AtRAE1 is involved in degradation of ABA receptor RCAR1 and negatively regulates ABA signalling in Arabidopsis

The phytohormone abscisic acid (ABA) plays an important role in regulating plant growth, development, and adaption to various environmental stresses. Regulatory components of ABA receptors (RCARs, also known as PYR/PYLs) sense ABA and initiate ABA signalling through inhibiting the activities of protein phosphatase 2C in Arabidopsis. However, the way in which ABA receptors are regulated is not well known. A DWD protein AtRAE1 (for RNA export factor 1 in Arabidopsis), which may act as a substrate receptor of CUL4–DDB1 E3 ligase, is an interacting partner of RCAR1/PYL9. The physical interaction between RCAR1 and AtRAE1 is confirmed in vitro and in vivo. Overexpression of AtRAE1 in Arabidopsis causes reduced sensitivity of plants to ABA, whereas suppression of AtRAE1 causes increased sensitivity to ABA. Analysis of protein stability demonstrates that RCAR1 is ubiquitinated and degraded in plant cells and AtRAE1 regulates the degradation speed of RCAR1. Our findings indicate that AtRAE1 likely participates in ABA signalling through regulating the degradation of ABA receptor RCAR1.     

siRNA介导的ERA1表达下调对拟南芥抗旱性的影响

ERA1是控制植物气孔开闭的一个重要基因,根据其保守域构建RNA干扰(RNAi)载体并转化拟南芥,考察转基因植株的生长、气孔导度、离体叶片失水率以及ERA1和相关基因表达,探讨siRNA介导的ERA1表达下调对拟南芥抗旱性的影响。结果表明:转基因拟南芥株系中ERA1的表达受到明显抑制,其离体叶片失水率低于野生型,但并未出现ERA1缺失突变体的负面生长表型;转基因株系对ABA处理比野生型更敏感,其ABA处理株的根长显著变短,气孔孔径更小;转基因株ABI1、ABI2、ATHB6的表达量降低,而RAB18、RD29B、ADH1的表达量升高,siRNA介导的ERA1表达下调可能会激活RAB18、RD29B等逆境响应元件。研究发现,采用RNAi技术可以有效下调ERA1表达,在没有过多负面生长表型的前提下提高拟南芥的抗旱性,且ERA1表达下调可能通过ABA途径正面影响拟南芥的抗旱性。     

Lectin activity of the nucleocytoplasmic EUL protein from Arabidopsis thaliana

The Euonymus lectin (EUL) domain was recognized as the structural motif for a novel class of putative carbohydrate binding proteins. Confocal microscopy demonstrated that the lectin from Euonymus europaeus (EEA) as well as the EUL protein from Arabidopsis thaliana (ArathEULS3) are located in the nucleocytoplasmic compartment of the plant cell. ArathEULS3 as well as its EUL domain were successfully expressed in Pichia pastoris and purified. The EUL domain from Arabidopsis interacts with glycan structures containing Lewis Y, Lewis X and lactosamine, indicating that it can be considered a true lectin domain. Despite the high sequence identity between the EUL domains in EEA and ArathEULS3, both domains recognize different carbohydrate structures.     

GsVAMP72, a novel Glycine soja R-SNARE protein, is involved in regulating plant salt tolerance and ABA sensitivity

Abiotic stress, especially high salinity, is a major threat to agricultural production. It has been well established that SNARE proteins sustain directed vesicle traffic to underpin plant growth and development, yet little is known about the role of SNARE protein in the capacity to withstand abiotic stress, especially in wild soybeans. Here we identified and characterized a GsCBRLK interacting protein, GsVAMP72, which is a putative vesicle-associated membrane protein in Glycine soja. GsVAMP72 protein has a longin domain at its N-terminus, belonging to R-SNARE family. Quantitative real-time (RT) PCR and beta-glucuronidase (GUS) activity assays revealed that the expression of GsVAMP72 was highly and rapidly induced by both high salt and ABA treatments. Overexpression of GsVAMP72 in Arabidopsis significantly reduced salt tolerance by modifying the ionic content and down-regulating expression of stress-responsive genes, including RD29A, COR47, KIN1, COR15A and RAB18. On the other hand, GsVAMP72 overexpression increased plant ABA sensitivity and altered the expression levels of ABA-responsive genes. Subcellular localization analysis showed that eGFP–GsVAMP72 fusion protein was observed on the plasma membrane-like and endosome-like structures but eGFP alone was distributing throughout the cytoplasm in Arabidopsis protoplasts and onion epidermal cells. GsVAMP72 promoter-controlled GUS activity was detected in both vegetative and reproductive organs, and was strongly induced by salt and ABA. In summary, we demonstrated that GsVAMP72 is a novel Glycine soja vesicle-associated membrane protein and is highly involved in regulating plant responses to salt and ABA stresses.     

Alleviation of Salt Stress by Enterobacter sp. EJ01 in Tomato and Arabidopsis Is Accompanied by Up-Regulation of Conserved Salinity Responsive Factors in Plants

Microbiota in the niches of the rhizosphere zones can affect plant growth and responses to environmental stress conditions via mutualistic interactions with host plants. Specifically, some beneficial bacteria, collectively referred to as Plant Growth Promoting Rhizobacteria (PGPRs), increase plant biomass and innate immunity potential. Here, we report that Enterobacter sp. EJ01, a bacterium isolated from sea china pink (Dianthus japonicus thunb) in reclaimed land of Gyehwa-do in Korea, improved the vegetative growth and alleviated salt stress in tomato and Arabidopsis. EJ01 was capable of producing 1-aminocy-clopropane-1-carboxylate (ACC) deaminase and also exhibited indole-3-acetic acid (IAA) production. The isolate EJ01 conferred increases in fresh weight, dry weight, and plant height of tomato and Arabidopsis under both normal and high salinity conditions. At the molecular level, short-term treatment with EJ01 increased the expression of salt stress responsive genes such as DREB2b, RD29A, RD29B, and RAB18 in Arabidopsis. The expression of proline biosynthetic genes (i.e. P5CS1 and P5CS2) and of genes related to priming processes (i.e. MPK3 and MPK6) were also up-regulated. In addition, reactive oxygen species scavenging activities were enhanced in tomatoes treated with EJ01 in stressed conditions. GFP-tagged EJ01 displayed colonization in the rhizosphere and endosphere in the roots of Arabidopsis. In conclusion, the newly isolated Enterobacter sp. EJ01 is a likely PGPR and alleviates salt stress in host plants through multiple mechanisms, including the rapid up-regulation of conserved plant salt stress responsive signaling pathways.     

Abscisic Acid Suppresses the Highly Occurred Somatic Homologous Recombination in Arabidopsis rfc1 Mutant

The phytohormone abscisic acid (ABA) regulates many aspects of plant growth, including seed germination, root growth and cell division. Previous study indicates that ABA treatment increases DNA damage and somatic homologous recombination (HR) in Arabidopsis abo4/pol ? (aba overly-sensitive 4 /DNA polymerase ?) mutants. DNA replication factor C (RFC) complex is required for loading PCNA (Proliferating Cell Nuclear Antigen) during DNA replication. The defect in RFC1, the largest subunit of RFC, causes the high HR and DNA damage sensitivity in Arabidopsis. Here we found that like pol ε/abo4, rfc1 is sensitive to ABA in both ABA-inhibiting seed germination and root growth. However, ABA treatment greatly reduces HR and also reduces the expression of the DNA-damaged marker genes in rfc1. These results suggest that RFC1 plays critical roles in ABA-mediated HR in Arabidopsis.     

Overexpression of maize mitogen-activated protein kinase gene, ZmSIMK1 in Arabidopsis increases tolerance to salt stress

Mitogen-activated protein kinase (MAPK) cascades play a remarkably crucial role in plants. It has been studied intensively in model plants Arabidopsis, tobacco and rice. However, the function of MAPKs in maize (Zea mays L.) has not been well documented. ZmSIMK1 (Zea mays salt-induced mitogen-activated protein kinase 1) is a previously identified MAPK gene in maize. In this research, we charactered ZmSIMK1 and showed that ZmSIMK1 was involved in Arabidopsis salt stress. The genomic organization of ZmSIMK1 gene and its expression in maize have been analyzed. In order to investigate the function of ZmSIMK1, we generated transgenic Arabidopsis constitutively overexpressing ZmSIMK1. Ectopic expression of ZmSIMK1 in Arabidopsis resulted in increased resistance against salt stress. Importantly, ZmSIMK1-overexpressing Arabidopsis exhibited constitutive expression of stress-responsive marker genes, RD29A and P5CS1. Furthermore, RD29A and P5CS1 were upregulated under salt stress. These results suggest that ZmSIMK1 may play an important role in plant salt stress.     

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.     

Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants

Summary We characterized the expression of genes that correspond to a cDNA clone, RD29, which is induced by desiccation, cold and high-salt conditions in Arabidopsis thaliana. Northern analysis of desiccation-induced expression revealed a two-step induction process. Early induction occurs within 20 min and secondary induction occurs 3 h after the start of desiccation. Exogenous abscisic acid (ABA) induces RD29 mRNA within 3 h. Two genes corresponding to RD29, rd29A and rd29B, are located in tandem in an 8 kb region of the Arabidopsis genome and encode hydrophilic proteins. Desiccation induces rd29A mRNA with two-step kinetics, while rd29B is induced only 3 h after the start of desiccation. The expression of both genes is stimulated about 3 h after application of ABA. It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to desiccation and the other induced by changes in osmotic potential. The -glucuronidase (GUS) reporter gene driven by the rd29A promoter was induced at significant levels by desiccation, cold, high-salt conditions and ABA in both transgenic Arabidopsis and tobacco. Histochemical analysis of GUS activity revealed that the rd29A promoter functions in almost all the organs and tissues of vegetative plants during water deficiency.     

Regulation and function of Arabidopsis AtGALK2 gene in abscisic acid response signaling

AtGALK2 belongs to galactokinase of GHMP family in Arabidopsis thaliana. Two homozygous T-DNA insertion mutants (Atgalk2-1 and Atgalk2-2) of the AtGALK2 gene were identified. The AtGALK2 gene was highly expressed in flowers and roots, but less in stems, leaves and petioles. It was found that the expression of AtGALK2 gene was induced by NaCl and ABA. The two Atgalk2 mutants showed higher germination activity when treated with ABA and NaCl than wild type (Col-0). Through comparing the results of seed germination, root growth, stomatal aperture, water loss, and proline accumulation between the Atgalk2 mutants and Col-0, it was found that Atgalk2 mutants showed less sensitive to ABA than Col-0. The expression levels of ABI1, ABI2, RAB18, ABF3, RD22, RD29A, and RD29B in the Atgalk2 mutants were higher than in Col-0. However, the expression level of OST1 in the Atgalk2 mutants was lower than in Col-0. Taken together, these results suggested AtGALK2 was required for abscisic acid regulation of seed germination, root growth and gene expression, and was involved in salt and osmotic stress response in the early development stage. This study provides important clues to galactokinase activities of GHMP family in ABA signaling and plant development.     

Cryptochrome 1b from Sweet Sorghum Regulates Photoperiodic Flowering,Photomorphogenesis, and ABA Response in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cryptochromes are blue/UV-A light receptors that mediate various aspects of plant growth and development. Here, we report the function and signal mechanism of cryptochrome 1b (SbCRY1b) from sweet sorghum [Sorghum bicolor (L.) Moench], a typical short-day cereal plant, to explore its potential for genetic improvement of sweet sorghum varieties. SbCRY1b mRNA enrichment showed almost 24-h diurnal rhythms in both short-day (SD) and long-day (LD) conditions. Overexpression of SbCRY1b rescued the late-flowering and the long hypocotyl phenotypes of cry1cry2 double mutant in the transgenic Arabidopsis. SbCRY1b mediated Arabidopsis FT mRNA expression in LD and HY5 protein accumulation in response to blue light. SbCRY1b protein was located in both the nucleus and cytoplasm and was degraded by 26S proteasomes in response to blue light. SbCRY1b interacted, respectively, with Arabidopsis suppressor of PHYA-1051 (AtSPA1), E3 ubiquitin ligase constitutive photomorphogenesis 1 (AtCOP1), and a putative COP1 from sweet sorghum (SbCOP1) instead of SbSPA1 in vitro in a blue light-dependent manner. The observations imply SbCRY1b functions as a major regulator of photoperiodic flowering and its function is more similar to that of Arabidopsis CRY2. Moreover, SbCRY1b-overexpressed transgenic Arabidopsis showed oversensitivity to abscisic acid (ABA) during seed germination and root development. The expression of abscisic acid-insensitive 4 (ABI4), ABI5, abscisic acid responsive element-binding 1 (ABF1), (sucrose non-fermenting 1)-related protein kinase (SnRK2.3), RD29A, and EM6 was upregulated in the transgenic Arabidopsis. The results demonstrated that SbCRY1b may integrate blue light and ABA signals to regulate plant development.