Transcription factors controlling stomatal movements and drought tolerance

Abstract

In the last years some efforts in the characterization of transcription factors involved in stomatal movements in plants have been undertaken. These findings provide new insights into the molecular mechanisms that plants adopt to cope with abiotic stress and offer new strategies to improve plant drought tolerance.

Stomata, from the Greek word “stoma” meaning mouth, are small pores distributed on the epidermis of plant leaves. They mediate the exchanges between the plant and the atmosphere: CO₂ enters through stomata as carbon source for photosynthesis, while water vapor is released by transpiration. Stomata are surrounded by a pair of highly specialized cells called guard cells. Variations in turgor pressure of these cells control the opening and closure of stomata: an increase in pressure enhances stomata aperture, while a decrease reduces opening. The activity of H⁺-ATPases (proton pumps coupled to ATP hydrolysis) stimulates the hyperpolarization of the guard cell plasma membrane, which allows the entry of K⁺ and initiates stomatal opening. Conversely, a reduction in turgor pressure, caused by the efflux of K⁺ and the release of organic acids, promotes stomatal closure.¹

The guard cell turgor is dynamically adjusted according to environmental and hormonal cues. In response to abiotic stresses like drought or high salinity one of the most rapid responses of plants is the closure of stomata to prevent excessive water loss by transpiration.¹ Stomatal closure is mediated by the hormone abscic acid (ABA), whose synthesis is induced when plants are exposed to a wide variety of abiotic stresses.¹ There are other environmental stimuli that promote stomatal opening and closure: light, particularly in the blue wavelengths, is an important signal triggering stomatal opening,² while darkness, high CO₂ concentration, and pathogen attack induce stomatal closure.¹

The guard cell has been recently defined as a “Rosetta stone” that promises to clarify the understanding of signal integration between these different stimuli through a multidisciplinary approach that includes the different languages of genetics, molecular biology and biophysics.²

Water scarcity is a serious problem that will be exacerbated by global climate change.³ Abiotic stresses, especially drought and increased salinity, are primary causes of crop loss worldwide. Moreover, agriculture currently uses over 70% (86% in developing countries) of available freshwater.^4,5 One of the approaches that may be adopted to save water in agriculture is the development of plants that use less water yet maintain high yields in conditions of water scarcity. As plants lose over 95% of their water via transpiration through stomata, the engineering of stomatal activity is a promising approach to reduce the water requirement of crops and to enhance productivity under stress conditions.⁶

For these reasons the understanding of the process that controls guard cell opening/closure is of fundamental importance not only for plant biologists, but also, at a more general level, to find the key factors that can be manipulated to improve water use efficiency.²

Different signaling components have been characterized for their involvement in the control of stomatal movements, including second messengers, protein kinases, protein phosphatases and phospholipases.¹ Gene expression has been traditionally believed as a late event respect to membrane transport in guard cell signaling controlling stomatal opening/closure.² In the last years it became clear that changes in gene expression patterns and in RNA processing are involved in stomatal movement. The first evidence in this sense was the demonstration that the application of transcriptional inhibitors has negative effects on stomatal opening⁷ and the involvement of two RNA-binding proteins in ABA-induced stomatal closure.^8,9 In the last years a great interest in the analysis of the guard cell transcriptomics in response to ABA¹⁰ and in the identification of transcritpion factors that control stomatal movements became evident, as summarized in Table 1 and extensively discussed below.

Table 1.

Plant transcriptional regulators with a function in stomatal movements

The NPX1 protein is not a transcription factor, but acts as a transcriptional repressor through interaction with other proteins. For AtERF7, SNAC1 and DST a role as transcription factors was demonstrated. For the other proteins activity as transcription factors was inferred by comparison to other proteins with sequence similarity and known function.

Because transcription factors naturally act as master regulators of cellular processes, they are expected to be excellent candidates for modifying complex traits in crop plants, such as response to abiotic stresses.¹¹ In fact, it has been shown that the engineering of a single gene encoding a specific stress protein does not always confer tolerance, because multiple and complex pathways are involved in controlling plant drought responses.¹² Moreover, a modification of a single enzyme in a biochemical pathway is usually contrasted by a tendency of plant cells to restore homeostasis.¹³ Targeting multiple steps in a pathway through the manipulation of a transcription factor has the potential of modifing metabolite fluxes in a more predictable manner. In particular, the manipulation of the activity of transcription factors that control guard cell movements is expected to be useful to develop plants able to consume less water and to maintain high yield in conditions of water scarcity.

In plants, transcription factor families are usually very large, as compared to microorganisms and animals.¹⁴ As sessile organisms, plants have evolved diverse strategies to respond to different environmental stimuli and their response pathways are highly regulated at the transcriptional level.

The majority of plant transcription factors so far characterized that have a role in stomatal movements is from the model species Arabidopsis thaliana, but there are also two proteins from rice (Table 1). A simplified model of the activity of all these regulators is presented in Figure 1.

Figure 1.

A simplified model representing the role of transcriptional regulators known to be involved in stomatal movements. SNAC1 and DST have been identified in rice, while the remaining transcription factors are from Arabidopsis thaliana. Orange and green boxes represent different stimuli that induce stomatal closure and opening, respectively. Transcriptional regulators in red contribute to stomatal closure, whereas green transcriptional regulators promote opening. Line arrows represent a positive regulation, while lines ending with a bar indicate a negative regulation. Dashed line corresponds to a not fully demonstrated repression. PP2Cs, serine/threonine protein phosphatases 2C; ABA, abscisic acid.

A first indication that a transcription factor could control guard cell movements appeared with the finding that the ectopic expression of the ABA-Insensitive 3 (ABI3) protein (an Arabidopsis ABI3/VP1 transcription factor belonging to the plant specific B3 DNA-binding superfamily¹⁵ and specifically expressed in the seed) affected ABA signaling in guard cells,¹⁶ which is controlled by ABI1, a well characterized protein phosphatase 2C.¹⁷ The abi1-1 mutant displays an improper stomatal regulation leading to increased transpiration. The constitutive overexpression of ABI3 in this mutant background has been shown to rescue the abi1-1 mutation.¹⁶

The first transcription factors for which a role in stomatal opening/closure has been clearly demonstrated were the Arabidopsis AtMYB60,¹⁸ and AtMYB61 proteins.¹⁹ They are members of the R2R3MYB family, a 126 member subgroup within the MYB superfamily that, with 198 proteins in Arabidopsis, represents the largest transcription factor group in Arabidopsis.²⁰

The expression of the AtMYB60 gene is specifically localized in guard cells.²¹ Its expression is upregulated by signals that induce stomatal opening, such as white and blue light, and negatively downregulated by darkness, desiccation and abscisic acid treatment, signals that promote stomatal closure.¹⁸ Leaves from the atmyb60-1 knock-out mutant displayed a reduction in the light-induced aperture of stomatal pores of approximately 30% compared to wild-type leaves. These data indicate that this transcription factor represents a positive regulator of stomatal opening that is silenced in stress conditions. It was clearly shown that the constitutively reduced opening in these plants helps to limit water loss during drought thus enhancing plant tolerance.¹⁸ Microarray expression data showed a differential expression between wild-type and atmyb60-1 in genes involved in the response to abiotic stresses and to pathogens. Based on these data it is intriguing to suggest a model in which AtMYB60 is the only transcriptional regulator known to be involved in stomatal movements that could integrate multiple signal transduction processes by modulating the expression of genes involved in guard cell responses to light and to biotic and abiotic stresses. In contrast to AtMYB60, the AtMYB61 gene is mainly expressed in guard cells in the darkness, when stomata are closed.¹⁹ The myb61 loss-of-function mutant had larger stomatal pores than wild-type, while the constitutive expression of AtMYB61 resulted in enhanced stomatal closure.¹⁹ Infrared thermography is extensively used to monitor stomatal function, as plants with more closed stomata result warmer because they lose less thermal energy by evaporative cooling.²² This technique revealed that the myb61 loss-of-function mutant plants were approximately 0.5°C cooler than wild-type plants, while the gain-of-function MYB61OE were approximately 0.5°C warmer.¹⁹ These findings suggest that constitutive expression of the gene results in more-closed-stomata, while loss of AtMYB61 activity results in more-open-stomata. These findings were confirmed by measuring stomatal aperture that revealed a difference of about 30% in opening between MYB61OE and myb61 stomatal pores. The activity of this transcription factor in regulating stomatal closure is apparently ABA-independent, as guard cells from both mutant and overexpressing lines are responsive to increasing ABA concentration. The authors proposed a model in which AtMYB61 has an active role in the dark in the inhibition of stomatal opening.¹⁹

Two other Arabidopsis R2R3MYB genes have been described for their involvement in guard cell movement: AtMYB44,²³ and AtMYB15.²⁴ AtMYB44 gene expression was induced by ABA and by different abiotic stresses. The gene was highly expressed in guard cells. Transgenic Arabidopsis plants overexpressing the gene are more tolerant to drought and high salinity than the wild-type. Conversely, the atmyb44 knock-out mutant showed an opposite phenotype. AtMYB44 negatively regulates the expression of genes encoding a group of serine/threonine protein phosphatases 2C (PP2Cs) that have been previously described as negative regulators of the ABA signaling. The higher response to ABA and the consequent more rapid ABA-induced stomatal closure in the overexpressing lines correlate with the hypothesized AtMYB44 role in the regulation of PP2Cs genes.²³

Based on results obtained from transgenic lines overexpressing the AtMYB15 gene, Ding and collaborators²⁴ suggested a role for this transcription factor in the regulation of stomatal closure. In fact, AtMYB15 overexpressor lines are more sensitive to ABA-induced stomatal closure and show improved drought and high salinity tolerance compared to the wild-type. This transcription factor positively regulates the expression of different genes upregulated in response to abiotic stresses.²⁴

In the model species Arabidopsis three other transcriptional regulators involved in stomatal movements have been characterized till now: AtERF7,²⁵ NFYA5,²⁶ and NPX1.²⁷

AtERF7 belongs to the APETALA2/ethylene-responsive element binding proteins (AP2/EREBP) family of transcription factors that is unique to plants and has 147 members in Arabidopsis.²⁸ This protein binds to the GCC box located in the promoter of its target genes and acts as a repressor of transcription.²⁵ It is able to interact with AtSin3, a homolog of a human protein that acts as a corepressor of transcription in concern with the histone deacetylase HDA19.²⁵ The promoter of this gene has a strong activity in guard cell, as shown by fusion to the GUS reporter gene. The aterf7 RNAi lines displayed guard cells with increased sensitivity to ABA compared to the wild-type and showed enhanced drought tolerance. Transgenic Arabidopsis plants overexpressing AtERF7 showed the opposite phenotype. The authors hypothesized that AtERF7 may be part of a transcription repression complex in the ABA-dependent signaling.²⁵

NFYA5 is a member of the Arabidopsis NF-YA family.²⁶ Nuclear factor Y (NF.Y) is a transcription factor that binds to the CCAAT box, a cis-element present in about one fourth of eukaryotic gene promoters. In animals and yeast there are single genes encoding the three subunits, while in Arabidopsis there are 10 NF-YAs, 13 NF-YBs and 13 NF-YCs.²⁹ The expression of NFYA5 is upregulated by ABA and drought. The gene is highly expressed in vascular tissues and guard cells. NFYA5 contains a target site in its 3′UTR for the microRNA miR169 that negatively regulates NFYA5 mRNA accumulation.²⁶ The nfya5 knock-out mutant plants and lines overexpressing miR169 had stomata more open than wild-type plants and were consequently more drought sensitive. On the contrary, transgenic Arabidopsis overexpressing NFYA5 showed enhanced stomatal closure and increased tolerance to water deficit. This transcription factor positively regulated the expression of different drought-responsive genes. The authors suggested that the double regulation of NFYA5 expression in response to drought stress, both at the transcriptional and at the post-transcriptional level, is consistent with a critical importance of this transcription factor as a positive regulator of drought stress resistance.²⁶

The most recently identified Arabidopsis transcriptional regulator involved in stomatal movement is NPX1 (Nuclear Protein X1).²⁷ This protein is a nuclear factor, without a functional DNAbinding motif. It has some limited similarities to the global transcription factor group E8 that contains a bromodomain. It acts as a negative regulator of transcription probably through the interaction with other proteins that bind DNA, such as, for example, the TIP protein, identified by Kim and collaborators,²⁷ which encodes a NAC transcription factor (a description of this family is included below). Different genes involved in ABA synthesis and signaling were differentially expressed in the npx1 null mutant and in transgenic Arabidopsis lines overexpressing the NPX1 gene. Mutant plants had stomata more closed than wild-type in response to ABA and were more drought tolerant, while NPX1 overexpressor lines showed the opposite phenotypes.²⁷

Two transcription factors, SNAC1 and DST, involved in the regulation of stomatal movements have been identified in a crop of worldwide relevance like rice.^30,31 SNAC1 (STRESS RESPONSIVE NAC1,³⁰) is a member of the plant-specific NAC (NAM, ATAF and CUC) family of transcription factors that include 149 members in rice.³² The SNAC1 gene expression is induced in response to abiotic stresses and is predominant in guard cells.³⁰ Transgenic rice plants that overexpressed SNAC1 had improved drought resistance and salt tolerance; they showed a 22–34% increase in seed-setting during severe in-the-field drought conditions at the reproductive stage, compared to control.³⁰ Such plants showed enhanced sensitivity to abscisic acid, and had more stomata closed under both normal and drought conditions than wild-type plants. This transcription factor positively regulated the expression of different stress-responsive genes.³⁰

A second rice transcription factor recently described for its involvement in stomatal movements regulation is DST (drought and salt tolerance), a C₂H₂-type Zinc finger-containing protein, belonging to a family that includes 77 members in rice.³² The DST transcription factor is unique in that its single Zinc finger motif is required for both its DNA-binding and transactivation (the latter function of the motif was not known for any other protein belonging to this family). Huang and colleagues³¹ isolated the dst mutant, which is characterized by markedly reduced wilting during drought treatment and enhanced tolerance to high salt stress. They found that this phenotype was dependent on increased stomatal closure under both normal and stressed conditions. They observed higher levels of hydrogen peroxide (H₂O₂), an important second messenger in ABA signaling that induces stomatal closure. Huang and colleagues found that DST is involved in a novel H₂O₂-mediated pathway for stomatal closure that is ABA-independent. Based on these data, the authors proposed a model in which DST activates the transcription of some genes whose products are related to the homeostasis of H₂O₂. When DST is in its active form, H₂O₂ accumulation is inhibited and, consequently, stomata are open; when DST is in its inactive state, the H₂O₂-scavenging genes are silent, allowing for high levels of H₂O₂ to accumulate in guard cells, the stomata are closed, and the plants are stress tolerant.

The precise definition of the transcriptional network acting in guard cells is central to understanding plant response mechanisms to dehydration and to improve crop tolerance to abiotic stresses. As some key regulators have been identified only recently, progress in this area has been relatively limited.³¹ As can be seen in Figure 1, the majority of the identified transcriptional regulators controlling stomatal movements are involved in closure. This may be due to the efforts of researchers to isolate genes that, when overexpressed, confer drought tolerance, instead of genes that have to be silenced to have the same effect. In fact, the overexpression strategy is simpler to transfer from a model species as Arabidopsis to an agriculturally relevant crop than the silencing strategy is. In many of the examples reported in Figure 1 specific genes have been chosen because they were upregulated in response to abiotic stresses and thus hypothesized to promote tolerance when overexpressed.^23–27,30 The full characterization of the transcription factors described here, and of other not yet isolated, will surely help in the engineering of stomatal response to reduce water loss, thus providing a powerful tool for the enhancement of drought tolerance in crops.⁶