Cis-acting Research Articles

Bioinformatics Analysis of the Panax ginseng Cyclophilin Gene and Its Anti-Phytophthora cactorum Activity.

In this paper, Panax ginseng cyclophilin (PgCyP) was successfully obtained through a genetic engineering technique. A bioinformatics method was used to analyze the physicochemical properties and structure of PgCyP. The results showed that PgCyP belongs to the cyclophilin gene family. The protein encoded by the PgCyP gene contains the active site of PPIase (R62, F67, and H133) and a binding site for cyclosporine A (W128). The relative molecular weight of PgCyP is 187.11 bp; its theoretical isoelectric point is 7.67, and it encodes 174 amino acids. The promoter region of PgCyP mainly contains the low-temperature environmental stress response (LTR) element, abscisic acid-responsive cis-acting element (ABRE), and light-responsive cis-acting element (G-Box). PgCyP includes a total of nine phosphorylation sites, comprising four serine phosphorylation sites, three threonine phosphorylation sites, and two tyrosine phosphorylation sites. PgCyP was recombined and expressed in vitro, and its recombinant expression was investigated. Furthermore, it was found that the recombinant PgCyP protein could effectively inhibit the germination of Phytophthora cactorum spores and the normal growth of Phytophthora cactorum mycelia in vitro. Further experiments on the roots of susceptible Arabidopsis thaliana showed that the PgCyP protein could improve the resistance of arabidopsis to Phytophthora cactorum. The findings of this study provide a basis for the use of the PgCyP protein as a new type of green biopesticide.

Genome-Wide Identification and Analysis of the WRKY Transcription Factor Family Associated with Leaf Senescence in Alfalfa

Leaves are the most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar ’XinjiangDaye’ using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources.

Massively parallel characterization of insulator activity across the genome

A key question in regulatory genomics is whether cis-regulatory elements (CREs) are modular elements that can function anywhere in the genome, or whether they are adapted to certain genomic locations. To distinguish between these possibilities we develop MPIRE (Massively Parallel Integrated Regulatory Elements), a technology for recurrently assaying CREs at thousands of defined locations across the genome in parallel. MPIRE allows us to separate the intrinsic activity of CREs from the effects of their genomic environments. We apply MPIRE to assay three insulator sequences at thousands of genomic locations and find that each insulator functions in locations with distinguishable properties. All three insulators can block enhancers, but each insulator blocks specific enhancers at specific locations. However, only ALOXE3 appears to block heterochromatin silencing. We conclude that insulator function is highly context dependent and that MPIRE is a robust method for revealing the context dependencies of CREs.

Characterization, Evolution, Expression and Functional Divergence of the DMP Gene Family in Plants.

The DMP (DOMAIN OF UNKNOWN FUNCTION 679 membrane protein) domain, containing a family of membrane proteins specific to green plants, is involved in numerous biological functions including physiological processes, reproductive development and senescence in Arabidopsis, but their evolutionary relationship and biological function in most crops remains unknown. In this study, we scrutinized phylogenetic relationships, gene structure, conserved domains and motifs, promoter regions, gene loss/duplication events and expression patterns. Overall, 240 DMPs were identified and analyzed in 24 plant species selected from lower plants to angiosperms. Comprehensive evolutionary analysis revealed that these DMPs underwent purifying selection and could be divided into five groups (I-V). DMP gene structure showed that it may have undergone an intron loss event during evolution. The five DMP groups had the same domains, which were distinct from each other in terms of the number of DMPs; group III was the largest, closely followed by group V. The DMP promotor region with various cis-regulatory elements was predicted to have a potential role in development, hormone induction and abiotic stresses. Based on transcriptomic data, expression profiling revealed that DMPs were primarily expressed in reproductive organs and were moderately expressed in other tissues. Evolutionary analysis suggested that gene loss events occurred more frequently than gene duplication events among all groups. Overall, this genome-wide study elucidates the potential function of the DMP gene family in selected plant species, but further research is needed in many crops to validate their biological roles.

A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Screening and Identification of Target Gene of StTCP7 Transcription Factor in Potato

TCP transcription factors are involved in the regulation of plant growth and development and response to stress. Previous studies showed that StTCP7 was involved in the abiotic stress response of potato and positively regulated plant tolerance to drought stress. On the basis of previous studies, this study verified the downstream target genes of StTCP7 transcription factor binding through yeast one hybridization, double luciferase and other technologies, and conducted a preliminary analysis of the downstream target genes. The results showed that the StTCP7 transcription factor could bind the promoter region of StDAM5 and StGOLS2 and regulate the expression of their genes. qRT-PCR analysis showed that the expression level of StDAM5 gene was the highest in flower stalk tissue and the lowest in leaf stalk. The expression of StGOLS2 gene was the highest in stem, the second in stalk, and the lower in root. Both StDAM5 and StGOLS2 genes responded to abiotic stress treated with 200 mM NaCl, 20% PEG-6000 and 100 µM ABA. The expression levels of target genes StDAM5 and StGOLS2 were up-regulated in StTCP7 interfered plants. The protein encoded by the target gene StDAM5 belongs to the Type II MADS-box protein, which contains 238 amino acids and is an acidic hydrophilic protein. The analysis of StDAM5 promoter region showed that the promoter region of StDAM5 gene contained cis-acting elements such as light response and abscisic acid. Subcellular localization showed that StDAM5 protein was expressed in both nucleus and cytoplasm.

Genome-Wide Identification of the Remorin Gene Family in Poplar and Their Responses to Abiotic Stresses.

The Remorin (REM) gene family is a plant-specific, oligomeric, filamentous family protein located on the cell membrane, which is important for plant growth and stress responses. In this study, a total of 22 PtREMs were identified in the genome of Populus trichocarpa. Subcellular localization analysis showed that they were predictively distributed in the cell membrane and nucleus. Only five PtREMs members contain both Remorin_C- and Remorin_N-conserved domains, and most of them only contain the Remorin_C domain. A total of 20 gene duplication pairs were found, all of which belonged to fragment duplication. Molecular evolutionary analysis showed the PtREMs have undergone purified selection. Lots of cis-acting elements assigned into categories of plant growth and development, stress response, hormone response and light response were detected in the promoters of PtREMs. PtREMs showed distinct gene expression patterns in response to diverse stress conditions where the mRNA levels of PtREM4.1, PtREM4.2 and PtREM6.11 were induced in most cases. A co-expression network centered by PtREMs was constructed to uncover the possible functions of PtREMs in protein modification, microtube-based movement and hormone signaling. The obtained results shed new light on understanding the roles of PtREMs in coping with environmental stresses in poplar species.

Transcription factor gene TaWRKY76 confers plants improved drought and salt tolerance through modulating stress defensive-associated processes in Triticum aestivum L.

WRKY transcription factor (TF) family acts as essential regulators in plant growth and abiotic stress responses. This study reported the function of TaWRKY76, a member of WRKY TF family in Triticum aestivum L., in regulating plant osmotic stress tolerance. TaWRKY76 transcripts were significantly upregulated upon drought and salt signaling, with dose extent- and stress temporal-dependent manners. Plant GUS activity assays suggested that stress responsive cis-acting elements, such as DRE and ABRE, exert essential roles in defining gene transcription under osmotic stress conditions. The TaWRKY76 protein targeted onto nucleus and possessed ability interacting with TaMYC2, a MYC TF member of wheat. TaWRKY76 and TaMYC2 positively regulated plant drought and salt adaptation by modulating osmotic stress-related physiological indices, including osmolyte contents, stomata movement, root morphology, and reactive oxygen species (ROS) homeostasis. Yeast one-hybrid assay indicated the binding ability of TaWRKY76 with promoters of TaDREB1;1, TaNCEB3, and TaCOR15;4. ChIP-PCR analysis confirmed that the osmotic stress genes are transcriptionally regulated by TaWRKY76. Moreover, the transgenic lines with knockdown of these stress-response genes displayed lowered plant biomass together with worsened root growth traits, decreased proline contents, and elevated ROS amounts. These results suggested that these stress defensive genes contributed to TaWRKY76-modulated osmotic stress tolerance. Highly positive correlations were observed between yield and the transcripts of TaWRKY76 in a wheat variety panel under field drought condition. A major haplotype TaWRKY76 Hap1 conferred improved drought tolerance. Our results suggested that TaWRKY76 is essential in plant drought and salt adaptation and a valuable target for molecular breeding stress-tolerant cultivars in Triticum aestivum L..

Long-range transcription factor binding sites clustered regions may mediate transcriptional regulation through phase-separation interactions in early human embryo

In mammals, during the post-fertilization pre-implantation phase, the expression of cell type-specific genes is crucial for normal embryonic development, which is regulated by cis-regulatory elements (CREs). TFs control gene expression by interacting with CREs. Research shows that transcription factor binding sites (TFBSs) reflect the general characteristics of the regulatory genome. Here, we identified TFBSs from chromatin accessibility data in five stages of early human embryonic development, and quantified transcription factor binding sites-clustered regions (TFCRs) and their complexity (TC). Assigning TC values to TFCRs has made it possible to assess the functionality of these regulatory elements in a more quantitative way. Our findings reveal a robust correlation between TFCR complexity and gene expression starting from the 8Cell stage, which is when the zygotic genome is activated in humans. Furthermore, we have defined long-range TFCRs (LR-TFCRs) and conjecture that LR-TFCRs may regulate gene expression through phase-separation mechanisms during the early stages of human embryonic development.

Genome-Wide Identification of the bHLH Gene Family in Rhododendron delavayi and Its Expression Analysis in Different Floral Tissues.

The bHLH genes play a crucial role in plant growth, development, and stress responses. However, there is currently limited research on bHLH genes in the important horticultural plant Rhododendron delavayi Franch. In this study, we conducted a comprehensive genome-wide identification and in-depth analysis of the bHLH gene family in R. delavayi using bioinformatics approaches. A total of 145 bHLH family members were identified, encoding proteins ranging from 98 to 3300 amino acids in length, with molecular weights ranging from 11.44 to 370.51 kDa and isoelectric points ranging from 4.22 to 10.80. These 145 bHLH genes were unevenly distributed across 13 chromosomes, with three bHLH genes located on contig 52. Chromosome 8 contained the highest number of bHLH family members with 19 genes, while chromosomes 9 and 13 had the lowest, with 7 genes each. Phylogenetic analysis revealed a close evolutionary relationship between bHLH genes in R. delavayi and Arabidopsis thaliana. Subcellular localization analysis indicated that most bHLH genes were located in the nucleus. Promoter analysis of R. delavayi bHLH genes revealed the presence of various cis-regulatory elements associated with light responses, methyl jasmonate responses, low-temperature responses, and coenzyme responses, suggesting that bHLH genes are involved in multiple biological processes in R. delavayi. Through transcriptome analysis, we identified three key functional genes-Rhdel02G0041700, Rhdel03G0013600, and Rhdel03G0341200-that may regulate flower color in R. delavayi. In conclusion, our study comprehensively identified and analyzed the bHLH gene family in R. delavayi and identified three bHLH genes related to flower color, providing a foundation for molecular biology research and breeding in R. delavayi.

Genome-wide analysis of the CCT gene family and functional characterization of SlCCT6 in response to drought stress in tomato

CCT transcription factors are important for photoperiod and abiotic stress regulation in Arabidopsis and rice. However, the CCT gene family has not been reported in tomato. Here, we systematically analyzed this. Thirty-one SlCCT genes were identified and divided into five groups (CMF, TIFY, PRR, S8, and COL), with members unevenly distributed across 12 chromosomes and the third chromosome exhibiting the most distribution. SlCCT was found to interact with an interacting protein (SlGI), transcription factor (MYB), and non-coding RNA (sly-miR156-5p) to jointly regulate the tomato stress response. cis-Acting element analysis of the SlCCT promoter region indicated large stress- and hormone-response elements in this family. Real-time PCR results indicated that SlPRR subfamily genes respond to various abiotic stresses and hormones. Tissue expression analysis revealed that several PRR subfamily genes are highly expressed in flowers, and subcellular localization analysis indicated an SlCCT6 nuclear location. Notably, SlCCT6 expression was significantly induced by drought, and its silencing reduced drought stress tolerance. Moreover, SlCCT6 overexpression enhanced tomato drought resistance by increasing antioxidant enzyme activity and activating stress-related genes, whereas SlCCT6 knockout decreased drought resistance. In conclusion, this provides valuable insights for future research on SlCCT functions.

Genome-wide identification of clock-associated genes and circadian rhythms in Fragaria × ananassa seedlings

Flowering time in plants is regulated by a photoperiod-responsive mechanism. Some plant species use a circadian clock-based control mechanism to adapt to variable environments. Strawberry is a horticultural crop that responds to certain photoperiods and temperatures to induce flowering. However, clock-associated genes in octoploid cultivated strawberry (Fragaria × ananassa) have not been defined, and their regulatory mechanism for responding to photoperiods is unclear. We herein targeted 12 clock-associated genes reported in other plant species and performed a genome-wide analysis and expression comparison in F. × ananassa seedlings. Seventy-eight sequences were selected from the F. × ananassa genome. The major domains and cis-acting elements were conserved in each sequence. Transcripts were clearly expressed under continuous light conditions in F. × ananassa seedlings (‘Yotsuboshi’) acclimated to long days. Among them, 9 genes maintained their unique autonomous circadian rhythms and may function as clock genes. LHY (LATE ELONGATED HYPOCOTYL) had the Myb domain and LHY expression peaked in the dawn. PRR (PSEUDO-RESPONSE REGULATOR) family members (PRR9, PRR7, PRR5, and TOC1 (TIMING OF CAB EXPRESSION 1)) had a pseudo-receiver domain and CCT domain, and peak expression times began sequentially from the afternoon for PRR9 to the evening for TOC1. LUX (LUXARRHYTHMO) had a Myb domain, and LUX expression peaked in evening with ELF3 (EARLY FLOWERING 3). FKF1 (FLAVIN-BINDING KELCH REPEAT F BOX 1) had PAS and F-box domains, and FKF1 expression peaked in the afternoon. GI (GIGANTEA) expression also peaked in the afternoon. F. × ananassa (‘Yotsuboshi’) appears to have multiple feedback loops comprising clock-associated genes. Although the rhythmic expression of CHE (CCA1 HIKING EXPEDITION) and ZTL (ZEITLUPE) was not observed, they had conserved domains, CHE with the TCP domain and ZTL with the PAS and F-box domains. The present results provide basic information on the circadian clock for the control of F. × ananassa flowering.

Genome-wide identification, expression pattern and interacting protein analysis of INDETERMINATE DOMAIN (IDD) gene family in Phalaenopsis equestris

The plant-specific INDETERMINATE DOMAIN (IDD) gene family is important for plant growth and development. However, a comprehensive analysis of the IDD family in orchids is limited. Based on the genome data of Phalaenopsis equestris, the IDD gene family was identified and analyzed by bioinformatics methods in this study. Ten putative P. equestris IDD genes (PeIDDs) were characterized and phylogenetically classified into two groups according to their full amino acid sequences. Protein motifs analysis revealed that overall structures of PeIDDs in the same group were relatively conserved. Its promoter regions harbored a large number of responsive elements, including light responsive, abiotic stress responsive elements, and plant hormone cis-acting elements. The transcript level of PeIDD genes under cold and drought conditions, and by exogenous auxin (NAA) and abscisic acid (ABA) treatments further confirmed that most PeIDDs responded to various conditions and might play essential roles under abiotic stresses and hormone responses. In addition, distinct expression profiles in different tissues/organs suggested that PeIDDs might be involved in various development processes. Furthermore, the prediction of protein-protein interactions (PPIs) revealed some PeIDDs (PeIDD3 or PeIDD5) might function via cooperating with chromatin remodeling factors. The results of this study provided a reference for further understanding the function of PeIDDs.

Definition of regulatory elements and transcription factors controlling porcine immune cell gene expression at single cell resolution using single nucleus ATAC-seq

The transcriptome of porcine peripheral blood mononuclear cells (PBMC) at single cell (sc) resolution is well described, but little is understood about the cis-regulatory mechanism behind scPBMC gene expression. Here, we profiled the open chromatin landscape of porcine PBMC that define cis-regulatory elements and mechanism contributing to the transcription using single nucleus ATAC sequencing (snATAC-seq). Approximately 22 % of the identified peaks overlapped with annotated transcription start sites (TSS). Using clustering based on open chromatin pattern similarity, we demonstrate that cell type annotations using snATAC-seq are highly concordant to that reported for sc RNA sequencing (scRNA-seq). The differentially accessible peaks (DAPs) for each cell type were characterized and the pattern of accessibility of the DAPs near cell type markers across cell types was similar to that of the average gene expression level of corresponding marker genes. Additionally, we found that peaks identified in snATAC-seq have the potential power to predict the cell type specific transcription starting site (TSS). We identified both transcription factors (TFs) whose binding motif were enriched in cell type DAPs of multiple cell types and cell type specific TFs by conducting transcription factor binding motif (TFBM) analysis. Furthermore, we identified the putative enhancer or promoter regions bound by TFs for each differentially expressed gene (DEG) with a DAP that overlapped with its TSS by generating cis-co-accessibility networks (CCAN). To predict the regulators of such DEGs, TFBM analysis was performed for each CCAN. The regulator TF-target DEG pairs predicted in this way were largely consistent with the results reported in the ENCODE Transcription Factor Targets Dataset (TFTD). This snATAC-seq approach provides insights into the regulation of chromatin accessibility landscape of porcine PBMCs and enables discovery of TFs predicted to control DEG through binding regulatory elements whose chromatin accessibility correlates with the DEG promoter region.

Comprehensive, Genome-Wide Identification and Expression Analyses of Phenylalanine Ammonia-Lyase Family under Abiotic Stresses in Brassica oleracea.

Phenylalanine ammonia-lyase (PAL) acts as the rate-limiting enzyme for anthocyanin biosynthesis through the phenylpropanoid pathway, a crucial component of plant secondary metabolism. The PAL gene family plays a crucial role in plants' defense and stress responses, but its in silico identification and expression analyses in Brassica oleracea under different abiotic stresses remain unexplored. In this study, nine BolPAL, seven BrPAL, four AtPAL, and seventeen BnPAL genes were obtained from the genomes of B. oleracea, Brassica rapa, Arabidopsis thaliana, and Brassica napus, respectively. Segmental duplication and purifying selection are the causes of the BolPAL gene's amplification and evolution. The BolPAL genes with comparable intron-exon architectures and motifs were grouped together in the same clade. Three categories comprised the cis-regulatory elements: abiotic stressors, phytohormones, and light. According to the results of the qRT-PCR experiments, the majority of the BolPAL genes were expressed highly under MeJA, a low temperature, and a high temperature, and they were downregulated under ABA. Under white light (100 µmol m-2 s-1) with 50, 100, or 150 µmol m-2 s-1 far-red (FR), only a small number of the PAL genes were expressed at 50 and 100 µmol m-2 s-1 FR, while the majority of the PAL genes were slightly elevated at 150 µmol m-2 s-1 FR. This work offers a theoretical foundation for molecular breeding research to investigate the role of BolPAL genes and their role in anthocyanin biosynthesis.

A Genome-Wide Identification and Expression Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family in Melon (Cucumis melo L.)

The xyloglucan endotransglucosylase/hydrolase (XTH) family is an important multigene family in plants that plays a key role in cell wall reconstruction and stress tolerance. However, the specific traits of XTH genes and their expression patterns under different stresses have not been systematically studied in melon. In this study, based on the genomic data of Cucumis melon, 29 XTH genes were identified; most of these genes contain two conserved domains (Glyco_hydro_16 and XET_C domains). Based on neighbor-joining phylogenetic analysis, the CmXTHs were divided into four subfamilies, I/II, IIIA, and IIIB, which are distributed across nine chromosomes of melon. Collinearity analysis showed that the melon XTH genes have an evolutionary history consistent with three species: Arabidopsis, tomato, and cucumber. The promoter regions of the CmXTH genes contain numerous cis-acting elements, which are associated with plant growth, hormonal response, and stress responses. RNA-Seq analysis indicated that CmXTH genes exhibit different expression patterns under drought and salt stress treatments, suggesting that this gene family plays an important role under abiotic stress. This study provides a theoretical basis for further studies on the molecular function of XTH genes in melon.

Investigating the cis-regulatory basis of C3 and C4 photosynthesis in grasses at single-cell resolution

While considerable knowledge exists about the enzymes pivotal for C4 photosynthesis, much less is known about the cis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4 enzymes for five different grass species. This study spans four C4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-dependent malic enzyme), Panicum miliaceum (NAD-dependent malic enzyme), Urochloa fusca (phosphoenolpyruvate carboxykinase), along with the C3 outgroup Oryza sativa. We studied the cis-regulatory landscape of enzymes essential across all C4 species and those unique to C4 subtypes, measuring cell-type-specific biases for C4 enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4 evolution. Besides promoter proximal ACRs, we found that, on average, C4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4 evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis-regulation at these C4 loci. This study illuminates the dynamic and complex nature of cis-regulatory elements evolution in C4 photosynthesis, particularly highlighting the intricate cis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3 crop performance under changing climatic conditions.

Identification of the ribosomal protein L18 (RPL18) gene family reveals that TaRPL18-1 positively regulates powdery mildew resistance in wheat

The Ribosomal protein L18 (RPL18) protein gene family plays an important role in plant growth, development and stress response. Although the RPL18 genes have been identified in several plant species, the RPL18 gene family in wheat (Triticum aestivum) is still unexplored. This study found 8 TaRPL18 genes, each of which has a significantly different gene sequence length and is evenly distributed on the chromosome; Additionally, these proteins have similar physicochemical characteristics as well as secondary and tertiary structures. 17 RPL18 genes in 4 species (wheat, Arabidopsis, rice, and maize) were classified into 5 groups, and the TaRPL18 genes within the same group showed similar structures and conserved motifs. Analysis of the cis-acting elements in the TaRPL18 genes promoter regions revealed the presence of developmental and stress-responsive elements in the majority of the genes. Through yeast two-hybrid (Y2H) experiments, it was confirmed that the powdery mildew resistance protein TaPm46 physically interacts with the Class IV TaRPL18-1. Functional analysis indicated that TaRPL18-1-silenced wheat plants show reduced resistance to powdery mildew compared to the wild type (WT), with decreased expression levels of PAL and PPO genes, and increased expression levels of the PR gene. The findings of this study provide a basis for clarifying the function of the TaRPL18 genes and will be useful for the selection of disease-resistant varieties of wheat.

Expression of Cystathionine-β-Synthase (CBS) Genes in Grape under Various Abiotic Stresses and Subcellular Localization of VvCBS27

The cystathionine-β-synthase (CBS) domain is present in the proteins of all living organisms and functions as an energy sensor, regulating protein activity through the binding capacity of its adenosine ligands. The role of the CBS gene in plant growth and development, as well as tolerance to abiotic stresses, remains largely unknown, especially in grapevine. In our study, 32 members of the CBS gene family were obtained that were distributed on 15 chromosomes. The results of the structural and evolutionary tree analyses indicated that the VvCBS gene family exhibits diverse intron-exon patterns and highly conserved motifs. Furthermore, the phylogenetic classification of the VvCBS genes revealed the presence of three subfamilies. Subcellular localization analyses showed that the VvCBS genes are mainly located in the plasma membrane region. The secondary structure of the VvCBS protein mainly consists of α-helices, extended strands, β-turns, and random coils. The VvCBS gene family exhibited four co-linear gene pairs, while the numbers for Arabidopsis thaliana and rice were 21 and 7, respectively. The promoter cis-acting element analysis revealed the presence of light-responsive, hormone-responsive, stress-responsive, and growth- and development-related elements in the VvCBS family. The expression characterization demonstrated that 12 VvCBS genes exhibited high expression levels in all grape tissues. Additionally, the RT-qPCR expression analyses showed that the 32 VvCBS exhibited different responses to a variety of abiotic stresses (cold, drought, salt), suggesting that they were functionally differentiated. VvCBS27 was cloned from ‘Pinot Noir’ of grapevine with a coding sequence of 624 bp. Subcellular localization showed that VvCBS27 protein was mainly located in the cytoplasm, cell membrane, and nucleus. This study lays a foundation for elucidating the function of grape CBS protein.

Identification of transcription factor binding sites of <i>HSP60</i> and <i>HSP90B1</i> Genes’ promoter in Bubalus bubalis

The gene expression determines cellular homeostasis while spatio-temporal nature of gene expression is regulated by several cis regulatory elements, such as promoter regions which bind to transcription factors. The ever-changing global climatic conditions have raised a necessity to scan heat tolerant livestock especially the riverine buffalo in tropics. The study focuses on characterization of the promoter region of HSP60 and HSP90B1 and looking for the binding sites of transcription factors in Bubalus bubalis and comparison with the nearest relative Bos taurus to gauge the interspecies variations. The results indicated no transcription binding factors in HSP90B1 promoter (550 nucleotides) within the region analyzed except for the Elk-1 and COMP1 in the cattle while promoter region of HSP60 gene (971 nucleotides) varied highly. Nine and seven transcription factor binding sites were specifically unique in Bubalus bubalis and in Bos taurus respectively. Occurrence of 1-Oct, BR-C Z4, Hairy, and HSF was higher in buffalo. Overall, this study provided the framework to explore cis regulatory elements and contributes to decipher the transcriptional regulatory elements of HSP60 and HSP90B1 genes in Bubalus bubalis.