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author:("Wu, changbai")
1.  Salt-induced transcription factor MYB74 is regulated by the RNA-directed DNA methylation pathway in Arabidopsis  
Journal of Experimental Botany  2015;66(19):5997-6008.
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.
Salt stress is one of the major abiotic stresses in agriculture worldwide that causes crop failure by interfering with the profile of gene expression and cell metabolism. Transcription factors and RNA-directed DNA methylation (RdDM) play an important role in the regulation of gene activation under abiotic stress in plants. This work characterized AtMYB74, a member of the R2R3-MYB gene family, which is transcriptionally regulated mainly by RdDM as a response in salt stress in Arabidopsis. Bisulphite sequencing indicated that 24-nt siRNAs target a region approximately 500bp upstream of the transcription initiation site of AtMYB74, which is heavily methylated. Levels of DNA methylation in this region were significantly reduced in wild type plants under salt stress, whereas no changes were found in RdDM mutants. Northern blot and quantitative real-time reverse transcription PCR analysis showed that the accumulation of 24-nt siRNAs was decreased in WT plants under salt stress. Further promoter deletion analysis revealed that the siRNA target region is essential for maintaining AtMYB74 expression patterns. In addition, transgenic plants overexpressing AtMYB74 displayed hypersensitivity to NaCl during seed germination. These results suggest that changes in the levels of the five 24-nt siRNAs regulate the AtMYB74 transcription factor via RdDM in response to salt stress.
PMCID: PMC4566987  PMID: 26139822
Arabidopsis; AtMYB74; RNA-directed DNA methylation; salt stress; siRNA; transcription; transcription factor.
2.  SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis  
Journal of Experimental Botany  2015;66(15):4683-4697.
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.
Skp1–Cullin–F-box (SCF) E3 ligases are essential to the post-translational regulation of many important factors involved in cellular signal transduction. In this study, we identified an F-box protein from Arabidopsis thaliana, AtPP2-B11, which was remarkably induced with increased duration of salt treatment in terms of both transcript and protein levels. Transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibited obvious tolerance to high salinity, whereas the RNA interference line was more sensitive to salt stress than wild-type plants. Isobaric tag for relative and absolute quantification analysis revealed that 4311 differentially expressed proteins were regulated by AtPP2-B11 under salt stress. AtPP2-B11 could upregulate the expression of annexin1 (AnnAt1) and function as a molecular link between salt stress and reactive oxygen species accumulation in Arabidopsis. Moreover, AtPP2-B11 influenced the expression of Na+ homeostasis genes under salt stress, and the AtPP2-B11 overexpressing lines exhibited lower Na+ accumulation. These results suggest that AtPP2-B11 functions as a positive regulator in response to salt stress in Arabidopsis.
PMCID: PMC4507775  PMID: 26041321
AnnAt1; E3 AtPP2-B11; ligases; Na+ homeostasis; ROS.
3.  Overexpression of tomato SlNAC1 transcription factor alters fruit pigmentation and softening 
BMC Plant Biology  2014;14:351.
Fruit maturation and ripening are genetically regulated processes that involve a complex interplay of plant hormones, growth regulators and multiple biological and environmental factors. Tomato (Solanum lycopersicum) has been used as a model of biological and genetic studies on the regulation of specific ripening pathways, including ethylene, carotenoid and cell wall metabolism. This model has also been used to investigate the functions of upstream signalling and transcriptional regulators. Thus far, many ripening-associated transcription factors that influence fruit development and ripening have been reported. NAC transcription factors are plant specific and play important roles in many stages of plant growth and development, such as lateral root formation, secondary cell wall synthesis, and embryo, floral organ, vegetative organ and fruit development.
Tissue-specific analysis by quantitative real-time PCR showed that SlNAC1 was highly accumulated in immature green fruits; the expression of SlNAC1 increased with fruit ripening till to the highest level at 7 d after the breaker stage. The overexpression of SlNAC1 resulted in reduced carotenoids by altering carotenoid pathway flux and decreasing ethylene synthesis mediated mainly by the reduced expression of ethylene biosynthetic genes of system-2, thus led to yellow or orange mature fruits. The results of yeast one-hybrid experiment demonstrated that SlNAC1 can interact with the regulatory regions of genes related lycopene and ethylene synthesis. These results also indicated that SlNAC1 inhibited fruit ripening by affecting ethylene synthesis and carotenoid accumulation in SlNAC1 overexpression lines. In addition, the overexpression of SlNAC1 reduced the firmness of the fruits and the thickness of the pericarp and produced more abscisic acid, resulting in the early softening of fruits. Hence, in SlNAC1 overexpression lines, both ethylene-dependent and abscisic acid-dependent pathways are regulated by SlNAC1 in fruit ripening regulatory network.
SlNAC1 had a broad influence on tomato fruit ripening and regulated SlNAC1 overexpression tomato fruit ripening through both ethylene-dependent and abscisic acid-dependent pathways. Thus, this study provided new insights into the current model of tomato fruit ripening regulatory network.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0351-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4272553  PMID: 25491370
Abscisic acid; Ethylene; Fruit ripening; Gene expression; SlNAC1; Tomato
4.  TM6, a Novel Nuclear Matrix Attachment Region, Enhances Its Flanking Gene Expression through Influencing Their Chromatin Structure 
Molecules and Cells  2013;36(2):127-137.
Nuclear matrix attachment regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase II-binding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two tobacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription.
PMCID: PMC3887953  PMID: 23852133
chromatin accessibility; DNA methylation; matrix attachment regions; transcription activation; transformation
5.  GhWRKY40, a Multiple Stress-Responsive Cotton WRKY Gene, Plays an Important Role in the Wounding Response and Enhances Susceptibility to Ralstonia solanacearum Infection in Transgenic Nicotiana benthamiana 
PLoS ONE  2014;9(4):e93577.
WRKY transcription factors form one of the largest transcription factor families and function as important components in the complex signaling processes that occur during plant stress responses. However, relative to the research progress in model plants, far less information is available on the function of WRKY proteins in cotton. In the present study, we identified the GhWRKY40 gene in cotton (Gossypium hirsutum) and determined that the GhWRKY40 protein is targeted to the nucleus and is a stress-inducible transcription factor. The GhWRKY40 transcript level was increased upon wounding and infection with the bacterial pathogen Ralstonia solanacearum. The overexpression of GhWRKY40 down-regulated most of the defense-related genes, enhanced the wounding tolerance and increased the susceptibility to R. solanacearum. Consistent with a role in multiple stress responses, we found that the GhWRKY40 transcript level was increased by the stress hormones salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET). Moreover, GhWRKY40 interacted with the MAPK kinase GhMPK20, as shown using yeast two-hybrid and bimolecular fluorescence complementation systems. Collectively, these results suggest that GhWRKY40 is regulated by SA, MeJA and ET signaling and coordinates responses to wounding and R. solanacearum attack. These findings highlight the importance of WRKYs in regulating wounding- and pathogen-induced responses.
PMCID: PMC3991585  PMID: 24747610
6.  Arabidopsis SAG protein containing the MDN1 domain participates in seed germination and seedling development by negatively regulating ABI3 and ABI5 
Journal of Experimental Botany  2013;65(1):35-45.
SAG encodes a MDN1 domain containing protein in Arabidopsis. Seeds of a T-DNA insertion line of this gene exhibited hypersensitivity to ABA, mannitol and NaCl during seed germination and early seedling development. SAG was genetically epistatic to ABI3 and ABI5
Three proteins containing a midasin homologue 1 (MDN1) domain from the yeast Solanum chacoense and Arabidopsis thaliana have important functions in yeast survival, seed development, and female gametogenesis. In this study, a novel protein containing the MDN1 domain from Arabidopsis negatively regulated abscisic acid (ABA) signalling during seed germination. Seeds of a T-DNA insertion line of this gene exhibited increased sensitivity to ABA during seed germination and seedling development (named sag). By contrast, seeds with overexpressed AtSAG (OX2) were less sensitive to ABA. The seeds of the sag mutant showed similar sensitivity to high concentrations of mannitol and NaCl during these stages. AtSAG was also highly expressed in germinating seeds. However, ABA-induced AtSAG expression remained almost unchanged. ABA-responsive marker genes, including ABI3, ABI5, Em1, Em6, RD29A, and RAB18, were upregulated in sag mutants but were downregulated in OX2. Genetic analyses indicated that the function of AtSAG in ABA signalling depended on ABI3 and ABI5. The expression of some target genes of ABI3 and ABI5, such as seed storage protein and oleosin genes, was induced higher by ABA in sag mutants than in wild-type germinated seeds, even higher than in abi5 mutants. This finding indicated that other regulators similar to ABI3 or ABI5 played a role during these stages. Taken together, these results indicate that AtSAG is an important negative regulator of ABA signalling during seed germination and seedling development.
PMCID: PMC3883281  PMID: 24163287
Abscisic acid; AtSAG; drought; MDN1 domain; salt; seed germination
7.  The Mitochondrial Phosphate Transporters Modulate Plant Responses to Salt Stress via Affecting ATP and Gibberellin Metabolism in Arabidopsis thaliana 
PLoS ONE  2012;7(8):e43530.
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.
PMCID: PMC3427375  PMID: 22937061
8.  GhWRKY15, a member of the WRKY transcription factor family identified from cotton (Gossypium hirsutum L.), is involved in disease resistance and plant development 
BMC Plant Biology  2012;12:144.
As a large family of regulatory proteins, WRKY transcription factors play essential roles in the processes of adaptation to diverse environmental stresses and plant growth and development. Although several studies have investigated the role of WRKY transcription factors during these processes, the mechanisms underlying the function of WRKY members need to be further explored, and research focusing on the WRKY family in cotton crops is extremely limited.
In the present study, a gene encoding a putative WRKY family member, GhWRKY15, was isolated from cotton. GhWRKY15 is present as a single copy gene, and a transient expression analysis indicated that GhWRKY15 was localised to the nucleus. Additionally, a group of cis-acting elements associated with the response to environmental stress and plant growth and development were detected in the promoter. Consistently, northern blot analysis showed that GhWRKY15 expression was significantly induced in cotton seedlings following fungal infection or treatment with salicylic acid, methyl jasmonate or methyl viologen. Furthermore, GhWRKY15-overexpressing tobacco exhibited more resistance to viral and fungal infections compared with wild-type tobacco. The GhWRKY15-overexpressing tobacco also exhibited increased RNA expression of several pathogen-related genes, NONEXPRESSOR OF PR1, and two genes that encode enzymes involved in ET biosynthesis. Importantly, increased activity of the antioxidant enzymes POD and APX during infection and enhanced expression of NtAPX1 and NtGPX in transgenic tobacco following methyl viologen treatment were observed. Moreover, GhWRKY15 transcription was greater in the roots and stems compared with the expression in the cotyledon of cotton, and the stems of transgenic plants displayed faster elongation at the earlier shooting stages compared with wide type tobacco. Additionally, exposure to abiotic stresses, including cold, wounding and drought, resulted in the accumulation of GhWRKY15 transcripts.
Overall, our data suggest that overexpression of GhWRKY15 may contribute to the alteration of defence resistance to both viral and fungal infections, probably through regulating the ROS system via multiple signalling pathways in tobacco. It is intriguing that GhWRKY15 overexpression in tobacco affects plant growth and development, especially stem elongation. This finding suggests that the role of the WRKY proteins in disease resistance may be closely related to their function in regulating plant growth and development.
PMCID: PMC3489871  PMID: 22883108
GhWRKY15; Cotton; Disease resistance; SA; ROS; Plant development
9.  GhMPK16, a novel stress-responsive group D MAPK gene from cotton, is involved in disease resistance and drought sensitivity 
BMC Molecular Biology  2011;12:22.
Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Members of group A and B have been extensively characterized, but little information on the group D MAPKs has been reported.
In this study, we isolated and characterised GhMPK16, the first group D MAPK gene found in cotton. Southern blot analysis suggests GhMPK16 is single copy in the cotton genome, and RNA blot analysis indicates that GhMPK16 transcripts accumulate following pathogen infection and treatment with multiple defense-related signal molecules. The analysis of the promoter region of GhMPK16 revealed a group of putative cis-acting elements related to stress responses. Subcellular localization analysis suggests that GhMPK16 acts in the nucleus. Transgenic Arabidopsis overexpressing GhMPK16 displayed significant resistance to fungi (Colletotrichum nicotianae and Alternaria alternata) and bacteria (Pseudomonas solanacearum) pathogen, and the transcripts of pathogen-related (PR) genes were more rapidly and strongly induced in the transgenic plants. Furthermore, transgenic Arabidopsis showed reduced drought tolerance and rapid H2O2 accumulation.
These results suggest that GhMPK16 might be involved in multiple signal transduction pathways, including biotic and abiotic stress signaling pathways.
PMCID: PMC3117701  PMID: 21575189
Cotton (Gossypium hirsutum); GhMPK16; Pathogen resistance; Drought sensitivity
10.  Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast 
Journal of Experimental Botany  2008;60(1):339-349.
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.
PMCID: PMC3071772  PMID: 19033550
Abiotic stress; antioxidant; GhMT3a; ROS; transgenic tobacco; yeast
11.  Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice 
BMC Genomics  2008;9:44.
Genes 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.
In 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.
Our 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.
PMCID: PMC2267713  PMID: 18221561

Results 1-11 (11)