Identification and functional roles of CaDIN1 in abscisic acid signaling and drought sensitivity

Plants frequently face challenges caused by various abiotic stresses, including drought, and have evolved defense mechanisms to counteract the deleterious effects of these stresses. The phytohormone abscisic acid (ABA) is involved in signal transduction pathways that mediate defense responses of plants to abiotic stress. Here, we report a new function of the CaDIN1 protein in defense responses to abiotic stress. The CaDIN1 gene was strongly induced in pepper leaves exposed to ABA, NaCl, and drought stresses. CaDIN1 proteins share high sequence homology with other known DIN1 proteins and are localized in chloroplasts. We generated CaDIN1-silenced peppers and overexpressing transgenic Arabidopsis plants and evaluated their response to ABA and drought stress. Virus-induced gene silencing of CaDIN1 in pepper plants conferred enhanced tolerance to drought stress, which was accompanied by low levels of lipid peroxidation in dehydrated leaves. CaDIN1-overexpressing transgenic plants exhibited reduced sensitivity to ABA during seed germination and seedling stages. Transgenic plants were more vulnerable to drought than that by the wild-type plants because of decreased expression of ABA responsive stress-related genes and reduced stomatal closure in response to ABA. Together, these results suggest that CaDIN1 modulates drought sensitivity through ABA-mediated cell signaling.     

Overexpression of a novel salt stress-induced glycine-rich protein gene from alfalfa causes salt and ABA sensitivity in Arabidopsis

Key message

We cloned a novel salt stress-induced glycine-rich protein gene ( MsGRP ) from alfalfa. Its overexpression retards seed germination and seedling growth of transgenic Arabidopsis after salt and ABA treatments.

Abstract

Since soil salinity is one of the most significant abiotic stresses, salt tolerance is required to overcome salinity-induced reductions in crop productivity. Many glycine-rich proteins (GRPs) have been implicated in plant responses to environmental stresses, but the function and importance of some GRPs in stress responses remain largely unknown. Here, we report on a novel salt stress-induced GRP gene (MsGRP) that we isolated from alfalfa. Compared with some glycine-rich RNA-binding proteins, MsGRP contains no RNA recognition motifs and localizes in the cell membrane or cell wall according to the subcellular localization result. MsGRP mRNA is induced by salt, abscisic acid (ABA), and drought stresses in alfalfa seedlings, and its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants confers salinity and ABA sensitivity compared with WT plants. MsGRP retards seed germination and seedling growth of transgenic Arabidopsis plants after salt and ABA treatments, which implies that MsGRP may affect germination and growth through an ABA-dependent regulation pathway. These results provide indirect evidence that MsGRP plays important roles in seed germination and seedling growth of alfalfa under some abiotic stress conditions.     

Nitric oxide suppresses the inhibitory effect of abscisic acid on seed germination by S-nitrosylation of SnRK2 proteins

Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.     

Arabidopsis acyl‐CoA‐binding protein ACBP1 participates in the regulation of seed germination and seedling development

A family of six genes encoding acyl‐CoA‐binding proteins (ACBPs), ACBP1–ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1‐over‐expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over‐production in 12‐day‐old seedlings up‐regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress‐responsive genes: ABA‐RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH‐TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12‐day‐old seedlings of ACBP1‐over‐expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two‐hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA‐mediated seed germination and seedling development.     

The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis

Calcium plays a pivotal role in plant responses to several stimuli, including pathogens, abiotic stresses, and hormones. However, the molecular mechanisms underlying calcium functions are poorly understood. It is hypothesized that calcium serves as second messenger and, in many cases, requires intracellular protein sensors to transduce the signal further downstream in the pathways. The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plant cells. Here, we report our analysis of the CBL9 member of this gene family. Expression of CBL9 was inducible by multiple stress signals and abscisic acid (ABA) in young seedlings. When CBL9 gene function was disrupted in Arabidopsis thaliana plants, the responses to ABA were drastically altered. The mutant plants became hypersensitive to ABA in the early developmental stages, including seed germination and post-germination seedling growth. In addition, seed germination in the mutant also showed increased sensitivity to inhibition by osmotic stress conditions produced by high concentrations of salt and mannitol. Further analyses indicated that increased stress sensitivity in the mutant may be a result of both ABA hypersensitivity and increased accumulation of ABA under the stress conditions. The cbl9 mutant plants showed enhanced expression of genes involved in ABA signaling, such as ABA-INSENSITIVE 4 and 5. This study has identified a calcium sensor as a common element in the ABA signaling and stress-induced ABA biosynthesis pathways.     

Interactions between ethylene,abscisic acid and cytokinin during germination and seedling establishment in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to investigate the interaction of the plant hormones ethylene, abscisic acid (ABA) and cytokinin in seed germination and early seedling development, we studied germination in ethylene-related mutants of Arabidopsis. Mutations in the genes etr1 and ein2, which reduce ethylene responses, showed increased dormancy and a delay in germination in comparison with wild type. Mutations in etr1, ein2 and ein6 also resulted in increased sensitivity to ABA with respect to inhibition of germination. Conversely, mutations in ctr1 and eto3, which lead to an increased ethylene response and overproduction of ethylene, respectively, decreased sensitivity to ABA during germination. Increased ABA sensitivity was also effected in wild type seeds by the presence during germination of AgNO₃, an inhibitor of ethylene action. The addition of the cytokinin N-6 benzyl adenine (BA) reversed the increased sensitivity of ethylene-resistant mutants to ABA. The action of cytokinin in reversing increased ABA sensitivity of ethylene-resistant mutants also suggests that at least part of the action of cytokinin in promoting germination is independent of its role in stimulating ethylene production. These observations further extend the evidence in support of interaction between ethylene, ABA and cytokinin signalling in controlling seed germination and early seedling development in Arabidopsis.     

Identification of Quantitative Trait Loci for ABA Sensitivity at Seed Germination and Seedling Stages in Rice

Abscisic acid (ABA) is one of the important plant hormones, which plays a critical role in seed development and adaptation to abiotic stresses. The sensitivity of rice (Oryza sativa L.) to exogenous ABA at seed germination and seedling stages was investigated in the recombinant inbred line (RIL) population derived from a cross between irrigated rice Zhenshan 97 and upland rice IRAT109, using relative germination vigor (RGV), relative germination rate (RGR) and leaf rolling scores of spraying (LRS) or culturing (LRC) with ABA as sensitivity indexes. The phenotypic correlation analysis revealed that only RGV at germination stage was positively correlated to ABA sensitivity at seedling stage. QTL detection using composite interval mapping (CIM) and mixed linear model was conducted to dissect the genetic basis of ABA sensitivity, and the single-locus QTLS detected by both methods are in good agreement with each other. Five single QTLs and six pairs of epistatic QTLs were detected for ABA sensitivity at germination stage. Eight single QTLs and five pairs of epistatic QTLs were detected for ABA sensitivity at seedling stage. Two QTLs were common between LRS and LRC; and one common QTL was detected for RGV, LRS and LRC simultaneously. These results indicated that both single and epistatic loci were involved in the ABA sensitivity in rice, and the genetic basis of ABA sensitivity at seed germination and seedling stage was largely different.     

OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.     

<Emphasis Type="Italic">CKB1</Emphasis> is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.     

Seed enhancement with cytokinins: changes in growth and grain yield in salt stressed wheat plants

Cytokinins are often considered abscisic acid (ABA) antagonists and auxins antagonists/synergists in various processes in plants. Seed enhancement (seed priming) with cytokinins is reported to increase plant salt tolerance. It was hypothesized that cytokinins could increase salt tolerance in wheat plants by interacting with other plant hormones, especially auxins and ABA. The present studies were therefore conducted to assess the effects of pre-sowing seed treatment with varying concentrations (100, 150 and 200 mg l⁻¹) of cytokinins (kinetin and benzylaminopurine (BAP)) on germination, growth, and concentrations of free endogenous auxins and ABA in two hexaploid spring wheat (Triticum aestivum L.) cultivars. The primed and non-primed seeds of MH-97 (salt-intolerant) and Inqlab-91 (salt-tolerant) were sown in both Petri dishes in a growth room and in the field after treatment with 15 dS m⁻¹ NaCl salinity. Both experiments were repeated during 2002 and 2003. Among priming agents, kinetin was effective in increasing germination rate in the salt-intolerant and early seedling growth in the salt-tolerant cultivar when compared with hydropriming under salt stress. Thus, during germination and early seedling growth, the cytokinin-priming induced effects were cultivar specific. In contrast, kinetin-priming showed a consistent promoting effect in the field and improved growth and grain yield in both cultivars under salt stress. The BAP-priming did not alleviate the inhibitory effects of salinity stress on the germination and early seedling growth in both cultivars. The increase in growth and grain yield in both cultivars was positively correlated with leaf indoleacetic acid concentration and negatively with ABA concentration under both saline and non-saline conditions. The decrease in ABA concentration in the plants raised from kinetin-primed seeds might reflect diminishing influence of salt stress. However, the possibility of involvement of other hormonal interactions is discussed.