was not investigated directly [20]. The results of our genetic screen revealed that two gene products, both previously known from investigations of ABA signaling, are also involved in the guard cell response to reductions in atmospheric RH. As one of these, ABA2, is involved in ABA biosynthesis [15–17], it seems reasonable to assume that ABA is involved in the stomatal response to a reduction in RH. In the light of this finding, it is perhaps not surprising that we found OST1, which is known to be intimately involved in guard cell ABA signaling [10, 11], was also involved in guard cell atmospheric RH signal transduction. Our investigations present a clear case for the involvement of both ABA and a component of the ABA-signaling network in the stomatal response to RH, and thus they appear to contradict the findings of Assmann, Snyder, and Lee [8]. These researchers observed wildtype stomatal responses to reduced RH in an ABA biosynthesis mutant (aba1) and mutants in two genes that encode closely related components of the ABA intracellular signaling network (abi1-1 and abi2-1). The balance of the available evidence makes it very likely that the OST1 protein is involved in stomatal humidity signaling. This is because the allelic srk2e mutant also shows a wilty phenotype in response to low atmospheric RH and the SRK2e protein kinase is activated by lowhumidity stress [13, 14]. The situation regarding the type 2C phosphoprotein phosphatases encoded by the ABI1 and ABI2 genes is less clear, as Yoshida et al. [14] report, on the basis of a simple gravimetric assay on detached Arabidopsis seedling rosette leaves, that a low-RH treatment results in more water loss in abi1-1 compared to wt. Although measuring water loss from detached leaves is far less direct indicator of stomatal behavior than stomatal conductance measurements carried out on intact plants [8], the results are clearly at odds with the observations of the Assmann group [8].
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Figure 4. ABA Signaling in Mutant and WildType Plants (A) Effect of withdrawing water on endogenous ABA levels in wt, ost1-4, and aba2-13. ABA levels were measured in 6-week-old plants (five from each genotype) from which water had been withheld (gray bars) for the final 6 days of the experiment and in plants (five from each genotype) that had been well watered throughout the experiment (black bars). Data are the mean 6 SE of two separate experiments. Methods are as follows: wild-type and mutant plants were grown until 6 weeks old. Water was then withheld from five plants of each genotype for 6 days, while a further five plants from each genotype received water during this period. On day 6, the aerial parts of all plants were harvested individually. The concentration of ABA in leaf tissue was determined by radioimmunoassay as described in Quarrie et al. [25]. In brief, freeze-dried leaves were ground to a fine powder and extracted with distilled water 1:20 (leaf DW:solvent) overnight at 5ºC– 10ºC. Each sample (50 ml) was incubated for 1 hr with 100 ml of DL-cis,trans-[G-3H]ABA (TRK644; Amersham Biosciences, UK) and 100 ml of MAC 252 monoclonal antibody against (S)-cis,trans-ABA (obtained from Dr. Geoff Butcher, Babraham Institute, Cambridge, UK). Excess label was removed by washing the bound complex twice with 100% and then 50% saturated ammonium sulfate. The pellet was resuspended in 100 ml of water with 1.5 ml of Ecoscint-H scintillation cocktail (National Diagnostics, Hull, UK) for counting (Tri-Carb 1600TR, Packard Instrument Company, Meriden, CT). The concentration of ABA in samples was calculated by interpolation of radioactive counts from a curve of standards that had been linearized by plotting the logit-transformation of the data against the ln of unlabeled ABA. The experiment was repeated twice. (B) The effect of exogenous ABA on stomatal aperture. The ability of ABA to inhibit stomatal opening in wt (white bars), aba2-13 (gray bars), and ost1-4 (black bars). Plants were grown and the inhibition of stomatal opening on isolated epidermal strips from 5- to 6week-old plants was investigated by the procedures described by Webb and Hetherington [23]. The experiment was repeated three times and the results are the means 6 SE of 120 stomata. Stomatal density determinations were carried out with the dental impression procedure described in Gray et al. [26].
Taking all these results together, the simplest conclusion that can be drawn is that some, but not all, components of the guard cell ABA signaling network are involved in the stomatal RH signaling response. It is more difficult to explain the differences in response of the aba2-13 plants to a reduction in atmospheric RH (Figures 1 and 2) with the wt response reported for the aba1 mutant [8]. However, it should be noted that, subject to the same caveat mentioned above, Yoshida et al. [14] reported that aba2-1 seedlings lost more water than wild-type after exposure to reduced RH. Both aba1 and aba2 are predicted to have reduced ABA biosynthesis, and we confirmed that the aba2-13 mutant failed to
accumulate as much ABA as wild-type when droughted (Figure 4A). These data support the suggestion made by Bunce [21] that ABA is involved in the stomatal response to reductions in RH and are also in line with other work showing that reductions in atmospheric RH result in increased levels of leaf ABA. Bauerle et al. [22], working with two ecotypes of Acer rubrum (red maple), reported that the imposition of a 5 kPa vapor pressure deficit (a reduction in RH) resulted in increases in leaf ABA levels in both ecotypes. They also showed that ABA levels fell on transfer of the material to higher RH. One possible explanation for the conflicting results of Assmann et al. [8] is that there was sufficient ABA present in the aba1 mutant
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to permit stomatal RH signaling to occur, whereas in the case of the aba2-13 mutant this was not the case. However, it is not possible to address this question because ABA levels were not measured in the aba1 study [8]. The simplest explanation for our observation that the ost1-4 and aba2-13 mutants are partially impaired in their ability to respond to reductions in RH is that both ABA and at least one component of the guard cell ABA signaling network are involved in this process. However, the fact that both mutants are only partially impaired in the ability to respond to RH raises some additional issues. The results from the aba2-13 mutant could be interpreted to suggest that part of the stomatal response to reduced RH is ABA independent [8, 14]. Alternatively, it is possible that the aba2-13 mutant contains sufficient ABA to permit a partial RH response. The current data do not provide an answer to this question or allow us to address whether additional ABA biosynthesis or simply ABA redistribution is required in the stomatal RH response. In the case of OST1, it is possible that the residual RH-induced closure response occurs due to the operation of a separate OST1-independent signaling network. Alternatively, it may not be necessary to invoke the existence of a separate OST1-independent network, as RH signaling may be sufficiently robust to tolerate or otherwise compensate for the loss of a single component. Finally, and in more general terms, one interesting fact to emerge from these studies is that the OST1 protein kinase is involved in both guard cell RH and ABA signaling. It will be very interesting to determine the full extent of overlap between the guard cell RH and ABA signaling pathways. Taking the OST1 results together with data showing that ABI1 and ABA2 are common elements in guard cell ABA and CO2 signaling [23] suggests that within the guard cell signaling network there is a repertoire of components that function during multiple responses. It will be interesting to establish the proportion of a cells complement of signaling components that function to transduce multiple signals, as this may have a bearing on the evolution of signaling networks. Supplemental Data The one supplemental figure can be found with this article online at http://www.current-biology.com/cgi/content/full/16/9/882/DC1/. Acknowledgments The authors are grateful to the UK Biotechnology and Biological Sciences Research Council and the EU for financial support. The authors declare no competing financial interest. Received: January 17, 2006 Revised: March 3, 2006 Accepted: March 3, 2006 Published: May 8, 2006 References 1. Raven, J.A. (2002). Selection pressure on stomatal evolution. New Phytol. 153, 371–386. 2. Hetherington, A.M., and Woodward, F.I. (2003). The role of stomata in sensing and driving environmental change. Nature 424, 901–908. 3. Darwin, F. (1898). Observations on stomata. Philos. Trans. R. Soc. Lond. B Biol. Sci. 190, 531–622.
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