Guodong Yang

Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice

BackgroundGenes in the CCCH family encode zinc finger proteins containing the motif with three cysteines and one histidine residues. They have been known to play important roles in RNA processing as RNA-binding proteins in animals. To date, few plant CCCH proteins have been studied functionally.ResultsIn this study, a comprehensive computational analysis identified 68 and 67 CCCH family genes in Arabidopsis and rice, respectively. A complete overview of this gene family in Arabidopsis was presented, including the gene structures, phylogeny, protein motifs, and chromosome locations. In addition, a comparative analysis between these genes in Arabidopsis and rice was performed. These results revealed that the CCCH families in Arabidopsis and rice were divided into 11 and 8 subfamilies, respectively. The gene duplication contributed to the expansion of the CCCH gene family in Arabidopsis genome. Expression studies indicated that CCCH proteins exhibit a variety of expression patterns, suggesting diverse functions. Finally, evolutionary analysis showed that one subfamily is higher plant specific. The expression profile indicated that most members of this subfamily are regulated by abiotic or biotic stresses, suggesting that they could have an effective role in stress tolerance.ConclusionOur comparative genomics analysis of CCCH genes and encoded proteins in two model plant species provides the first step towards the functional dissection of this emerging family of potential RNA-binding proteins.

Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast.

A cDNA clone encoding a 64-amino acid type 3 metallothionein protein, designated GhMT3a, was isolated from cotton (Gossypium hirsutum) by cDNA library screening. Northern blot analysis indicated that mRNA accumulation of GhMT3a was up-regulated not only by high salinity, drought, and low temperature stresses, but also by heavy metal ions, abscisic acid (ABA), ethylene, and reactive oxygen species (ROS) in cotton seedlings. Transgenic tobacco (Nicotiana tabacum) plants overexpressing GhMT3a showed increased tolerance against abiotic stresses compared with wild-type plants. Interestingly, the induced expression of GhMT3a by salt, drought, and low-temperature stresses could be inhibited in the presence of antioxidants. H2O2 levels in transgenic tobacco plants were only half of that in wild-type (WT) plants under such stress conditions. According to in vitro assay, recombinant GhMT3a protein showed an ability to bind metal ions and scavenge ROS. Transgenic yeast overexpressing GhMT3a also showed higher tolerance against ROS stresses. Taken together, these results indicated that GhMT3a could function as an effective ROS scavenger and its expression could be regulated by abiotic stresses through ROS signalling.

GhZFP1, a novel CCCH‐type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5

* Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

Stress-Induced Alternative Splicing Provides a Mechanism for the Regulation of MicroRNA Processing in Arabidopsis thaliana

MicroRNAs (miRNAs) have emerged as a class of regulators of gene expression through posttranscriptional degradation or translational repression in living cells. Increasing evidence points to the important relationship between miRNAs and environmental stress responses, but the regulatory mechanisms in plants are poorly understood. Here, we found that Arabidopsis thaliana intronic miR400 was cotranscribed with its host gene (At1g32583) and downregulated by heat treatment. Intriguingly, an alternative splicing (AS) event that occurred in the intron (306 bp) where MIR400 was located was specifically induced by heat stress. A 100 bp fragment was excised, and the remaining 206 bp intron containing MIR400 transcripts was retained in the host gene. The stress-induced AS event thus resulted in greater accumulation of miR400 primary transcripts and a low level of mature miR400. Together, these results provide the direct evidence that AS acts as a regulatory mechanism linking miRNAs and environmental stress in plants.

The mitochondrial phosphate transporters modulate plant responses to salt stress via affecting ATP and gibberellin metabolism in Arabidopsis thaliana.

The mitochondrial phosphate transporter (MPT) plays crucial roles in ATP production in plant cells. Three MPT genes have been identified in Arabidopsis thaliana. Here we report that the mRNA accumulations of AtMPTs were up-regulated by high salinity stress in A. thaliana seedlings. And the transgenic lines overexpressing AtMPTs displayed increased sensitivity to salt stress compared with the wild-type plants during seed germination and seedling establishment stages. ATP content and energy charge was higher in overexpressing plants than those in wild-type A. thaliana under salt stress. Accordingly, the salt-sensitive phenotype of overexpressing plants was recovered after the exogenous application of atractyloside due to the change of ATP content. Interestingly, Genevestigator survey and qRT-PCR analysis indicated a large number of genes, including those related to gibberellin synthesis could be regulated by the energy availability change under stress conditions in A. thaliana. Moreover, the exogenous application of uniconazole to overexpressing lines showed that gibberellin homeostasis was disturbed in the overexpressors. Our studies reveal a possible link between the ATP content mediated by AtMPTs and gibberellin metabolism in responses to high salinity stress in A. thaliana.

GhDREB1 enhances abiotic stress tolerance, delays GA‐mediated development and represses cytokinin signalling in transgenic Arabidopsis

Plants vary significantly in their ability to tolerate low temperatures. The CBF/DREB1 cold response pathway has been identified in many plant species and plays a pivotal role in low temperature tolerance. Here, we show that GhDREB1 is a functional homologue and elevates the freezing, salt and osmotic stress tolerance of transgenic Arabidopsis. The constitutive expression of GhDREB1 in Arabidopsis caused dwarfism and late flowering phenotypes, which could be rescued by exogenous application of GA(3). Endogenous bioactive GA contents were significantly lower in GhDREB1 overexpressing Arabidopsis than in wild-type plants. RT-PCR analyses revealed that the transcript levels of the GA synthase genes were higher in transgenics than in wild-type plants, whereas the GA deactivating genes were lower. Flowering related genes in different regulatory pathways were also affected by GhDREB1, which may account for the flowering delay phenotype. Moreover, the GhDREB1 overexpressing Arabidopsis exhibited decreased sensitivity to cytokinin (CK) which is associated with repression of expression of type-B and type-A ARRs, two key components in the CK-signalling pathway.

Salt-induced transcription factor MYB74 is regulated by the RNA-directed DNA methylation pathway in Arabidopsis

Highlight AtMYB74, a R2R3-MYB gene, is transcriptionally regulated through RdDM for response to salt stress. The accumulation of siRNA targeting to the AtMYB74 promoter region is essential for maintaining AtMYB74 expression.

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.

Seed-specific overexpression of antioxidant genes in Arabidopsis enhances oxidative stress tolerance during germination and early seedling growth.

Enzymatic and non-enzymatic antioxidants play important roles in the tolerance of abiotic stress. To increase the resistance of seeds to oxidative stress, At2S3 promoter from Arabidopsis was used to achieve overexpression of the antioxidants in a seed-specific manner. This promoter was shown to be capable of driving the target gene to have a high level of expression in seed-related organs, including siliques, mature seeds, and early seedlings, thus making its molecular farming applications in plants possible. Subsequently, genes encoding Mn-superoxide dismutase (MSD1), catalase (CAT1), and homogentisate phytyltransferase (HPT1, responsible for the first committed reaction in the tocopherol biosynthesis pathway) were overexpressed in Arabidopsis under the control of the At2S3 promoter. Double overexpressers co-expressing two enzymes and triple overexpressers were produced by cross pollination. Mn-SOD and total CAT activities, as well as gamma-tocopherol content, significantly increased in the corresponding overproduction lines. Moreover, single MSD1-transgene, double, and triple overexpressers displayed remarkably enhanced oxidative stress tolerance compared to wild type during seed germination and early seedling growth. Interestingly, an increase in the total CAT activity was also observed in the single MSD1-transgenic lines as a result of MSD1 overexpression. Together, the combined increase in Mn-SOD and CAT activities in seeds plays an essential role in the improvement of antioxidant capacity at early developmental stage in Arabidopsis.

SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis.

Highlight AtPP2-B11, an F-box protein, enhances the salt stress tolerance by regulating AnnAt1 expression, repressing reactive oxygen species production, and disrupting Na+ homeostasis in Arabidopsis.