Cis-acting Research Articles

Developing a pipeline for identification, characterization and molecular editing of cis-regulatory elements: a case study in potato

AbstractCrop breeding requires a balance of tradeoffs among key agronomic traits caused by gene pleiotropy. The molecular manipulation of genes can effectively improve target traits, but this may not reduce gene pleiotropy, potentially leading to undesirable traits or even lethal conditions. However, molecular editing of cis-regulatory elements (CREs) of target genes may facilitate the dissection of gene pleiotropy to fine-tune gene expression. In this study, we developed a pipeline, in potato, which employs open chromatin to predict candidate CREs, along with both transient and genetic assays to validate the function of CREs and CRISPR/Cas9 to edit candidate CREs. We used StCDF1 as an example, a key gene for potato tuberization and identified a 288 bp-core promoter region, which showed photoperiodic inducibility. A homozygous CRISPR/Cas9-editing line was established, with two deletions in the core promoter, which displayed a reduced expression level, resulting in late tuberization under both long-day and short-day conditions. This pipeline provides an alternative pathway to improve a specific trait with limited downside on other phenotypes.

Genome-Wide Identification, Characterization and Expression Patterns of the DBB Transcription Factor Family Genes in Wheat.

Double B-box (DBB) proteins are plant-specific transcription factors (TFs) that play crucial roles in plant growth and stress responses. This study investigated the classification, structure, conserved motifs, chromosomal locations, cis-elements, duplication events, expression levels, and protein interaction network of the DBB TF family genes in common wheat (Triticum aestivum L.). In all, twenty-seven wheat DBB genes (TaDBBs) with two conserved B-box domains were identified and classified into six subgroups based on sequence features. A collinearity analysis of the DBB family genes among wheat, Arabidopsis, and rice revealed some duplicated gene pairs and highly conserved genes in wheat. An expression pattern analysis indicated that wheat TaDBBs were involved in plant growth, responses to drought stress, light/dark, and abscisic acid treatment. A large number of cis-acting regulatory elements related to light response are enriched in the predicted promoter regions of 27 TaDBBs. Furthermore, some of TaDBBs can interact with COP1 or HY5 based on the STRING database prediction and yeast two-hybrid (Y2H) assay, indicating the potential key roles of TaDBBs in the light signaling pathway. Conclusively, our study revealed the potential functions and regulatory mechanisms of TaDBBs in plant growth and development under drought stress, light, and abscisic acid.

Genome-wide analysis of calmodulin binding Protein60 candidates in the important crop plants.

Efficient management of environmental stresses is essential for sustainable crop production. Calcium (Ca²⁺) signaling plays a crucial role in regulating responses to both biotic and abiotic stresses, particularly during host-pathogen interactions. In Arabidopsis thaliana, calmodulin-binding protein 60 (CBP60) family members, such as AtCBP60g, AtCBP60a, and AtSARD1, have been well characterized for their involvement in immune regulation. However, a comprehensive understanding of CBP60 genes in major crops remains limited. In this study, we utilized the Phytozome v12.1 database to identify and analyze CBP60 genes in agriculturally important crops. Expression patterns of a Oryza sativa (rice) CBP60 gene, OsCBP60bcd-1, were assessed in resistant and susceptible rice genotypes in response to infection by the bacterial pathogen Xanthomonas oryzae. Localization of CBP60 proteins was analyzed to predict their functional roles, and computational promoter analysis was performed to identify stress-responsive cis-regulatory elements. Phylogenetic analysis revealed that most CBP60 genes in crops belong to the immune-related clade. Expression analysis showed that OsCBP60bcd-1 was significantly upregulated in the resistant rice genotype upon pathogen infection. Subcellular localization studies suggested that the majority of CBP60 proteins are nuclear-localized, indicating a potential role as transcription factors. Promoter analysis identified diverse stress-responsive cis-regulatory elements in the promoters of CBP60 genes, highlighting their regulatory potential under stress conditions. The upregulation of OsCBP60bcd-1 in response to Xanthomonas oryzae and the presence of stress-responsive elements in its promoter underscore the importance of CBP60 genes in pathogen defense. These findings provide a basis for further investigation into the functional roles of CBP60 genes in crop disease resistance, with implications for enhancing stress resilience in agricultural species.

MIKC*-type MADS transcription factors control JINGUBANG expression and the degree of pollen dormancy in Arabidopsis.

While pollen dormancy has been proposed to play a necessary role in sexual reproduction, it remains poorly understood. Here, we used traditional pollen germination assays to characterize dormancy. Our results underscore variation in the degree of dormancy between individual pollen grains. In addition, we provide evidence that JINGUBANG (JGB), previously defined as a negative regulator of pollen germination in Arabidopsis (Arabidopsis thaliana), is responsible for the uneven degrees of pollen dormancy, as asynchronous pollen germination in vitro reflected varied expression levels of JGB. We identified five cis-acting elements, including four CArG-boxes and the previously uncharacterized element ERE7, as essential for the initiation and enhancement of JGB expression. A 10-bp sequence between CArG-box 3 and ERE7, likely the result of an inverse DNA loop formed between CArG-box 3 and CArG-box 4, was required for robust gene expression. In addition, the pollen-specific AtMIKC*-type MADS transcription factors AGAMOUS-LIKE 30 (AGL30), AGL65, AGL66, AGL94, and AGL104 activated JGB transcription. Notably, the transactivation levels differed among the obligate AtMIKC* heterodimers tested. Our results indicate that distinct AtMIKC* complexes formed in individual pollen grains direct pollen dormancy to uneven degrees, which is likely an adaptive trait that ensures broader pollen dispersal under adverse environmental conditions.

Membrane Interactions of GET1 and GET2 Facilitate Fiber Cell Initiation through the Guided Entry of the TA Protein Pathway in Cotton.

The guided entry of TA proteins (GET) pathway, which is responsible for the post-translational targeting and insertion of the tail-anchored (TA) protein into the endoplasmic reticulum (ER), plays an important role in physiological processes such as protein sorting, vesicle trafficking, cell apoptosis, and enzymatic reactions in which the GET1/2 complex is indispensable. However, a comprehensive study of the GET1 and GET2 genes and the GET pathway in cotton has not yet been carried out. Here, 12 GET1 and 21 GET2 genes were identified in nine representative plant species, and the phylogenetic relationships, gene structures, protein motifs, cis-regulatory elements (CREs), and temporal and spatial expression profiles were analyzed thoroughly. Our study indicated that GhGET1s and GhGET2s might be localized on ER membranes. According to expression profiling and CREs analysis, GhGET2-A02 was identified as a promising candidate for fiber cell development, interacting with two GhGET1s in the membrane, with a binding bias toward GhGET1-A06. Silencing of GhGET1-A06 or GhGET2-A02 reduced fiber initiation and elongation. In summary, our research provides important evidence for understanding the gene families and functions of GET1 and GET2 in cotton and provides clues for molecular breeding of high-quality cotton fiber varieties.

Genome-wide identification and expression analysis of two-component system genes in switchgrass (Panicum virgatum L.)

The two-component system (TCS) consists of histidine kinase (HK), histidine phosphate transfer protein (HP), and response regulatory factor (RR). It is one of the most crucial components of signal transduction in plants, playing a significant role in regulating plant growth, development, and responses to various abiotic stresses. Although TCS genes have been extensively identified in a variety of plants, the genome-wide recognition and examination of TCS in switchgrass remain unreported. Accordingly, this study identified a total of 87 TCS members in the genome of switchgrass, comprising 20 HK(L)s, 10 HPs, and 57 RRs. Detailed analyses were also conducted on their gene structures, conserved domains, and phylogenetic relationships. Moreover, this study analysed the gene expression profiles across diverse organs and investigated their response patterns to adverse environmental stresses. Results revealed that 87 TCS genes were distributed across 18 chromosomes, with uneven distribution. Expansion of these genes in switchgrass was achieved through both fragment and tandem duplication. PvTCS members are relatively conservative in the evolutionary process, but the gene structure varies significantly. Various cis-acting elements, varying in types and amounts, are present in the promoter region of PvTCSs, all related to plant growth, development, and abiotic stress, due to the TCS gene structure. Protein-protein interaction and microRNA prediction suggest complex interactions and transcriptional regulation among TCS members. Additionally, most TCS members are expressed in roots and stems, with some genes showing organ-specific expression at different stages of leaf and inflorescence development. Under conditions of abiotic stress such as drought, low temperature, high temperature, and salt stress, as well as exogenous abscisic acid (ABA), the expression of most TCS genes is either stimulated or inhibited. Our systematic analysis could offer insight into the characterization of the TCS genes, and further the growth of functional studies in switchgrass.

Genome-wide identification and expression analysis of the OSC gene family in Platycodon grandiflorus

Platycodon grandiflorus stands as one of the most extensively utilized traditional Chinese medicinal herbs, with triterpenoids and their derivatives serving as its primary medicinal components. Oxido squalene cyclase (OSC), serving as a crucial enzyme in the triterpenoid synthesis pathway, has the capability to enzymatically generate significant quantities of sterols and triterpenoid intermediates. While the OSC gene family has been identified in numerous species, bioinformatics research on this family remains scant. Presently, the specific members of this gene family in Platycodon grandiflorus have yet to be definitively determined. In this study, we successfully identified a total of 15 PgOSC genes within the genome of Platycodon grandiflorus by conducting homology comparisons. These genes were discovered to be unevenly distributed across the five chromosomes of the species, organized in the form of gene clusters. Subsequently, we conducted a thorough analysis of the OSC gene family’s evolutionary relationship by constructing a phylogenetic tree. Other characteristics of PgOSC family members, including gene structure, conserved motifs, protein three-dimensional structure, subcellular localization, and cis-acting elements were thoroughly characterized. Furthermore, We analyzed the expression of PgOSC gene in different tissues of Platycodon grandiflorus by qRT-PCR, and found that the expression of PgOSC genes in root was higher than that in stem and leaf. Upon comparing the effects of salt, heat, and drought treatments, we observed a significant induction of PgOSC gene expression in Platycodon grandiflorus specifically under salt stress conditions. In summary, this study comprehensively identified and analyzed the OSC gene family, aiming to provide basic biological information for exploring the members of PgOSC gene family.

Genome-wide identification of alcohol dehydrogenase (ADH) gene family in oilseed rape (Brassica napus L.) and BnADH36 functional verification under salt stress

BackgroundAlcohol dehydrogenase (ADH) is an enzyme that binds to zinc, facilitating the interconversion of ethanol and acetaldehyde or other corresponding alcohols/aldehydes in the pathway of ethanol fermentation. It plays a pivotal role in responding to environmental stress. However, the response of the ADH family to abiotic stress remains unknown in rapeseed.ResultIn this study, we conducted a comprehensive genome-wide investigation of the ADH family in rapeseed, encompassing analysis of their gene structure, replication patterns, conserved motifs, cis-acting elements, and response to stress. A total of 47 ADH genes were identified within the rapeseed genome. Through phylogenetic analysis, BnADHs were classified into four distinct clades (I, II, IV, V). Prediction of protein domains revealed that all BnADH members possessed a GroES-like (ADH_N) domain and a zinc-bound (ADH_zinc_N) domain. Analysis of promoter sequences demonstrated that BnADHs contained numerous cis-acting elements associated with hormone and stress responses, indicating their widespread involvement in various biological regulatory processes. Expression profiling under different concentrations of salt stress treatments (0%, 0.4%, 0.8%, 1.0% NaCl) further highlighted the significant role played by the BnADH family in abiotic stress response mechanisms. Overexpression of BnADH36 in rapeseed significantly improved the salt tolerance of rapeseed.ConclusionThe features of the BnADH family in rapeseed was comprehensively characterized in this study, which could provide reference to the research of BnADHs in abiotic stress response.

Genome-Wide Identification and Expression Profiling Analysis of the CCT Gene Family in Solanum lycopersicum and Solanum melongena

CCT family genes play crucial roles in photoperiodic flowering and environmental stress response; however, there are limited reports in Solanum species with considerable edible and medicinal value. In this study, we conducted genome-wide characterization and expression profiling analysis of the CCT gene family in two Solanum species: tomato (Solanum lycopersicum L.) and eggplant (Solanum melongena L.). A total of 27 SlCCT and 29 SmCCT genes were identified in the tomato and eggplant genomes, respectively. Phylogenetic analysis showed that the CCT gene family could be divided into six subgroups (COL I, COL II, COL III, PRR, CMF I, and CMF II) in Oryza sativa and Arabidopsis thaliana. The similarity in the distribution of exon–intron structures and conserved motifs within the same subgroup indicated the conservation of SlCCT and SmCCT genes during evolution. Intraspecies collinearity analysis revealed that six pairs of SlCCT genes and seven pairs of SmCCT genes showed collinear relationships, suggesting that segmental duplication played a vital role in the expansion of the SlCCT and SmCCT family genes. Cis-acting element prediction indicated that SlCCT and SmCCT were likely to be involved in multiple responses stimulated by light, phytohormones, and abiotic stress. RT-qPCR analysis revealed that SmCCT15, SlCCT6/SlCCT14, and SlCCT23/SmCCT9 responded significantly to salt, drought, and cold stress, respectively. Our comprehensive analysis of the CCT gene family in tomato and eggplant provides a basis for further studies on its molecular role in regulating flowering and resistance to abiotic stress, and provides valuable candidate gene resources for tomato and eggplant molecular breeding.

Oryza sativa Stress Associated Protein (OsSAP) Promoter Modulates Gene Expression in Response To Abiotic Stress by Utilizing Cis Regulatory Elements Within The Promoter Region

The occurrence of extreme weather patterns induced by climate change has resulted in abiotic stress problems impacting the growth and productivity of plants. Rice (Oryza sativa), a staple food source for most Asians, is similarly affected by these challenges. Previous studies have identified the Oryza sativa Stress Associated Protein (OsSAP) genes to play a significant role in responding to abiotic stress. Among the 18 Stress Associated Protein members, OsSAP4 was highly expressed during drought and salinity conditions. Therefore, further experiments have been conducted, focusing specifically on the promoter region, to comprehend its regulation in response to abiotic stresses. Various types of cis-elements binding sites have been identified within the OsSAP4 promoter, encompassing MYB, CAMTA, CPP, C3H, HDZIP, bZIP, WRKY, and ERF. However, promoter analysis revealed that the distribution of the Cis-Regulatory elements bound by the Ethylene Response Factor (ERF) was the most prominent in the OsSAP4 promoter. Consequently, an analysis of promoter regulation was conducted using GUS reporter in Arabidopsis thaliana (A. thaliana) on two different sizes of OsSAP4 promoter sequences, each containing different quantities of ERF transcription factor binding sites. A noticeable difference in GUS staining activity was observed between pOsSAP4(1524 pb)::GUS and pOsSAP4(460 pb)::GUS, where pOsSAP4(1524 pb)::GUS exhibited higher GUS staining activity than pOsSAP4(460 pb)::GUS. The differences in GUS staining analysis are evident at the vegetative stage (leaf), silique, and inflorescence stages. This implies the participation of various other cis-element binding sites that influence the expression pattern of the OsSAP4 promoter during abiotic stress.

The Potential Role of PeMAP65-18 in Secondary Cell Wall Formation in Moso Bamboo.

Microtubule-associated proteins (MAPs) play a pivotal role in the assembly and stabilization of microtubules, which are essential for plant cell growth, development, and morphogenesis. A class of plant-specific MAPs, MAP65, plays largely unexplored roles in moso bamboo (Phyllostachys edulis). This study identified 19 PeMAP65 genes in moso bamboo, systematically examining their phylogenetic relationships, conserved motifs, gene structures, collinearity, and cis-acting elements. Analysis of gene expression indicated that PeMAP65s exhibit tissue-specific expression patterns. Functional differentiation was investigated among the members of different PeMAP65 subfamilies according to their expression patterns in different development stages of bamboo shoots. The expression of PeMAP65-18 was positively correlated with the expression of genes involved in secondary cell wall (SCW) biosynthesis. Y1H and Dual-LUC assays demonstrated that the transcription of PeMAP65-18 was upregulated by PeMYB46, a key transcription factor of SCW biosynthesis. The result of subcellular localization showed that PeMAP65-18 was located in cortical microtubules. We speculate that PeMAP65-18 may play a crucial role in the SCW deposition of moso bamboo. This comprehensive analysis of the MAP65 family offers novel insights into the roles of PeMAP65s in moso bamboo, particularly in relation to the formation of SCWs.

Identification of lineage-specific cis-trans regulatory networks related to kiwifruit ripening initiation.

Previous research on the ripening process of many fruit crop varieties typically involved analyses of the conserved genetic factors among species. However, even for seemingly identical ripening processes, the associated gene expression networks often evolved independently, as reflected by the diversity in the interactions between transcription factors (TFs) and the targeted cis-regulatory elements (CREs). In this study, explainable deep learning (DL) frameworks were used to predict expression patterns on the basis of CREs in promoter sequences. We initially screened potential lineage-specific CRE-TF interactions influencing the kiwifruit ripening process, which is triggered by ethylene, similar to the corresponding processes in other climacteric fruit crops. Some novel regulatory relationships affecting ethylene-induced fruit ripening were identified. Specifically, ABI5-like bZIP, G2-like, and MYB81-like TFs were revealed as trans-factors modulating the expression of representative ethylene signaling/biosynthesis-related genes (e.g., ACS1, ERT2, and ERF143). Transient reporter assays and DNA affinity purification sequencing (DAP-Seq) analyses validated these CRE-TF interactions and their regulatory relationships. A comparative analysis with co-expression networking suggested that this DL-based screening can identify regulatory networks independently of co-expression patterns. Our results highlight the utility of an explainable DL approach for identifying novel CRE-TF interactions. These imply that fruit crop species may have evolved lineage-specific fruit ripening-related cis-trans regulatory networks.

Identification of the HbZAR1 Gene and Its Potential Role as a Minor Gene in Response to Powdery Mildew and Anthracnose of Hevea brasiliensis

Powdery mildew and anthracnose are the main diseases of rubber trees. In recent years, there have been large outbreaks in the rubber-planting areas of Asia, seriously affecting the yield and quality of rubber latex. ZAR1 is a conserved and distinctive coiled-coil nucleotide-binding leucine-rich (CNL) repeat in the plant kingdom, playing a crucial role in disease-resistance processes. To elucidate the function of the HbZAR1 gene in rubber trees (Hevea brasiliensis), three candidate HbZAR1 genes were identified using bioinformatics methods and comprehensively analyzed. The results indicate that the HbZAR1 protein is conserved in different plant species. Examination of cis-regulatory element sequences of HbZAR1genes reveals that the HbZAR1 gene promoter exhibits a remarkable enrichment of stress, light, and hormone elements. An expression analysis shows that the expression levels of the three HbZAR1 genes are highest in the bark and lowest in latex. Three HbZAR1 genes can respond to both rubber tree Erysiphe quercicola and Colletotrichum siamense infection; especially, HbZAR1.1 and HbZAR1.2 show significant upregulation in expression levels during the early stages of infection. These findings suggest that the three HbZAR1 genes may be involved in rubber tree susceptibility to E. quercicola and C. siamense through different immune mechanisms. Subcellular localization results indicate that the HbZAR1 genes are expressed in the nucleus and plasma membrane. This study also shows that the three HbZAR1 genes and activated mutant HbZAR1.1D481V do not induce stable ROS production and cell death, suggesting possible gene degradation, functional redundancy, or acting as minor genes in disease resistance. This research provides valuable insights for further studying the function of HbZAR1 genes in rubber trees and the mechanisms of immune molecules.

Identification of Potato StPIN Gene Family and Regulation of Root Development by StPIN4.

The growth hormone export PIN-FORMED (PIN) is an important carrier for regulating the polar transport of plant growth hormones and plays an important role in plant growth and development. However, little is known about the characteristics and functions of PINs in potatoes. In this study, 10 PIN members were identified from potatoes and named StPIN1, StPIN2, StPIN3, StPIN4, StPIN5, StPIN6, StPIN7, StPIN8, StPIN9, and StPIN10 according to their positions in the potato chromosome In addition, the expression of 10 StPINs was analyzed by qRT-PCR during potato root development. The results showed that the StPIN4 gene plays an important regulatory role in potato root development, and its tissue expression varied greatly. Several cis-regulatory elements related to growth factors were also detected in the promoter region of the StPIN gene. The transgenic overexpressing StPIN4 in potato showed suppressed growth in root length and lateral root number, and StPIN4-interfering plants showed the opposite. These results suggested that StPIN4 plays a key role in the regulation of the potato root architecture.

Characterization of Superoxide Dismutase (SOD) Gene Family and Their Responses to Salinity Stress and Fruit Development in Octoploid Strawberry

Superoxide dismutases (SODs), as the first line of defense against reactive oxygen species (ROS), play an essential role in protecting plants from adverse elicitors during plant growth and development. However, little is known about the SOD gene family and their response to salinity stress and fruit development in cultivated strawberries (Fragaria × ananassa). In this study, 32 SOD genes consisting of 16 Cu/ZnSODs, 11 FeSODs, and 5 MnSOD were identified, which presented three well-resolved clades in the phylogenetic tree. Each clade had similar motifs, and exon–intron structures, which in turn supported the evolutionary classification. Cis-acting element analysis suggested that FaSOD genes might be involved in the plant response to abiotic and biotic stresses, hormones, and light. The analysis of previously published transcriptome data revealed that FaSOD genes are expressed variably under salt stress. Among these SODs, FaMSD5 was expressed at relatively high levels in strawberry root and leaf, and its transcript abundance significantly increased after salt treatment. Some transcription factors related to photomorphogenesis, hormone signaling pathways, and hyperosmotic salinity response were predicted to bind to the FaMSD5 promoter. These outcomes implied that FaMSD5 might play an important role in protection against salt stress. In addition, the comprehensive transcriptome analysis of FaSOD genes in strawberry fruit showed that almost all FaCSDs and FaMSDs were more highly expressed than FaFSDs at different developmental stages, and the expression patterns of FaCSD1, FaCSD2, FaCSD7, FaCSD8, and FaCSD10 suggested that they were likely to be involved in fruit development and ripening. This study provides a basis for further exploration of the function of the FaSOD gene family in strawberry and provides candidate FaSOD genes for enhancing salinity tolerance and regulating fruit development and ripening.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

Genome-wide computational analysis of the dirigent gene family in Solanum lycopersicum

BackgroundDirigent (DIR) genes play a key role in the development of organic products in plants. They confer conformational influence on processes that lack stereoselectivity and regioselectivity through processes that are mostly understood. They are required to produce lignans, which are a unique and widely distributed family of plant secondary metabolites with intriguing pharmacological characteristics and potential role in plant development. DIR genes are implicated in the process of lignification and protect plants from environmental stresses, including biotic and abiotic stresses. Nevertheless, no research has been performed on the DIR gene family in Solanum lycopersicum. This study provides detailed information on the DIR gene family in S. lycopersicum.Methods and resultsThe conserved domain analysis, phylogenetic analysis, evolutionary adaptation, cis-acting elements, proteomic analysis, signal peptide detection, transmembrane potential analysis, sequence identity and similarity analysis, gene assembly, genomic localization, duplication of gene analysis, and evolutionary linkage of 31 potential DIR genes were studied. All these analyses provide a deep understanding of DIR genes in the S. lycopersicum genome that will provide a useful reference for further functional analysis of the DIR genes in S. lycopersicum.ConclusionThis research provides an in-depth and comprehensive explanation of the detailed process and structural characterization of DIR genes in the genome of S. lycopersicum, laying the groundwork for future plant genetic engineering and crop development exploration. This work will provide valuable information for identifying DIR genes in higher plants and support future research on the DIR gene family.

Identification of AP2/ERF Transcription Factors and Characterization of AP2/ERF Genes Related to Low-Temperature Stress Response and Fruit Development in Luffa

Plant-specific APETALA2/Ethylene-Responsive Factor (AP2/ERF) transcription factors are involved in the regulation of genes associated with the growth and developmental processes of numerous plants. Although AP2/ERF proteins from other species have been intensively studied, no studies have been reported on the AP2/ERF family of Luffa cylindrica, an important vegetable of the cucurbit family, and one of the most popular vegetables in the world. In this study, 133 genes (315–6696 bp) encoding LcAP2/ERF proteins with complete AP2/ERF domains were identified according to the luffa P93075 genome. These LcAP2/ERF genes were subsequently classified and analyzed for their gene structures, chromosomal distribution locations, promoter cis-acting elements, conserved structural domains of encoded proteins, and responses to abiotic stresses. The LcAP2/ERF genes were identified and divided into five phylogenetic groups (AP2, DREBs, ERFs, RAV, and soloists). These genes were unevenly distributed across 13 chromosomes. An analysis of gene structures indicated the LcAP2/ERF genes contained 0–11 introns (average of 4.4). Additionally, 16 motifs were identified in the LcAP2/ERF proteins that were conserved across different phylogenetic groups. Moreover, 11 cis-acting elements associated with response to the environment were analyzed in a 2000 bp region upstream of the LcAP2/ERF gene promoters. A transcriptome analysis involving RNA-seq data revealed tissue-specific LcAP2/ERF expression profiles and the diversity in LcAP2/ERF expression. The effects of low-temperature stress on LcAP2/ERF expression were determined. Furthermore, fruit-development-related and low-temperature-induced expressional changes were verified by RT-qPCR analyses of 14 differentially expressed LcAP2/ERF genes in luffa. Our findings will help clarify the evolution of the luffa AP2/ERF family, while also providing valuable insights for future studies on AP2/ERF functions.

Terpene synthase gene family in Jasminum sambac var. Fuzhou bifoliatum: genome-wide analysis and expression pattern in response to methyl jasmonate

Terpene synthases (TPSs) play a crucial role in the synthesis of terpenoids that contribute to the scent profiles of flowers. However, few studies report the genome-wide analysis of TPSs gene in Jasminum sambac var. Fuzhou bifoliatum and their expression pattern in response to methyl jasmonate (MeJA). In this study, we employed bioinformatics tools for genome-wide analysis of the J. sambac TPS (DJTPS) gene family and determined the physical and chemical properties, subcellular location, protein-protein interactions, phylogenetic relationship, subfamily classification, chromosomal location and collinearity, gene structure, conserved motifs, and promoter cis-acting elements. The expression patterns of DJTPSs in different tissues and in response to MeJA treatment were analyzed based on the transcriptome data combined with quantitative real-time PCR (qRT-PCR). We identified 32 intact DJTPS genes in the genome of J. sambac, which presented uneven distribution across nine chromosomes. All the deduced proteins were hydrophilic, predominantly localized in the cytoplasm. The phylogenetic analysis classified the DJTPS genes into five subfamilies: TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. The results of the collinearity analysis showed a total of 10 sets of replication events in DJTPSs, most of which underwent purifying selection. A comparative analysis of TPS homologous gene pairs was performed among J. sambac var. Fuzhou bifoliatum and other six species, which revealed different number of homologous gene pairs. The number of exons and motifs was conserved within the same subfamily. DJTPS genes carried multiple elements that may be involved in the response to MeJA. In addition, the transcriptome and qRT-PCR data unveiled that several TPS genes exhibited tissue-specific expression patterns, and the genes with specific expression in flowers were the most. Upon exposure to MeJA, 14 TPS genes showcased upregulated expression 5 h or 6 h post-treatment, and DJTPS03, DJTPS04 and DJTPS21 showed significantly increased expression levels after MeJA treatment. This study provides preliminary evidence that MeJA possesses the ability to enhance the expression of DJTPS genes during the critical flowering stage, which will facilitate the synthesis of terpenoids and improve the quality of floral fragrance.

Genomic Identification and Expression Analysis of Regulator of Chromosome Condensation 1-Domain Protein Family in Maize.

Abiotic stress affects the growth and development of maize (Zea mays). The regulator of chromosome condensation 1 (RCC1)-containing proteins (RCPs) plays crucial roles in plant growth and development and response to abiotic stresses. However, a comprehensive analysis of the maize RCP family has not been reported in detail. This study presents a systematic bioinformatics analysis of the ZmRCP family, identifying a total of 30 members distributed across nine chromosomes. The physicochemical properties and cis-acting elements in the promoters of ZmRCP members are predicted. The results of subcellular localization showed that ZmRCP3 and ZmRCP10 are targeted to mitochondria and ZmRCP2 is localized in the nucleus. A heatmap of expression levels among family members under abiotic stress conditions revealed varying degrees of induced expression, and the expression levels of 10 ZmRCP members were quantified using RT-qPCR under abiotic stress and plant hormone treatments. The results showed that ZmRCP members exhibit induced or inhibited responses to these abiotic stresses and plant hormones. These results contribute to a better understanding of the evolutionary history and potential role of the ZmRCP family in mediating responses to abiotic stress in maize.