VQ Protein Research Articles

Structural basis for the regulation of plant transcription factor WRKY33 by the VQ protein SIB1

The WRKY transcription factors play essential roles in a variety of plant signaling pathways associated with biotic and abiotic stress response. The transcriptional activity of many WRKY members are regulated by a class of intrinsically disordered VQ proteins. While it is known that VQ proteins interact with the WRKY DNA-binding domains (DBDs), also termed as the WRKY domains, structural information regarding VQ-WRKY interaction is lacking and the regulation mechanism remains unknown. Herein we report a solution NMR study of the interaction between Arabidopsis WRKY33 and its regulatory VQ protein partner SIB1. We uncover a SIB1 minimal sequence neccessary for forming a stable complex with WRKY33 DBD, which comprises not only the consensus “FxxhVQxhTG” VQ motif but also its preceding region. We demonstrate that the βN-strand and the extended βN-β1 loop of WRKY33 DBD form the SIB1 docking site, and build a structural model of the complex based on the NMR paramagnetic relaxation enhancement and mutagenesis data. Based on this model, we further identify a cluster of positively-charged residues in the N-terminal region of SIB1 to be essential for the formation of a SIB1-WRKY33-DNA ternary complex. These results provide a framework for the mechanism of SIB1-enhanced WRKY33 transcriptional activity.

Overexpression of the potato VQ31 enhances salt tolerance in Arabidopsis.

Plant-specific VQ proteins have crucial functions in the regulation of plant growth and development, as well as in plant abiotic stress responses. Their roles have been well established in the model plant Arabidopsis thaliana; however, the functions of the potato VQ proteins have not been adequately investigated. The VQ protein core region contains a short FxxhVQxhTG amino acid motif sequence. In this study, the VQ31 protein from potato was cloned and functionally characterized. The complete open reading frame (ORF) size of StVQ31 is 672 bp, encoding 223 amino acids. Subcellular localization analysis revealed that StVQ31 is located in the nucleus. Transgenic Arabidopsis plants overexpressing StVQ31 exhibited enhanced salt tolerance compared to wild-type (WT) plants, as evidenced by increased root length, germination rate, and chlorophyll content under salinity stress. The increased tolerance of transgenic plants was associated with increased osmotic potential (proline and soluble sugars), decreased MDA accumulation, decreased total protein content, and improved membrane integrity. These results implied that StVQ31 overexpression enhanced the osmotic potential of the plants to maintain normal cell growth. Compared to the WT, the transgenic plants exhibited a notable increase in antioxidant enzyme activities, reducing cell membrane damage. Furthermore, the real-time fluorescence quantitative PCR analysis demonstrated that StVQ31 regulated the expression of genes associated with the response to salt stress, including ERD, LEA4-5, At2g38905, and AtNCED3. These findings suggest that StVQ31 significantly impacts osmotic and antioxidant cellular homeostasis, thereby enhancing salt tolerance.

TaVQ22 Interacts with TaWRKY19-2B to Negatively Regulate Wheat Resistance to Sheath Blight.

Wheat sheath blight caused by the necrotic fungal pathogen Rhizoctonia cerealis is responsible for severe damage to bread wheat. Reactive oxygen species (ROS) are vital for stress resistance by plants and their homeostasis plays an important role in wheat resistance to sheath blight. Valine-glutamine (VQ) proteins play important roles in plant growth and development, and responses to biotic and abiotic stresses. However, the functional mechanism mediated by wheat VQ protein in response to sheath blight via ROS homeostasis regulation is unclear. In this study, we identified TaVQ22 protein containing the VQ motif and clarified the functional mechanisms involved in the defense of wheat against R. cerealis. TaVQ22 silencing reduced the accumulation of ROS and enhanced the resistance of wheat to R. cerealis. In addition, we showed that TaVQ22 regulated ROS generation by interacting with the WRKY transcription factor TaWRKY19-2B, thereby indicating that TaVQ22 and TaWRKY19-2B formed complexes in the plant cell nucleus. Yeast two-hybrid analysis showed that the VQ motif in TaVQ22 is crucial for the interaction, where it inhibits the transcriptional activation function of TaWRKY19-2B. In summary, TaVQ22 interacts with TaWRKY19-2B to regulate ROS homeostasis and negatively regulate the defense response to R. cerealis infection. This study provides novel insights into the mechanism that allows VQ protein to mediate the immune response in plants.

Characterization of the wheat VQ protein family and expression of candidate genes associated with seed dormancy and germination

BackgroundSeed dormancy and germination determine wheat resistance to pre-harvest sprouting and thereby affect grain yield and quality. Arabidopsis VQ genes have been shown to influence seed germination; however, the functions of wheat VQ genes have not been characterized.ResultsWe identified 65 TaVQ genes in common wheat and named them TaVQ1–65. We identified 48 paralogous pairs, 37 of which had Ka/Ks values greater than 1, suggesting that most TaVQ genes have experienced positive selection. Chromosome locations, gene structures, promoter element analysis, and gene ontology annotations of the TaVQs showed that their structures determined their functions and that structural changes reflected functional diversity. Transcriptome-based expression analysis of 62 TaVQ genes and microarray analysis of 11 TaVQ genes indicated that they played important roles in diverse biological processes. We compared TaVQ gene expression and seed germination index values among wheat varieties with contrasting seed dormancy and germination phenotypes and identified 21 TaVQ genes that may be involved in seed dormancy and germination.ConclusionsSixty-five TaVQ proteins were identified for the first time in common wheat, and bioinformatics analyses were used to investigate their phylogenetic relationships and evolutionary divergence. qRT-PCR data showed that 21 TaVQ candidate genes were potentially involved in seed dormancy and germination. These findings provide useful information for further cloning and functional analysis of TaVQ genes and introduce useful candidate genes for the improvement of PHS resistance in wheat.

The poplar VQ1 gene confers salt tolerance and pathogen resistance in transgenic Arabidopsis plants via changes in hormonal signaling.

The VQ protein family is plant-specific, and is involved in growth, development, and biotic and abiotic stress responses. In this study, we found that the gene expression of poplar VQ1(Potri.001G029700) from Populus trichocarpa varied remarkably under salt stress and hormones associated with disease. A subcellular localization experiment showed that VQ1 was localized in the nucleus and cytomembrane in tobacco. The overexpression of VQ1 in Arabidopsis thaliana enhanced its resistance to salt stress and disease, and was also responsive to it through abscisic acid. Compared with wild-type, transgenic Arabidopsis lines had significantly increased levels of abscisic acid and salicylic acid. The expression of some stress-related genes, such as MPK6, NPR1, and PDF1.2, was significantly up-regulated by salt in transgenic plants, while WRKY70, ABI1, KUP6, and NCED2 were significantly down-regulated by Pseudomonas syringae pv. tomato DC3000 in transgenic plants. Together, these results demonstrate that VQ1 modulates hormonal signaling to confer multiple biotic and abiotic stress responses in transgenic Arabidopsis plants.

Genome-wide identification and expression analysis of the VQ gene family in Cucurbita pepo L.

VQ protein is a plant specific protein, which plays an important role in plant growth and development and biological and abiotic stress response. This study aimed to systematically analyze for the first time the VQ of Cucurbita pepo and understand their expression patterns in response to different stimuli. Herein, 44 VQ genes were identified, which were divided into eight groups (I–VIII) based on phylogenetic analysis. Two genes (CpVQ1 and CpVQ2) could not be located on the chromosome, whereas the remaining CpVQ genes were randomly distributed on the chromosomes, except for chromosomes 15 and 18. Noteworthy, the main event driving the expansion of the VQ gene family was chromosome fragment duplication. Based on qRT-PCR analysis, VQ genes are expressed in different tissues, and VQ genes are differentially regulated under a variety of abiotic stresses and powdery mildew stress, indicating that they play an important role in plant stress response and other aspects. This report presents the first systematic analysis of VQ genes from C. pepo and provides a solid foundation for further research of the specific functions of VQ proteins.

Valine-Glutamine Proteins in Plant Responses to Oxygen and Nitric Oxide.

Multigene families coding for valine-glutamine (VQ) proteins have been identified in all kind of plants but chlorophytes. VQ proteins are transcriptional regulators, which often interact with WRKY transcription factors to regulate gene expression sometimes modulated by reversible phosphorylation. Different VQ-WRKY complexes regulate defense against varied pathogens as well as responses to osmotic stress and extreme temperatures. However, despite these well-known functions, new regulatory activities for VQ proteins are still to be explored. Searching public Arabidopsis thaliana transcriptome data for new potential targets of VQ-WRKY regulation allowed us identifying several VQ protein and WRKY factor encoding genes that were differentially expressed in oxygen-related processes such as responses to hypoxia or ozone-triggered oxidative stress. Moreover, some of those were also differentially regulated upon nitric oxide (NO) treatment. These subsets of VQ and WRKY proteins might combine into different VQ-WRKY complexes, thus representing a potential regulatory core of NO-modulated and O2-modulated responses. Given the increasing relevance that gasotransmitters are gaining as plant physiology regulators, and particularly considering the key roles exerted by O2 and NO in regulating the N-degron pathway-controlled stability of transcription factors, VQ and WRKY proteins could be instrumental in regulating manifold processes in plants.

Sugarcane mosaic virus mediated changes in cytosine methylation pattern and differentially transcribed fragments in resistance-contrasting sugarcane genotypes.

Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it is mainly controlled by using resistant cultivars. Studies on the changes in sugarcane transcriptome provided the first insights about the molecular basis underlying the genetic resistance to SMD; nonetheless, epigenetic modifications such as cytosine methylation is also informative, considering its roles in gene expression regulation. In our previous study, differentially transcribed fragments (DTFs) were obtained using cDNA-amplified fragment length polymorphism by comparing mock- and SCMV-inoculated plants from two sugarcane cultivars with contrasting responses to SMD. In this study, the identification of unexplored DTFs was continued while the same leaf samples were used to evaluate SCMV-mediated changes in the cytosine methylation pattern by using methylation-sensitive amplification polymorphism. This analysis revealed minor changes in cytosine methylation in response to SCMV infection, but distinct changes between the cultivars with contrasting responses to SMD, with higher hypomethylation events 24 and 72 h post-inoculation in the resistant cultivar. The differentially methylated fragments (DMFs) aligned with transcripts, putative promoters, and genomic regions, with a preponderant distribution within CpG islands. The transcripts found were associated with plant immunity and other stress responses, epigenetic changes, and transposable elements. The DTFs aligned with transcripts assigned to stress responses, epigenetic changes, photosynthesis, lipid transport, and oxidoreductases, in which the transcriptional start site is located in proximity with CpG islands and tandem repeats. Real-time quantitative polymerase chain reaction results revealed significant upregulation in the resistant cultivar of aspartyl protease and VQ protein, respectively, selected from DMF and DTF alignments, suggesting their roles in genetic resistance to SMD and supporting the influence of cytosine methylation in gene expression. Thus, we identified new candidate genes for further validation and showed that the changes in cytosine methylation may regulate important mechanisms underlying the genetic resistance to SMD.

Genome-Wide Identification of the VQ Protein Gene Family of Tobacco (Nicotiana tabacum L.) and Analysis of Its Expression in Response to Phytohormones and Abiotic and Biotic Stresses.

VQ motif-containing proteins (VQ proteins) are transcriptional regulators that work independently or in combination with other transcription factors (TFs) to control plant growth and development and responses to biotic and abiotic stresses. VQ proteins contain a conserved FxxhVQxhTG amino acid motif that is the main element of its interaction with WRKY TFs. We identified 59 members of the tobacco (Nicotiana tabacum L.) NtVQ gene family by in silico analysis and examined their differential expression in response to phytohormonal treatments and following exposure to biotic and abiotic stressors. NtVQ proteins clustered into eight groups based upon their amino acid sequence and presence of various conserved domains. Groups II, IV, V, VI, and VIII contained the largest proportion of NtVQ gene family members differentially expressed in response to one or more phytohormone, and NtVQ proteins with similar domain structures had similar patterns of response to different phytohormones. NtVQ genes differentially expressed in response to temperature alterations and mechanical wounding were also identified. Over half of the NtVQ genes were significantly induced in response to Ralstonia solanacearum infection. This first comprehensive characterization of the NtVQ genes in tobacco lays the foundation for further studies of the NtVQ-mediated regulatory network in plant growth, developmental, and stress-related processes.

Characterization of VQ motif-containing protein family and their expression patterns under phytohormones and abiotic stresses in melon (Cucumis melo L.)

Many studies have showed plant VQ gene family plays an important part in response to abiotic and biotic stresses. At present, VQ protein family has been found successively in many plants, however, there have no detailed characterization and expression profiles of VQ gene family in muskmelon. Thus, the author probed into genome-wide identification, characterization and expression pattern under various hormones and abiotic stresses. Thirty CmVQ genes were classified ten groups according to orthologous of Arabidopsis VQ genes and dispersedly distributed on 11 chromosomes except for chromosomes 5 and 9. Majority of CmVQ members contained 300 amino acids or less, and were predicted to localize in the nucleus and had no intron. Many cis-elements in response to different stresses and hormones were found in the promoters of CmVQ genes. Gene ontology analysis displayed that 60% CmVQ genes participated in response to stimuli. Quantitative real-time PCR displayed that numerous CmVQ genes showed either positively or negatively regulated expression patterns under hormonal treatments and abiotic stresses. In summary, this research offers a general introduction to melon VQ genes and will contribute to the molecular breeding for resistance to stresses, as well as deeper inquiry on the biological functions of the VQ proteins.

Genome-Wide Classification and Evolutionary and Functional Analyses of the VQ Family

VQ protein, contain a conserved and short FxxxVQxLTG amino acid sequence motif. Some VQ proteins play important roles in development process and abiotic/biotic stress in plants. However, there has not been a systematic study of the VQ family in plants. The results revealed that Group I genes are conserved in plant development in angiosperms; Group IV and Group X in eudicots and Group I, Group VI and Group VII in monocots are involved in drought response; Group I and Group IV are the most important groups and conservational in response to salt stress in eudicots but not in monocots; VQ genes in Group II and Group IV which responded to cold stress were overlapped in angiosperms; Group IV, Group V, Group VI and Group IX in eudicots and Group VI in monocots play key roles in response to ABA treatment; Group IV in eudicots while Group V and Group VI are irreplaceable in response to biotic stress in monocots. We propose that this study provides solid foundations for the investigation of the functions and evolution of VQ genes in angiosperms.

Comparison of gene co-networks reveals the molecular mechanisms of the rice (Oryza sativa L.) response to Rhizoctonia solani AG1 IA infection

Rhizoctonia solani causes rice sheath blight, an important disease affecting the growth of rice (Oryza sativa L.). Attempts to control the disease have met with little success. Based on transcriptional profiling, we previously identified more than 11,947 common differentially expressed genes (TPM > 10) between the rice genotypes TeQing and Lemont. In the current study, we extended these findings by focusing on an analysis of gene co-expression in response to R. solani AG1 IA and identified gene modules within the networks through weighted gene co-expression network analysis (WGCNA). We compared the different genes assigned to each module and the biological interpretations of gene co-expression networks at early and later modules in the two rice genotypes to reveal differential responses to AG1 IA. Our results show that different changes occurred in the two rice genotypes and that the modules in the two groups contain a number of candidate genes possibly involved in pathogenesis, such as the VQ protein. Furthermore, these gene co-expression networks provide comprehensive transcriptional information regarding gene expression in rice in response to AG1 IA. The co-expression networks derived from our data offer ideas for follow-up experimentation that will help advance our understanding of the translational regulation of rice gene expression changes in response to AG1 IA.

Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean.

Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution.

The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators.

The VQ motif-containing proteins (designated as VQ proteins) are a class of plant-specific proteins with a conserved and single short FxxhVQxhTG amino acid sequence motif. VQ proteins regulate diverse developmental processes, including responses to biotic and abiotic stresses, seed development, and photomorphogenesis. In this Update, we summarize and discuss recent advances in our understanding of the regulation and function of VQ proteins and the role of the VQ motif in mediating transcriptional regulation and protein-protein interactions in signaling pathways. Based on the accumulated evidence, we propose a general mechanism of action for the VQ protein family, which likely defines a novel class of transcriptional regulators specific to plants.