Identification of novel cis-elements bound by BplMYB46 involved in abiotic stress responses and secondary wall deposition

Transcription factors （TFs） play vital roles in various biological processes by binding to cis-acting elements to control expressions of their target genes. The MYB TF BplMYB46, from Betula platyphylla, is involved in abiotic stress responses and secondary wall deposition. In the present study, we used a TF-centered yeast onehybrid technology （TF-centered YIH） to identify the cis- acting elements bound by BplMYB46. We screened a shortinsert random library and identified three cis-elements bound by BplMYB46： an E-box （CA（A/T/C）（A/G/C）TG） and two novel motifs, a TO-box （T（GIA）TCG（C/G）） and a GT-box （A（G/T）T（AIC）GT（T/G）C）. Chromatin immunoprecipitation （CHIP） and effector-reporter coexpression assays inNicotiana tabacum confirmed binding of BplMYB46 to the TC-box, GT-box, and E-box motifs in the promoters of the phenylalanine ammonia lyase （PAL）, peroxidase （POD）, and superoxide dismutase （SOD） genes, which function in abiotic stress tolerance and secondary wall biosynthesis. This finding improves our understanding of potential regulatory mechanisms in the response to abiotic stress and secondary wall deposition of BplMYB46 in B. platyphylla.

Identification and characterization of genes related to m^(6)A modification in kiwifruit using RNA-seq and ATAC-seq

N6-methyladenosine(m^(6)A)RNA modification is a conserved mechanism that regulates the fate of RNA across eukaryotic organisms.Despite its significance,a comprehensive analysis of m^(6)A-related genes in non-model plants,such as kiwifruit,is lacking.Here,we identified 36 m^(6)A-related genes in the kiwifruit genome according to homology and phylogenetic inference.We performed bioinformatics and evolutionary analyses of the writer,eraser,and reader families of m^(6)A modification.Reanalysis of public RNA-seq data collected from samples under various biotic and abiotic stresses indicated that most m^(6)A-related genes were remarkably expressed under different conditions.Through construction of gene co-expression networks,we found significant correlations between several m^(6)A-related genes and transcription factors(TFs)as well as receptor-like genes during the development and ripening of kiwifruit.Furthermore,we performed ATAC-seq assays on diverse kiwifruit tissues to investigate the regulatory mechanisms of m^(6)A-related genes.We identified 10 common open chromatin regions that were present in at least two tissues,and these regions might serve as potential binding sites for MADS protein,C2H2 protein,and other predicted TFs.Our study offers comprehensive insights into the gene family of m^(6)A-related components in kiwifruit,which will lay foundation for exploring mechanisms of post-transcriptional regulation involved in development and adaptation of kiwifruit.

Expression Activity of CLCuV Bidirectional Promoter in Agrobacterium tumefaciens

Cotton leaf curl virus (CLCuV) belongs to the subgroup III of geminiviruses with single strand DNA genome. Study demonstrated that the bidirectional promoter of CLCuV had activity in Agrobacterium tumefaciens (Smith et Townsend) Conn. This is the first report for the activity of the bidirectional promoter of geminivirus in A. tumefaciens. Results showed that the activity of the complementary sense promoter was stronger than that of virion sense promoter, and was detected 2-fold higher than that of CaMV 35S promoter in A. tumefaciens. Moreover, the promoter 5' deletion analysis indicated that the mean GUS activity driven by a 287 nucleotides complementary sense promoter fragment (from-287 to the translation initiation site) is 4 times higher than that driven by the whole complementary sense promoter in A. tumefaciens. This result suggested that there might exist negative regulatory elements in this deleted fragment. The function of other cis-elements included in CLCuV complementary sense promoter was also discussed in this paper.

Genome-wide identification,molecular evolution, and expression divergence of the hexokinase gene family in apple

Hexokinase(HXK)is the first irreversible catalytic enzyme in the glycolytic pathway,which not only provides energy for plant growth and development but also serves as a signaling molecule in response to environmental changes.However,the evolutionary pattern of the HXK gene family in apple remains unknown.In this study,a total of nine HXK genes were identified in the Malus×domestica genome GDDH13 v1.1.The physiological and biochemical properties,exonintron structures,conserved motifs,and cis-elements of the MdHXK genes were determined.Predicted subcellular localization indicated that the MdHXK genes were mainly distributed in the mitochondria,cytoplasm,and nucleus.Gene duplication revealed that whole-genome duplication(WGD)and segmental duplication played vital roles in MdHXK gene family expansion.Theωvalues of pairwise MdHXK genes indicated that this family was subjected to strong purifying selection during apple domestication.Additionally,five subfamilies were classified,and recent/old duplication events were identified based on phylogenetic tree analysis.Different evolutionary rates were estimated among the various HXK subfamilies.Moreover,divergent expression patterns of the MdHXK genes in four source-sink tissues and at five different apple fruit developmental stages indicated that they play vital roles in apple fruit development and sugar accumulation.Our study provides a theoretical basis for future elucidation of the biological functions of the MdHXK genes during apple fruit development.

Role of Activator Protein-1 in the Transcription of Interleukin-5 Gene Regulated by Protein Kinase C Signal in Asthmatic Human TLymphocytes

Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.

Characterization and Expression of Ammonium Transporter in Peach (Prunus persica) and Regulation Analysis in Response to External Ammonium Supply

As the preferred nitrogen(N)source,ammonium(NH_(4)^(+))contributes to plant growth and development and fruit quality.In plants,NH 4+uptake is facilitated by a family of NH_(4)^(+) transporters(AMT).However,the molecular mechanisms and functional characteristics of the AMT genes in peach have not been mentioned yet.In this present study,excess NH_(4)^(+) stress severely hindered shoot growth and root elongation,accompanied with reduced mineral accumulation,decreased leaf chlorophyll concentration,and stunned photosynthetic performance.In addition,we identified 14 putative AMT genes in peach(PpeAMT).Expression analysis showed that PpeAMT genes were differently expressed in peach leaves,stems and roots,and were distinctly regulated by external NH_(4)^(+) supplies.Putative cis-elements involved in abiotic stress adaption,Ca^(2+) response,light and circadian rhythms regulation,and seed development were observed in the promoters of the PpeAMT family genes.Phosphorylation analysis of residues within the C-terminal of PpeAMT proteins revealed many conserved phosphorylation residues in both the AMT1 and AMT2 subfamily members,which could potentially play roles in controlling the NH 4+transport activities.This study provides gene resources to study the biological function of AMT proteins in peach,and reveals molecular basis for NH_(4)^(+) uptake and N nutrition mechanisms of fruit trees.

A Fragment Substitution in Promoter of MS92/PTC1 Causes Male Sterility in Rice

Persistent tapetal cell1(PTC1) plays a curial role in pollen development, and is thought to function as a transcriptional activator in rice. However, the molecular mechanism of PTC1 in regulating pollen development and its cis-elements are not well understood. We identified a novel weak male sterility mutant(ms92) which exhibited expanded tapetum and shrink pollen grains. Map-based cloning and allelic analysis suggested that the male sterility of ms92 was caused by a DNA fragment substitution in the promoter of PTC1. The decreased expression of MS92/PTC1 in ms92 and cis-element analysis indicated that the substituted sequence contained several potential binding cis-element of negative feedback. MS92/PTC1 was specifically expressed in tapetum and microspores at the young microspore stage, and its protein was localized in nucleus. We further found that MS92/PTC1 functions as a transcription activator by recognizing H3K4me3. Transcriptomic analysis revealed that a number of genes involved in tapetum degeneration and pollen wall formation were down-regulated in ms92, which are the potential targets of MS92/PTC1. The substitution fragment in MS92/PTC1 promoter was essential for pollen development, and we provided a novel mutant for further identifying the cis-elements in promoter and the molecular network of MS92/PTC1.

Villin Family Members Associated with Multiple Stress Responses in Cotton

Villin(VLN)is considered to be one of the most important actin-binding proteins,participates in modulating the actin cytoskeleton dynamics,plays essential role in plant development and resisting adverse environments.However,systematic studies of the VLN gene family have not been reported in cotton(Gossypium).In this study,14 GhVLNs were identified in G.hirsutum.These GhVLN genes were distributed in 6 A-subgenome chromosomes and 6 D-subgenome chromosomes of the allotetraploid upland cotton and classified into three phylogenetical groups based on the classification model of AtVLNs.In addition,the 14 GhVLN genes have highly conserved gene structure and motif architecture.The number of introns was ranged from 18 to 22 and the length of protein sequences was varied from 901 to 1077.Six gelsolin homology domains,G1–G6,and villin headpiece domain,VHP,were existed in all GhVLNs with the exception of two VLNs(GhVLN6 and GhVLN13)which lacked VHP.Cis-elements analysis revealed that the promoter regions of GhVLNs contained various light related components and also elements responsible for phytohormones and stresses response,indicating that,when subjected to those adverse environments,cotton plants may activate the response system by targeting VLN genes to survive the crisis.Heatmaps showed that the GhVLN genes exhibited various expression patterns,some were accumulated in certain tissues,root,petal,stamen or elongating fibers,and some were obviously induced by environmental changes.Especially GhVLN3 and GhVLN10 were highly and preferentially expressed in elongating fibers and distinctly upregulated by abiotic(salt,PEG,cold and heat)and biotic(Verticillium dahliae V991)stresses.This study may provide useful information for biological function identification of GhVLN genes and gene resources for creating high-quality and various resistant cotton germplasms.

An <i>in silico</i>Analysis of Upstream Regulatory Modules (URMs) of Tapetum Specific Genes to Identify Regulatory <i>cis</i>-Elements and Transcription Factors

The present work presents an iin silicoi analysis of Upstream Regulatory Modules (URMs) of genes expressed in tapetum specific manner in dicotyledon and monocotyledon plants. In the current analysis, we identified several motifs conserved in these URMs of which ten were observed to be part of known icisi-elements using tools and databases like MEME, PLACE, MAST and TFSEARCH. We also identified that binding sites for two transcription factors, DOF and WRKY71 were found to be present in majority of the URMs.

Calcium-Regulated Transcription in Plants

The past two decades revealed a plethora of Ca^2＋-responsive proteins and downstream targets in plants, of which several are unique to plants. More recent high-throughput ＇omics＂ approaches and bioinformatics are exposing Ca^2＋-responsive cis-elements and the corresponding Ca^2＋-responsive genes. Here, we review the current knowledge on Ca^2＋-signaling pathways that regulate gene expression in plants, and we link these to mechanisms by which plants respond to biotic and abiotic stresses.

A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity

Flavonoid biosynthetic genes are often coordinately regulated in a temporal manner during flower or fruit development, resulting in specific accumulation profiles of flavonoid compounds. R2R3-MYB-type transcription factors （TFs） ＂recruit＂ a set of biosynthetic genes to produce flavonoids, and, therefore, R2R3-MYBs are responsible for the coordinated expression of structural genes. Although a wealth of information regarding the identified and functionally characterized R2R3-MYBs that are involved in flavonoid accumulation is available to date, this is the first review on the global regulation of MYB factors in the flavonoid pathway. The data presented in this review demonstrate that anthocyanin, flavone/flavonol/3-deoxyflavonoid （FFD）, proanthocyanidin （PA）, and isoflavonoid are independently regulated by different subgroups of R2R3-MYBs. Furthermore, FFD-specific R2R3-MYBs have a preference for early biosynthetic genes （EBGs） as their target genes; anthocyanin-specific R2R3-MYBs from dicot species essentially regulate late biosynthetic genes （LBGs）; the remaining R2R3-MYBs have a wider range of target gene specificity. To elucidate the nature of the differential target gene specificity between R2R3-MYBs, we analyzed the DNA binding domain （also termed the MYB-domain） of R2R3-MYBs and the distribution of the recognition cis-elements. We identified four conserved amino acid residues located in or just before helix-3 of dicot anthocyanin R2R3-MYBs that might account for the different recognition DNA sequence and subsequently the different target gene specificity to the remaining R2R3-MYB TFs.

Identification of Target Genes and Transcription Factors Implicated in Translation-Dependent Retrograde Sig- naling in Arabidopsis

Changes in organellar gene expression （OGE） trigger retrograde signaling. The molecular dissection of OGE-dependent retrograde signaling based on analyses of mutants with altered OGE is complicated by compensatory responses that mask the primary signaling defect and by secondary effects that influence other retrograde signaling pathways. Therefore, to identify the earliest effects of altered OGE on nuclear transcript accumulation, we have induced OGE defects in adult plants by ethanol-dependent repression of PRORS1, which encodes a prolyl-tRNA synthetase located in chloroplasts and mitochondria. After 32 h of PRORS1 repression, the translational capacity of chloroplasts was reduced, and this effect subsequently intensified, while basic photosynthetic parameters were still unchanged at 51 h. Analysis of changes in whole-genome transcriptomes during exposure to ethanol revealed that induced PRORS1 silencing affects the expression of 1020 genes in all. Some of these encode photosynthesis-related proteins, including several down-regulated light-harvesting chlorophyll a/b binding （LHC） proteins. Interestingly, genes for presumptive endoplasmic reticulum pro- teins are transiently up-regulated. Furthermore, several NAC-domain-containing proteins are among the transcription factors regulated. Candidate cis-acting elements which may coordinate the transcriptional co-regulation of genes sets include both G-box variants and sequence motifs with no similarity to known plant c/s-elements.

The contribution of cis-regulatory elements to head-to-head gene pairs' co-expression pattern

Transcription regulation is one of the most critical pipelines in biological process,in which cis-elements play the role as gene expression regulators.We attempt to deduce the principles underlying the co-expression of "head-to-head" gene pairs by analyzing activities or behaviors of the shared cis-elements.A network component analysis was performed to estimate the impact of cis-elements on gene promoters and their activities under different conditions.Our discoveries reveal how biological system uses those regulatory elements to control the expression pattern of "head-to-head" gene pairs and the whole transcription regulation system.

GhKLCR1, a kinesin light chain-related gene, induces drought-stress sensitivity in Arabidopsis

Drought stress results in significant losses in agricultural production, and especially that of cotton. The molecular mechanisms that coordinate drought tolerance remain elusive in cotton. Here, we isolated a drought-response gene GhKLCR1, which is a close homolog of AtKLCR1, which encodes a kinesin light chain-related protein enriched with a tetratrico peptide-repeat region.A subcellular localization assay showed that GhKLCR1 is associated with the cell membrane. A tissue-specific expression profile analysis demonstrated that GhKLCR1 is a cotton root-specific gene. Further abiotic and hormonal stress treatments showed that GhKLCR1 was upregulated during abiotic stresses, especially after polyethylene glycol treatments. In addition, the glucuronidase(GUS) staining activity increased as the increment of mannitol concentration in transgenic Arabidopsis plants harboring the fusion construct PGhKLCR1::GUS. The root lengths of 35 S::GhKLCR1 lines were significantly reduced compared with that of wild type. Additionally, seed germination was strongly inhibited in 35 S::GhKLCR1 lines after 300-mmol L^(-1) mannitol treatments as compared with Columbia-0, indicating the sensitivity of GhKLCR1 to drought. These findings provide a better understanding of the structural, physiological and functional mechanisms of kinesin light chain-related proteins.

Transcriptional Regulatory Networks Activated by PI3K and ERK Transduced Growth Signals in Human Glioblastoma Cells

Determining how cells regulate their transcriptional response toextracellular signals is key to the understanding of complex eukaryotic systems. This study wasinitiated with the goals of furthering the study of mammalian transcriptional regulation andanalyzing the relative benefits of related computational methodologies. One dataset available forsuch an analysis involved gene expression profiling of the early growth factor response to plateletderived growth factor (PDGF) in a human glioblastoma cell line; this study differentiated geneswhose expression was regulated by signaling through the phosphoinositide-3-kinase (PI3K) versus theextracellular-signal regulated kinase (ERK) pathways. We have compared the inferred transcriptionfactors from this previous study with additional predictions of regulatory transcription factorsusing two alternative promoter sequence analysis techniques. This comparative analysis, in which thealgorithms predict overlapping, although not identical, sets of factors, argues for meticulousbenchmarking of promoter sequence analysis methods to determine the positive and negative attributesthat contribute to their varying results. Finally, we inferred transcriptional regulatory networksderiving from various signaling pathways using the CARRIE program suite. These networks not onlyincluded previously described transcriptional features of the response to growth signals, but alsopredicted new regulatory features for the propagation and modulation of the growth signal.

Deletional analysis of functional regions of complementary sense promoter from cotton leaf curl virus

Complementary sense promoter from cotton leaf curl virus (CLCuV) is a novel plant promoter for genetic engineering that could drive high-level foreign gene expression in plant. To determine the optimal promoter sequence for gene expression, CLCuV promoter was deleted from its 5' end to form promoter fragments with five different lengths, and chimeric gus genes were constructed using the promoterdeletion. These vectors were delivered into Agrobacterium and tobacco (Nicotiana tabacum L cv. Xanthi) plants which were transformed by leaf discs method. GUS activity of transgenic plants was measured. The results showed that GUS activities with the promoter deleted to -287 and -271 from the translation initiation site were respectively about five and three times that of full-length promoter. There exists a c/s-element which is important for the expressing activity in phloem from -271 to -176. Deletion from -176 to -141 resulted in a 20-30-fold reduction in GUS activity in leaves with weak activity in leaves and stems and losing GUS activity in roots. The functional domains of complementary sense gene promoter of CLCuV were firstly analyzed and compared. It was found that the promoter activity with the deletion of negative c/s-elements was much stronger than that of full-length promoter and was about twelve times on average that of CaMV 35S promoter, suggesting that the promoter has great application potential. Results also provide novel clues for understanding the mechanisms of geminivirus gene regulation and interaction between virus and plant.

Multifaceted roles of complementary sequences on circRNA formation

Background： Circular RNAs （circRNAs） from back-spliced exon（s） are characterized by the covalently closed loop feature with neither 5＇ to 3＇ polarity nor polyadenylated tail. By using specific computational approaches that identify reads mapped to back-splice junctions with a reversed genomic orientation, ten thousands of cireRNAs have been recently re-identified in various cell lines/tissues and across different species. Increasing lines of evidence suggest that back-splicing is catalyzed by the canonical spliceosomal machinery and modulated by cis-elements and trans-factors. Results： In this mini-review, we discuss our current understanding of circRNA biogenesis regulation, mainly focusing on the complex regulation of complementary sequences, especially Alus in human, on circRNA formation. Conclusions： Back-splicing can be significantly facilitated by RNA pair formed by orientation-opposite complementary sequences that juxtapose flanking introns of circularized exon（s）. RNA pair formed within individual introns competes with RNA pair formed across flanking introns in the same gene locus, leading to distinct choices for either canonical splicing or back-splicing. Multiple RNA pairs that bracket different circle-forming exons compete for alternative back-splicing selection, resulting in multiple circRNAs generated in a single gene locus.