Functional roles of the pepper antimicrobial protein gene, <Emphasis Type="Italic">CaAMP1</Emphasis>, in abscisic acid signaling,and salt and drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Biotic signaling molecules including abscisic acid (ABA) are involved in signal transduction pathways that mediate the defense response of plants to environmental stresses. The antimicrobial protein gene CaAMP1, previously isolated from pepper (Capsicum annuum), was strongly induced in pepper leaves exposed to ABA, NaCl, drought, or low temperature. Because transformation is very difficult in pepper, we overexpressed CaAMP1 in Arabidopsis. CaAMP1-overexpressing (OX) transgenic plants exhibited reduced sensitivity to ABA during the seed germination and seedling stages. Overexpression of CaAMP1 conferred enhanced tolerance to high salinity and drought, accompanied by altered expression of the AtRD29A gene, which is correlated with ABA levels and environmental stresses. The transgenic plants were also highly tolerant to osmotic stress caused by high concentrations of mannitol. Together, these results suggest that overexpression of the CaAMP1 transgene modulates salt and drought tolerance in Arabidopsis through ABA-mediated cell signaling. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession number AY548741.     

A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis>) overexpressing<Emphasis Type="Italic">CaLEA</Emphasis> or<Emphasis Type="Italic">otsA</Emphasis>

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.     

Differential protection of photosynthetic capacity in trehalose-and lea protein-producing transgenic plants under abiotic stresses

We previously demonstrated that both trehalose and LEA protein protect plants from damage by drought, salt, and heat. Here, we compared their effectiveness in preserving photosynthetic capacity under those abiotic stresses. Upon dehydration, the Pmax (maximal photosynthetic rate) of O₂ evolution decreased similarly in both nontransformants andotsA plants. Contrastingly, Pmax was maintained at a considerably higher level inCaLEA6 plants. However, no significant differences in Chl fluorescence parameters were observed between transformants and nontransformants. Under salinity stress,CaLEA6 plants were also better thanotsA plants in terms of their values for Pmax, photochemical efficiency of PSII(Fv/Fm), and photochemical quenching (qP). After heat bothotsA andCaLEA6 plants maintained a higher Pmax as well as more favorable Chl fluorescence parameters, although the latter transformant performed slightly better overall. Therefore, despite the comparable effectiveness of trehalose and LEA protein in enhancing tolerance against those abiotic stresses, they confer differential protection in maintaining photosynthetic capacity. Compared with trehalose, the CaLEA6 protein appears to be a more universal and effective agent under those stresses.     

Pepper protein phosphatase type 2C,CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response

Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum A BA and D rought‐I nduced P rotein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1‐OX) exhibited an ABA‐hyposensitive and drought‐susceptible phenotype. We used a yeast two‐hybrid screening assay to identify CaRLP1 (Capsicum annuum R CAR‐L ike P rotein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1‐OX plants, CaRLP1‐OX plants displayed an ABA‐hypersensitive and drought‐tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress‐responsive genes relative to those of wild‐type plants. In CaADIP1‐OX/CaRLP1‐OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1‐induced ABA hyposensitivity during the germinative and post‐germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA‐dependent defense signalling response to drought stress.     

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.     

Arabidopsis C3HC4‐RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress‐responsive abscisic acid signaling

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)‐insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4‐RING finger domain in its C‐terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T‐DNA insertion mutant atairp4 effectively recovered the ABA‐associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild‐type and atairp4 mutant plants. In addition, the expression levels of ABA‐ and drought‐induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild‐type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA‐mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis.     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.     

Knockout of AtDjB1, a J‐domain protein from Arabidopsis thaliana,alters plant responses to osmotic stress and abscisic acid

AtDjB1 is a member of the Arabidopsis thaliana J‐protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1‐1) exhibited greater ABA sensitivity compared with the wild‐type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA‐responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1‐1 plants exhibited higher glucose levels and greater glucose sensitivity in the post‐germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1‐1 than WT seedlings. Taken together, higher glucose levels in atj1‐1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.     

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1‐related protein kinase 2.10 in response to osmotic stress

SNF1‐related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA‐dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA‐non‐activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S‐segment is involved in the regulation of dehydrin subcellular localization in response to stress.