Weiming Cai

OsLEA3-2, an abiotic stress induced gene of rice plays a key role in salt and drought tolerance.

Late embryogenesis abundant (LEA) proteins are involved in tolerance to drought, cold and high salinity in many different organisms. In this report, a LEA protein producing full-length gene OsLEA3-2 was identified in rice (Oryza sativa) using the Rapid Amplification of cDNA Ends (RACE) method. OsLEA3-2 was found to be only expressed in the embryo and can be induced by abiotic stresses. The coding protein localizes to the nucleus and overexpression of OsLEA3-2 in yeast improved growth performance compared with control under salt- and osmotic-stress conditions. OsLEA3-2 was also inserted into pHB vector and overexpressed in Arabidopsis and rice. The transgenic Arabidopsis seedlings showed better growth on MS media supplemented with 150 mM mannitol or 100 mM NaCl as compared with wild type plants. The transgenic rice also showed significantly stronger growth performance than control under salinity or osmotic stress conditions and were able to recover after 20 days of drought stress. In vitro analysis showed that OsLEA3-2 was able to protect LDH from aggregation on freezing and inactivation on desiccation. These results indicated that OsLEA3-2 plays an important role in tolerance to abiotic stresses.

OsGA2ox5, a Gibberellin Metabolism Enzyme, Is Involved in Plant Growth, the Root Gravity Response and Salt Stress

Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C19-GA2oxs and a smaller class of C20-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C20-GA2oxs subfamily, a subfamily of GA2oxs acting on C20-GAs (GA12, GA53). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.

Quaternized chitosan oligomers as novel elicitors inducing protection against B. cinerea in Arabidopsis

Chitosan oligomers prepared from enzyme hydrolysis of chitosan have for many years been recognized as potent elicitors of plant innate immunity, but their efficacy is limited by the degree of polymerization and the degree of acetylation. In this study, we presented a new type of chitosan oligomers (COS), with the name of quaternized chitosan oligomers (QCOS) that were prepared by reacting COS with glycidyltrimethylammonium chloride and overcome these problems. First, QCOS clearly induced hydrogen peroxide accumulation and callose deposition in Arabidopsis seedlings. Second, we found that PAD3 expression was significantly upregulated more than 5-fold by QCOS as compared to COS. Further, PAD3 expression activated by QCOS was required for inducing Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, independent of salicylic acid signaling. These results demonstrate that quaternized modifications of COS possess better elicitor properties than the original COS and that QCOS stimulate plant protection against B. cinerea attack in Arabidopsis. Importantly, our work provides a novel and valuable strategy for enhancing elicitor activities of other types of oligosaccharides for plant innate immunity.

Microarray Analyses and Comparisons of Upper or Lower Flanks of Rice Shoot Base Preceding Gravitropic Bending

Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (Oryza sativa sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base.

AtDOF5.4/OBP4, a DOF Transcription Factor Gene that Negatively Regulates Cell Cycle Progression and Cell Expansion in Arabidopsis thaliana

In contrast to animals, plant development involves continuous organ formation, which requires strict regulation of cell proliferation. The core cell cycle machinery is conserved across plants and animals, but plants have developed new mechanisms that precisely regulate cell proliferation in response to internal and external stimuli. Here, we report that the DOF transcription factor OBP4 negatively regulates cell proliferation and expansion. OBP4 is a nuclear protein. Constitutive and inducible overexpression of OBP4 reduced the cell size and number, resulting in dwarf plants. Inducible overexpression of OBP4 in Arabidopsis also promoted early endocycle onset and inhibited cell expansion, while inducible overexpression of OBP4 fused to the VP16 activation domain in Arabidopsis delayed endocycle onset and promoted plant growth. Furthermore, gene expression analysis showed that cell cycle regulators and cell wall expansion factors were largely down-regulated in the OBP4 overexpression lines. Short-term inducible analysis coupled with in vivo ChIP assays indicated that OBP4 targets the CyclinB1;1, CDKB1;1 and XTH genes. These results strongly suggest that OBP4 is a negative regulator of cell cycle progression and cell growth. These findings increase our understanding of the transcriptional regulation of the cell cycle in plants.

A ginseng PgTIP1 gene whose protein biological activity related to Ser128 residue confers faster growth and enhanced salt stress tolerance in Arabidopsis

Water movement across cellular membranes is mostly regulated by aquaporins. A tonoplast intrinsic protein PgTIP1 from Panax ginseng has been found to play an important role in plant growth and development, and also in the response of plants to abiotic stress. However, the regulation of its function and activity remains unknown. To answer this question, mutated forms of PgTIP1 were made by replacing Ser(128) with Ala (named S128A) or Asp (named S128D), and also by replacing Thr(54) with Ala (named T54A) or Asp (named T54D). Then, wild type or mutated PgTIP1 was expressed in yeast and water transport was monitored in protoplasts. The substitution of Ser(128) abolished the water channel activity of PgTIP1, while the substitution of Thr(54) did not inhibit its activity. Moreover, the overexpression of PgTIP1 but not S128A or S128D in Arabidopsis significantly increased plant growth as determined by biomass production, it also had a beneficial effect on salt stress tolerance. Importantly, the overexpression of PgTIP1 led to the altered expression of stress-related genes, which made the plants more tolerant to salt stress. Our results demonstrated that PgTIP1 conferred faster growth and enhanced tolerance to salt in Arabidopsis, and that its biological activity related to Ser(128) residue.

Functional characterization of the BnNCED3 gene in Brassica napus

Abscisic acid (ABA) has been implicated in plant adaptation to various environmental stresses and the regulation of seed dormancy, leaf senescence and organ abscission progression. The cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) family proteins is a critical step in the regulation of abscisic acid (ABA) synthesis in plants. In the present study, the NCED family gene BnNCED3 was isolated from Brassica napus. BnNCED3 encodes a 592-amino acid protein with high amino acid sequence similarity to the Arabidopsis AtNCED3 protein. Expression pattern assays revealed that BnNCED3 is ubiquitously expressed at different levels in all the examined organs. Furthermore, the overexpression of BnNCED3 contributed to ABA accumulation and NO and ROS generation in transgenic Arabidopsis plants, thereby enhancing abiotic stress tolerance. These experiments also indicated the involvement of BnNCED3 in the control of plant development in transgenic Arabidopsis, such as the inhibition of seed germination, lateral root initiation, early phase changes and the enhancement of ABA-associated leaf senescence. Together, these results indicated that BnNCED3 is at least partly involved in both stress adaptation and plant development through the regulation of ABA biosynthesis.

Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings exposed to microgravity spaceflight conditions on board the SJ-10 recoverable satellite

DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many studies of DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in plants challenged by exposure to microgravity conditions. In this report, we mapped the Arabidopsis genome methylation pattern changes associated with microgravity conditions on board the Chinese recoverable scientific satellite SJ-10 at single-base resolution. Interestingly, we found epigenetic differences in Arabidopsis seedlings exposed to microgravity in that the Arabidopsis genome exhibits lower methylation levels in the CHG, CHH, and CpG contexts under microgravity conditions. Microgravity stimulation was related to altered methylation of a number of genes, including DNA methylation-associated genes, hormone signaling related genes, cell-wall modification genes and transposable elements (TEs). Relatively unstable DNA methylation of TEs was responsible for the induction of active transposons. These observations suggest that DNA demethylation within TEs may affect the transcription of transposons in response to microgravity conditions. In summary, the results of this investigation are beneficial for understanding the mechanism of plant adaptation to microgravity and improve strategies to allow plants to adapt to space.Plant biology: Spaceflight leaves its epigenetic mark on seedling DNASpaceflight alters the pattern of chemical tags that adorn DNA in plant seedlings. Weiming Cai and colleagues from the Shanghai Institute of Plant Physiology and Ecology, China, profiled the genome-wide epigenetic patterns of Arabidopsis thaliana seedlings that spent 60 h in the microgravity of low Earth orbit aboard the Shijian-10 recoverable satellite. They analyzed the distribution of methyl tags across the genome — an epigenetic mark that affects gene expression levels — and found that seedlings exposed to microgravity had lower methylation on average than control plants grown on the ground, although certain genes related to methylation, transcription factors and hormones tended to be more methylated. Epigenetic differences were also observed among genes involved in cell-wall modification and in transposable elements. The findings could help inform the design of plants optimized for growth in space.

The Small G Protein AtRAN1 Regulates Vegetative Growth and Stress Tolerance in Arabidopsis thaliana

The evolutionarily conserved small G-protein Ran plays important role in nuclear translocation of proteins, cell cycle regulation, and nuclear envelope maintenance in mammalian cells and yeast. Arabidopsis Ran proteins are encoded by a family of four genes and are highly conserved at the protein level. However, their biological functions are poorly understood. We report here that AtRAN1 plays an important role in vegetative growth and the molecular improvement of stress tolerance in Arabidopsis. AtRAN1 overexpression promoted vegetative growth and enhanced abiotic tolerance, while the atran1 atran3 double mutant showed higher freezing sensitivity than WT. The AtRAN1 gene is ubiquitously expressed in plants, and the expression levels are higher in the buds, flowers and siliques. Subcellular localization results showed that AtRAN1 is mainly localized in the nucleus, with some present in the cytoplasm. AtRAN1 could maintain cell division and cell cycle progression and promote the formation of an intact nuclear envelope, especially under freezing conditions.