Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2-1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1-1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.     

The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers

A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.     

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

A DEAD‐box RNA helicase,RH8, is critical for regulation of ABA signalling and the drought stress response via inhibition of PP2CA activity

Abscisic acid (ABA) is major plant hormone involved in regulating abiotic stress responses. Several studies have established that an ABA‐signalling transduction pathway—from ABA perception to response—functions in plant cells. The group A PP2Cs constitute core components of ABA signalling, and they negatively regulate ABA signalling and stress responses. Recent studies have identified and functionally analysed regulators of PP2C activity; however, the precise regulatory mechanisms remain unclear. In the present study, we used a yeast 2‐hybrid (Y2H) screening analysis to identify the DEAD‐box RNA helicase RH8, which interacted with PP2CA in the nucleus. rh8 knockout mutants exhibited ABA hyposensitivity and drought‐susceptible phenotypes characterized by high levels of transpirational water loss via reduced stomatal closure and decreased leaf temperatures. However, rh8/pp2ca double mutants showed ABA hypersensitivity and drought‐tolerant phenotypes, indicating that RH8 and PP2CA function in the same ABA‐signalling pathway in the drought stress response; moreover, RH8 functions upstream of PP2CA. In vitro phosphatase and kinase assays revealed that RH8 inhibits PP2CA phosphatase activity. Our data indicate that RH8 and its interacting partner PP2CA modulate the drought stress response via ABA‐dependent signalling.     

ABA inhibits entry into stomatal‐lineage development in Arabidopsis leaves

The number and density of stomata are controlled by endogenous and environmental factors. Despite recent advances in our understanding of stomatal development, mechanisms which prevent stomatal‐lineage entry remain unclear. Here, we propose that abscisic acid (ABA), a phytohormone known to induce stomatal closure, limits initiation of stomatal development and induces enlargement of pavement cells in Arabidopsis cotyledons. An ABA‐deficient aba2‐2 mutant had an increased number/proportion of stomata within a smaller cotyledon, as well as reduced expansion of pavement cells. This tendency was reversed after ABA application or in an ABA over‐accumulating cyp707a1cyp707a3 doublemutant. Our time course analysis revealed that aba2‐2 shows prolonged formation of meristemoids and guard mother cells, both precursors of stoma. This finding is in accordance with prolonged gene expression of SPCH and MUTE, master regulators for stomatal formation, indicating that ABA acts upstream of these genes. Only aba2‐2 mute, but not aba2‐2 spch double mutant showed additive phenotypes and displayed inhibition of pavement cell enlargement with increased meristemoid number, indicating that ABA action on pavement cell expansion requires the presence of stomatal‐lineage cells.     

ATHB17 is a positive regulator of abscisic acid response during early seedling growth

We performed activation tagging screen to isolate abscisic acid (ABA) response mutants. One of the mutants, designated ahs10 (ABA-hypersensitive 10), exhibited ABA-hypersensitive phenotypes. TAIL-PCR analysis of the mutant revealed that T-DNA was inserted in the promoter region of the Arabidopsis gene, At2g01430, which encodes a homeodomain-leucine zipper protein ATHB17. Subsequent expression analysis indicated that ATHB17 was activated in ahs10. To recapitulate the mutant phenotypes, we prepared ATHB17 OX lines and investigated their phenotypes. The results showed that ATHB17 confers ABA-hypersensitivity and drought tolerance. On the contrary, ATHB17 knockout lines were ABA-insensitive and drought-sensitive, further demonstrating that ATHB17 is involved in ABA and water-stress responses. Interestingly, the ATHB17 effect on seedling growth in the presence of ABA was observed only during the postgermination seedling establishment stage, suggesting that it functions during a narrow developmental window of early seedling growth.     

CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.     

Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis

To examine the cross talk between the abscisic acid (ABA) and ethylene signal transduction pathways, signaling events during ABA-induced stomatal closure were examined in Arabidopsis (Arabidopsis thaliana) wild-type plants, in an ethylene-overproducing mutant (eto1-1), and in two ethylene-insensitive mutants (etr1-1 and ein3-1). Using isolated epidermal peels, stomata of wild-type plants were found to close within a few minutes in response to ABA, whereas stomata of the eto1-1 mutant showed a similar but less sensitive ABA response. In addition, ABA-induced stomatal closure could be inhibited by application of ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, stomata of the etr1-1 and ein3-1 mutants were able to close in response to concomitant ABA and ACC application, although to a lesser extent than in wild-type plants. Moreover, expression of the ABA-induced gene RAB18 was reduced following ACC application. These results indicate that ethylene delays stomatal closure by inhibiting the ABA signaling pathway. The same inhibitive effects of ethylene on stomatal closure were observed in ABA-irrigated plants and the plants in drought condition. Furthermore, upon drought stress, the rate of transpiration was greater in eto1-1 and wild-type plants exposed to ethylene than in untreated wild-type control plants, indicating that the inhibitive effects of ethylene on ABA-induced stomatal closure were also observed in planta.     

Involvement of endogenous abscisic acid in methyl jasmonate-induced stomatal closure in Arabidopsis

In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ([Ca(2+)](cyt)) in guard cells using a Ca(2+)-reporter fluorescent protein, Yellow Cameleon 3.6. In wild-type guard cells, FLU inhibited MeJA-induced [Ca(2+)](cyt) elevation but not ABA-induced [Ca(2+)](cyt) elevation. The aba2-2 mutation did not affect ABA-elicited [Ca(2+)](cyt) elevation but suppressed MeJA-induced [Ca(2+)](cyt) elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 μm, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.     

The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar-insensitive (sis) mutants of Arabidopsis that, unlike wild-type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA-biosynthesis mutant aba2 and the ABA-insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose- and mannose-insensitive phenotypes. Wild-type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.     

Interplay Between Abscisic Acid and Jasmonic Acid and its Role in Water-oxidative Stress in Wild-type, ABA-deficient, JA-deficient, and Ascorbate-deficient Arabidopsis Plants

The interplay between jasmonic acid (JA) and abscisic acid (ABA) in plant responses to water stress and in water-stress-enhanced oxidative stress was investigated in Arabidopsis thaliana plants subjected to water stress by water deprivation. For this purpose a drought assay was conducted using Arabidopsis mutants impaired in ABA (aba2), JA (aos), and ascorbate (vtc1) biosynthesis. Our results show an interaction between ABA and JA during their biosynthesis. Moreover, the coordinated action of ABA and JA protected wild-type, aba2, and aos plants from the effects of stress. However, this effect was not observed in the vtc1 mutant, which showed a distinct decrease in the F _v/F _m ratio, concomitant with a marked fall in relative water content (RWC), despite high endogenous concentrations of JA and ABA. This finding indicates the relevance of ascorbate metabolism in plant acclimation to stress. Despite the interaction between the two phytohormones, drought-associated stomatal closure is regulated mainly by ABA and weakly by JA, whereas JA plays a role in the formation of antioxidants regulating ascorbate and glutathione metabolism. A time course analysis revealed the relevance of plant age and stress duration in the responses of the mutants compared to wild-type plants. Here we discuss the relationship between ABA, JA, ascorbate, and glutathione in plants under water stress.     

Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis.

Exogenous abscisic acid (ABA) induced the alcohol dehydrogenase gene (Adh) in Arabidopsis roots. Both the G-box-1 element and the GT/GC motifs (anaerobic response element) were required for Adh inducibility. Measurement of endogenous ABA levels during stress treatment showed that ABA levels increased during dehydration treatment but not following exposure to either hypoxia or low temperature. Arabidopsis ABA mutants (aba1 and abi2) displayed reduced Adh mRNA induction levels following either dehydration treatment or exogenous application of ABA. Low-oxygen response was slightly increased in the aba1 mutant but was unchanged in abi2. Low-temperature response was unaffected in both aba1 and abi2 mutants. Our results indicate that, although induction of the Adh gene by ABA, dehydration, and low temperature required the same cis-acting promoter elements, their regulatory pathways were at least partially separated in a combined dehydration/ABA pathway and an ABA-independent low-temperature pathway. These pathways were in turn independent of a third signal transduction pathway leading to low-oxygen response, which did not involve either ABA or the G-box-1 promoter element.     

ESKIMO1 disruption in Arabidopsis alters vascular tissue and impairs water transport

Water economy in agricultural practices is an issue that is being addressed through studies aimed at understanding both plant water-use efficiency (WUE), i.e. biomass produced per water consumed, and responses to water shortage. In the model species Arabidopsis thaliana, the ESKIMO1 (ESK1) gene has been described as involved in freezing, cold and salt tolerance as well as in water economy: esk1 mutants have very low evapo-transpiration rates and high water-use efficiency. In order to establish ESK1 function, detailed characterization of esk1 mutants has been carried out. The stress hormone ABA (abscisic acid) was present at high levels in esk1 compared to wild type, nevertheless, the weak water loss of esk1 was independent of stomata closure through ABA biosynthesis, as combining mutant in this pathway with esk1 led to additive phenotypes. Measurement of root hydraulic conductivity suggests that the esk1 vegetative apparatus suffers water deficit due to a defect in water transport. ESK1 promoter-driven reporter gene expression was observed in xylem and fibers, the vascular tissue responsible for the transport of water and mineral nutrients from the soil to the shoots, via the roots. Moreover, in cross sections of hypocotyls, roots and stems, esk1 xylem vessels were collapsed. Finally, using Fourier-Transform Infrared (FTIR) spectroscopy, severe chemical modifications of xylem cell wall composition were highlighted in the esk1 mutants. Taken together our findings show that ESK1 is necessary for the production of functional xylem vessels, through its implication in the laying down of secondary cell wall components.     

Barley strigolactone signalling mutant hvd14.d reveals the role of strigolactones in abscisic acid-dependent response to drought

Strigolactones (SLs) are a group of plant hormones involved in many aspects of plant development and stress adaptation. Here, we investigated the drought response of a barley (Hordeum vulgare L.) mutant carrying a missense mutation in the gene encoding the SL-specific receptor HvD14. Our results clearly showed that hvd14.d mutant is hyper-sensitive to drought stress. This was illustrated by a lower leaf relative water content (RWC), impaired photosynthesis, disorganization of chloroplast structure, altered stomatal density and slower closure of stomata in response to drought in the mutant compared to the wild type parent cultivar Sebastian. Although the content of abscisic acid (ABA) and its derivatives remained unchanged in the mutant, significant differences in expression of genes related to ABA biosynthesis were observed. Moreover, hvd14.d was insensitive to ABA during seed germination. Analysis of Arabidopsis thaliana mutant atd14-1 also demonstrated that mutation in the SL receptor resulted in increased sensitivity to drought. Our results indicate that the drought-sensitive phenotype of barley SL mutant might be caused by a disturbed ABA metabolism and/or signalling pathways. These results together uncovered a link between SL signalling and ABA-dependent drought stress response in barley.     

Role of abscisic acid (ABA) and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation

The mechanisms by which plants respond to reduced water availability (low water potential) include both ABA-dependent and ABA-independent processes. Pro accumulation and osmotic adjustment are two important traits for which the mechanisms of regulation by low water potential, and the involvement of ABA, is not well understood. The ABA-deficient mutant, aba2-1, was used to investigate the regulatory role of ABA in low water potential-induced Pro accumulation and osmotic adjustment in seedlings of Arabidopsis thaliana. Low water potential-induced Pro accumulation required wild-type levels of ABA, as well as a change in ABA sensitivity or ABA-independent events. Osmotic adjustment, in contrast, occurred independently of ABA accumulation in aba2-1. Quantification of low water potential-induced ABA and Pro accumulation in five ABA-insensitive mutants, abi1-1, abi2-1, abi3, abi4, and abi5, revealed that abi4 had increased Pro accumulation at low water potential, but a reduced response to exogenous ABA. Both of these responses were modified by sucrose treatment, indicating that ABI4 has a role in connecting ABA and sugar in regulating Pro accumulation. Of the other abi mutants, only abi1 had reduced Pro accumulation in response to low water potential and ABA application. It was also observed that abi1-1 and abi2-1 had increased ABA accumulation. The involvement of these loci in feedback regulation of ABA accumulation may occur through an effect on ABA catabolism or conjugation. These data provide new information on the function of ABA in seedlings exposed to low water potential and define new roles for three of the well-studied abi loci.     

Drought signal transduction in plants

Water deficit is one of the most common environmental limitations of crop productivity by affecting growth through alterations in metabolism and gene expression. The mechanisms involved in drought perception and signal transduction pathways are poorly understood. The participation of the plant hormone abscisic acid (ABA) has been well established. ABA levels increase when there are changes in the environment that result in cellular dehydration. Different approaches have been taken to understanding the molecular responses to desiccation and how ABA regulates gene expression. Recent efforts have identified particular topics of importance in the dissection of the signal transduction pathway which are summarized as follows: physiological approaches: identification of signalling molecules. Genetic approaches: the use of mutants, and Molecular approaches: promoter analysis.     

A hydraulic signal in root-to-shoot signalling of water shortage

Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.     

Overexpression of AtMYB52 confers ABA hypersensitivity and drought tolerance

We carried out activation tagging screen to isolate genes regulating abscisic acid (ABA) response. From the screen of approximately 10,000 plants, we isolated ca 100 ABA response mutants. We characterized one of the mutants, designated ahs1, in this study. The mutant is ABA-hypersensitive, and AtMYB52 was found to be activated in the mutant. Overexpression analysis to recapitulate the mutant phenotypes demonstrated that ATMYB confers ABA-hypersensitivity during postgermination growth. Additionally, AtMYB52 overexpression lines were drought-tolerant and their seedlings were salt-sensitive. Changes in the expression levels of a few genes involved in ABA response or cell wall biosynthesis were also observed. Together, our data suggest that AtMYB52 is involved in ABA response. Others previously demonstrated that AtMYB52 regulates cell wall biosynthesis; thus, our results imply a possible connection between ABA response and cell wall biosynthesis.