Involvement of RD20, a member of caleosin family,in ABA-mediated regulation of germination in Arabidopsis thaliana

The RD20 gene encodes a member of the caleosin family, which is primarily known to function in the mobilization of seed storage lipids during germination. In contrast to other caleosins, RD20 expression is early-induced by water deficit conditions and we recently provided genetic evidence for its positive role in drought tolerance in Arabidopsis. RD20 is also responsive to pathogen infection and is constitutively expressed in diverse tissues and organs during development suggesting additional roles for this caleosin. This addendum describes further exploration of phenotypic alterations in T-DNA insertional rd20 mutant and knock-out complemented transgenic plants in the context of early development and susceptibility to a phytopathogenic bacteria. We show that the RD20 gene is involved in ABA-mediated inhibition of germination and does not play a significant role in plant defense against Pseudomonas syringae.Key words: ABA, Arabidopsis thaliana, biotic stress, caleosin, germination, RD20     

Activation of abscisic acid biosynthesis in the leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> in response to water deficit

It is well known that endogenous abscisic acid (ABA) levels increase rapidly in response to drought stress and that this induces stomatal closure. In Arabidopsis thaliana, ABA levels increased rapidly in the leaves and roots when intact wild-type whole plants were exposed to drought stress. However, if the leaves and roots were separated and exposed to drought independently, the ABA level increased only in the leaves. These results suggest that, under our experimental conditions, ABA is synthesized mainly in the leaves in response to drought stress and that some of the ABA accumulated in the leaves is transported to the roots. Tracer experiments using isotope-labeled ABA indicate that the movement of ABA from leaves to roots is activated by water deficit in the roots. We also demonstrate that the endogenous ABA level in the leaves increased only when the leaves themselves were exposed to drought stress, suggesting that leaves play a major role in the production of ABA in response to acute water shortage.     

Abscisic Acid and Desiccation Tolerance in Mosses

In higher plants the phytohormone ABA is involved in processes that are connected to water deficit, like stomatal closure or desiccation tolerance. In bryophytes, also containing ABA in their tissues, physiological functions remained uncertain for a long time. Quite recently, several papers have shown different effects of exogenously applied ABA: stomatal closure in Anthoceros, drought hardening in Funaria and production of the landform in Riccia. In all these cases the relevant conditions (water deficit) enhance the endogenous ABA level significantly. For induced desiccation tolerance, ABA serves as a mediator to induce specific proteins (dehydrins) strongly connected with this tolerance. Therefore, it can be concluded that in bryophytes ABA has the same function as in higher plants. It acts as a mediator in stress conditions.     

Signaling mechanisms in abscisic acid-mediated stomatal closure

The plant hormone abscisic acid (ABA) plays a central role in the regulation of stomatal movements under water-deficit conditions. The identification of ABA receptors and the ABA signaling core consisting of PYR/PYL/RCAR ABA receptors, PP2C protein phosphatases and SnRK2 protein kinases has led to studies that have greatly advanced our knowledge of the molecular mechanisms mediating ABA-induced stomatal closure in the past decade. This review focuses on recent progress in illuminating the regulatory mechanisms of ABA signal transduction, and the physiological importance of basal ABA signaling in stomatal regulation by CO₂ and, as hypothesized here, vapor-pressure deficit. Furthermore, advances in understanding the interactions of ABA and other stomatal signaling pathways are reviewed here. We also review recent studies investigating the use of ABA signaling mechanisms for the manipulation of stomatal conductance and the enhancement of drought tolerance and water-use efficiency of plants.     

A stress-responsive caleosin-like protein, AtCLO4, acts as a negative regulator of ABA responses in Arabidopsis

Caleosins or related sequences have been found in a wide range of higher plants. In Arabidopsis, seed-specific caleosins are viewed as oil-body (OB)-associated proteins that possess Ca(2+)-dependent peroxygenase activity and are involved in processes of lipid degradation. Recent experimental evidence suggests that one of the Arabidopsis non-seed caleosins, AtCLO3, is involved in controlling stomatal aperture during the drought response; the roles of the other caleosin-like proteins in Arabidopsis remain largely uncharacterized. We have demonstrated that a novel stress-responsive and OB-associated Ca(2+)-binding caleosin-like protein, AtCLO4, is expressed in non-seed tissues of Arabidopsis, including guard cells, and down-regulated following exposure to exogenous ABA and salt stress. At the seed germination stage, a loss-of-function mutant (atclo4) was hypersensitive to ABA, salt and mannitol stresses, whereas AtCLO4-overexpressing (Ox) lines were more hyposensitive to those stresses than the wild type. In adult stage, atclo4 mutant and AtCLO4-Ox plants showed enhanced and decreased drought tolerance, respectively. Following exposure to exogenous ABA, the expression of key ABA-dependent regulatory genes, such as ABF3 and ABF4, was up-regulated in the atclo4 mutant, while it was down-regulated in AtCLO4-Ox lines. Based on these results, we propose that the OB-associated Ca(2+)-binding AtCLO4 protein acts as a negative regulator of ABA responses in Arabidopsis.     

Molecular tailoring of farnesylation for plant drought tolerance and yield protection

Protecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the beta-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the alpha- or beta-subunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the non-transgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficit-induced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

Uniconazole-induced tolerance of soybean to water deficit stress in relation to changes in photosynthesis, hormones and antioxidant system

This study investigated whether uniconazole confers drought tolerance to soybean and if such tolerance is correlated with changes in photosynthesis, hormones and antioxidant system of leaves. Soybean plants were foliar treated with uniconazole at 50 mg L-1 at the beginning of bloom and then exposed to water deficit stress at pod initiation for 7 d. Uniconazole promoted biomass accumulation and seed yield under both water conditions. Plants treated with uniconazole showed higher leaf water potential only in water-stressed condition. Water stress decreased the chlorophyll content and photosynthetic rate, but those of uniconazole-treated plants were higher than the stressed control. Uniconazole increased the maximum quantum yield of photosystemand ribulose-1,5-bisphosphate carboxylase/oxygenase activity of water-stressed plants. Water stress decreased partitioning of assimilated 14C from labeled leaf to the other parts of the plant. In contrast, uniconazole enhanced translocation of assimilated 14C from labeled leaves to the other parts, except stems, regardless of water treatment. Uniconazole-treated plants contained less GA3, GA4 and ABA under well-watered condition than untreated plants, while the IAA and zeatin levels were increased substantially under both water conditions, and ABA concentration was also increased under water stressed condition. Under water-stressed conditions, uniconazole increased the content of proline and soluble sugars, and the activities of superoxide dismutase and peroxidase in soybean leaves but not the malondialdehyde content or electrical conductivity. These results suggest that uniconazole-induced tolerance to water deficit stress in soybean was related to the changes of photosynthesis, hormones and antioxidant system of leaves.     

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H₂O₂-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.     

Effect of ethylene perception inhibitor on growth, water relations, and abscisic acid content in wheat plants under water deficit

The effects of treatment with 1-methylcyclopropene (1-MCP, the inhibitor of ethylene receptors) of 7-day-old wheat (Triticum durum Desf., cv. Bezenchukskaya 139) seedlings on growth characteristics, water relations, and the content of phytohormones during three days after watering cessation were studied. In treated seedlings, a decrease in the water content in the substrate resulted in a decrease in stomatal conductance in the leaves by one day earlier than in untreated seedlings. This could be related to the more rapid and substantial accumulation of ABA in treated plants. There was no clear relationship between changes in the content of cytokinins and water relations under the influence of 1-MCP under drought conditions. The role of ethylene and ABA in the regulation of growth and water relations in plants suffering from water deficit is discussed.     

F-box protein DOR functions as a novel inhibitory factor for abscisic acid-induced stomatal closure under drought stress in Arabidopsis,

Guard cells, which form stoma in leaf epidermis, sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. Under drought stress, plants synthesize abscisic acid (ABA), which in turn induces a rapid closing of stoma, to prevent water loss by transpiration. However, many aspects of the molecular mechanism for ABA-mediated stomatal closure are still not understood. Here, we report a novel negative regulator of guard cell ABA signaling, DOR, in Arabidopsis (Arabidopsis thaliana). The DOR gene encodes a putative F-box protein, a member of the S-locus F-box-like family related to AhSLF-S(2) and specifically interacting with ASK14 and CUL1. A null mutation in DOR resulted in a hypersensitive ABA response of stomatal closing and a substantial increase of drought tolerance; in contrast, the transgenic plants overexpressing DOR were more susceptible to the drought stress. DOR is strongly expressed in guard cells and suppressed by ABA treatment, suggesting a negative feedback loop of DOR in ABA responses. Double-mutant analyses of dor with ABA-insensitive mutant abi1-1 showed that abi1-1 is epistatic to dor, but no apparent change of phospholipase Dalpha1 was detected between the wild type and dor. Affymetrix GeneChip analysis showed that DOR likely regulates ABA biosynthesis under drought stress. Taken together, our results demonstrate that DOR acts independent of phospholipase Dalpha1 in an ABA signaling pathway to inhibit the ABA-induced stomatal closure under drought stress.     

Respiratory complex I deficiency induces drought tolerance by impacting leaf stomatal and hydraulic conductances

To investigate the role of plant mitochondria in drought tolerance, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant, which has low-efficient respiration and photosynthesis, high levels of amino acids and pyridine nucleotides, and increased antioxidant capacity. We show that the delayed decrease in relative water content after water withholding in CMSII, as compared to WT leaves, is due to a lower stomatal conductance. The stomatal index and the abscisic acid (ABA) content were unaffected in well-watered mutant leaves, but the ABA/stomatal conductance relation was altered during drought, indicating that specific factors interact with ABA signalling. Leaf hydraulic conductance was lower in mutant leaves when compared to WT leaves and the role of oxidative aquaporin gating in attaining a maximum stomatal conductance is discussed. In addition, differences in leaf metabolic status between the mutant and the WT might contribute to the low stomatal conductance, as reported for TCA cycle-deficient plants. After withholding watering, TCA cycle derived organic acids declined more in CMSII leaves than in the WT, and ATP content decreased only in the CMSII. Moreover, in contrast to the WT, total free amino acid levels declined whilst soluble protein content increased in CMSII leaves, suggesting an accelerated amino acid remobilisation. We propose that oxidative and metabolic disturbances resulting from remodelled respiration in the absence of Complex I activity could be involved in bringing about the lower stomatal and hydraulic conductances.     

Circadian clock-dependent gating in ABA signalling networks

Plant growth and development are intimately attuned to fluctuations in environmental variables such as light, temperature and water availability. A broad range of signalling and dynamic response mechanisms allows them to adjust their physiology so that growth and reproductive capacity are optimised for the prevailing conditions. Many of the response mechanisms are mediated by the plant hormones. The hormone abscisic acid (ABA) plays a dominant role in fundamental processes such as seed dormancy and germination, regulation of stomatal movements and enhancing drought tolerance in response to the osmotic stresses that result from water deficit, salinity and freezing. Whereas plants maintain a constant vigilance, there is emerging evidence that the capacity to respond is gated by the circadian clock so that it varies with diurnal fluctuations in light, temperature and water status. Clock regulation enables plants to anticipate regular diurnal fluctuations and thereby presumably to maximise metabolic efficiency. Circadian clock-dependent gating appears to regulate the ABA signalling network at numerous points, including metabolism, transport, perception and activity of the hormone. In this review, we summarise the basic principles and recent progress in elucidating the molecular mechanisms of circadian gating of the ABA response network and how it can affect fundamental processes in plant growth and development.     

Reciprocal grafting between clones with contrasting drought tolerance suggests a key role of abscisic acid in coffee acclimation to drought stress

The role of abscisic acid (ABA) in drought tolerance of Coffea canephora is unknown. To determine whether ABA is associated with drought tolerance and if the use of tolerant rootstocks could increase ABA and drought tolerance, we performed reciprocal grafting experiments between clones with contrasting tolerance to drought (clone 109, sensitive; and clone 120, tolerant). Plants were grown in large (120 L) pots in a greenhouse and subjected to drought stress by withholding irrigation. The non-grafted 120 plants and graft treatments with 120 as a rootstock showed a slower reduction of predawn leaf water potential (Ψ_pd) and a lower negative carbon isotopic composition ratio compared with the other grafting combinations in response to drought. The same 120 graft treatments also showed higher leaf ABA concentrations, lower levels of electrolyte leakage, and lower activities of ascorbate peroxidase and catalase under moderate (Ψ_pd?=???1.0 or ??1.5 MPa) and severe (Ψ_pd?=???3.0 MPa) drought. Root ABA concentrations were higher in plants with the 120 rootstocks regardless of watering regime. The 120 shoots could also contribute to drought tolerance because treatment with 120/109 rootstock/scion combination showed postponed dehydration, higher leaf ABA concentration, and lower leaf electrolyte leakage compared with the sensitive clone. We conclude that both the shoot and root systems of the tolerant clone can increase the concentrations of ABA in leaves in response to drought. This further suggests that ABA is associated with a delayed onset of severe water deficit and decreased oxidative damage in C. canephora.     

Does engineering abscisic acid biosynthesis in Nicotiana plumbaginifolia modify stomatal response to drought?

The consequences of manipulating abscisic acid (ABA) biosynthesis rates on stomatal response to drought were analysed in wild‐type, a full‐deficient mutant and four under‐producing transgenic lines of N. plumbaginifolia. The roles of ABA, xylem sap pH and leaf water potential were investigated under four experimental conditions: feeding detached leaves with varying ABA concentration; injecting exogenous ABA into well‐watered plants; and withholding irrigation on pot‐grown plants, either intact or grafted onto tobacco. Changes in ABA synthesis abilities among lines did not affect stomatal sensitivity to ABA concentration in the leaf xylem sap ([ABA]_xyl), as evidenced with exogenous ABA supplies and natural increases of [ABA]_xyl in grafted plants subjected to drought. The ABA‐deficient mutant, which is uncultivable under normal evaporative demand, was grafted onto tobacco stock and then presented the same stomatal response to [ABA]_xyl as wild‐type and other lines. This reinforces the dominant role of ABA in controlling stomatal response to drought in N. plumbaginifolia whereas roles of leaf water potential and xylem sap pH were excluded under all studied conditions. However, when plants were submitted to soil drying onto their own roots, stomatal response to [ABA]_xyl slightly differed among lines. It is suggested, consistently with all the results, that an additional root signal of soil drying modulates stomatal response to [ABA]_xyl.