Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis.

E. rutaecarpa var. officinalis is a traditional Chinese medicinal plant known for its therapeutic effects, which encompass the promotion of digestion, the dispelling of cold, the alleviation of pain, and the exhibition of anti-inflammatory and antibacterial properties. The principal active component of this plant, limonin, is a potent triterpene compound with notable pharmacological activities. Despite its significance, the complete biosynthesis pathway of limonin in E. rutaecarpa var. officinalis remains incompletely understood, and the underlying molecular mechanisms remain unexplored. The main purpose of this study was to screen the reference genes suitable for expression analysis in E. rutaecarpa var. officinalis, calculate the expression patterns of the genes in the limonin biosynthesis pathway, and identify the relevant enzyme genes related to limonin biosynthesis. The reference genes play a pivotal role in establishing reliable reference standards for normalizing the gene expression data, thereby ensuring precision and credibility in the biological research outcomes. In order to identify the optimal reference genes and gene expression patterns across the diverse tissues (e.g., roots, stems, leaves, and flower buds) and developmental stages (i.e., 17 July, 24 August, 1 September, and 24 October) of E. rutaecarpa var. officinalis, LC-MS was used to analyze the limonin contents in distinct tissue samples and developmental stages, and qRT-PCR technology was employed to investigate the expression patterns of the ten reference genes and eighteen genes involved in limonin biosynthesis. Utilizing a comprehensive analysis that integrated three software tools (GeNorm ver. 3.5, NormFinder ver. 0.953 and BestKeeper ver. 1.0) and Delta Ct method alongside the RefFinder website, the best reference genes were selected. Through the research, we determined that Act1 and UBQ served as the preferred reference genes for normalizing gene expression during various fruit developmental stages, while Act1 and His3 were optimal for different tissues. Using Act1 and UBQ as the reference genes, and based on the different fruit developmental stages, qRT-PCR analysis was performed on the pathway genes selected from the "full-length transcriptome + expression profile + metabolome" data in the limonin biosynthesis pathway of E. rutaecarpa var. officinalis. The findings indicated that there were consistent expression patterns of HMGCR, SQE, and CYP450 with fluctuations in the limonin contents, suggesting their potential involvement in the limonin biosynthesis of E. rutaecarpa var. officinalis. This study lays the foundation for further research on the metabolic pathway of limonin in E. rutaecarpa var. officinalis and provides reliable reference genes for other researchers to use for conducting expression analyses.

Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses.

BACKGROUND: Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide. RESULTS: In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron-exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants. CONCLUSIONS: This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

Genome-Wide Identification, Classification, and Expression Analyses of the CsDGAT Gene Family in Cannabis sativa L. and Their Response to Cold Treatment.

Hempseed is a nutrient-rich natural resource, and high levels of hempseed oil accumulate within hemp seeds, consisting primarily of different triglycerides. Members of the diacylglycerol acyltransferase (DGAT) enzyme family play critical roles in catalyzing triacylglycerol biosynthesis in plants, often governing the rate-limiting step in this process. As such, this study was designed to characterize the Cannabis sativa DGAT (CsDGAT) gene family in detail. Genomic analyses of the C. sativa revealed 10 candidate DGAT genes that were classified into four families (DGAT1, DGAT2, DGAT3, WS/DGAT) based on the features of different isoforms. Members of the CsDGAT family were found to be associated with large numbers of cis-acting promoter elements, including plant response elements, plant hormone response elements, light response elements, and stress response elements, suggesting roles for these genes in key processes such as development, environmental adaptation, and abiotic stress responses. Profiling of these genes in various tissues and varieties revealed varying spatial patterns of CsDGAT expression dynamics and differences in expression among C. sativa varieties, suggesting that the members of this gene family likely play distinct functional regulatory functions CsDGAT genes were upregulated in response to cold stress, and significant differences in the mode of regulation were observed when comparing roots and leaves, indicating that CsDGAT genes may play positive roles as regulators of cold responses in hemp while also playing distinct roles in shaping the responses of different parts of hemp seedlings to cold exposure. These data provide a robust basis for further functional studies of this gene family, supporting future efforts to screen the significance of CsDGAT candidate genes to validate their functions to improve hempseed oil composition.

Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiata.

Lycoris radiata, belonging to the Amaryllidaceae family, is a well-known Chinese traditional medicinal plant and susceptible to many stresses. WRKY proteins are one of the largest families of transcription factors (TFs) in plants and play significant functions in regulating physiological metabolisms and abiotic stress responses. The WRKY TF family has been identified and investigated in many medicinal plants, but its members and functions are not identified in L. radiata. In this study, a total of 31 L. radiata WRKY (LrWRKY) genes were identified based on the transcriptome-sequencing data. Next, the LrWRKYs were divided into three major clades (Group I-III) based on the WRKY domains. A motif analysis showed the members within same group shared a similar motif component, indicating a conservational function. Furthermore, subcellular localization analysis exhibited that most LrWRKYs were localized in the nucleus. The expression pattern of the LrWRKY genes differed across tissues and might be important for Lycoris growth and flower development. There were large differences among the LrWRKYs based on the transcriptional levels under drought stress and MeJA treatments. Moreover, a total of 18 anthocyanin components were characterized using an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis and pelargonidin-3-O-glucoside-5-O-arabinoside as well as cyanidin-3-O-sambubioside were identified as the major anthocyanin aglycones responsible for the coloration of the red petals in L. radiata. We further established a gene-to-metabolite correlation network and identified LrWRKY3 and LrWRKY27 significant association with the accumulation of pelargonidin-3-O-glucoside-5-O-arabinoside in the Lycoris red petals. These results provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in anthocyanin biosynthesis and in response to drought stress and MeJA treatment.

Identification and expression analysis of YABBY family genes in Platycodon grandiflorus.

Platycodon grandiflorus set ornamental, edible, and medicinal plant with broad prospects for further application development. However, there are no reports on the YABBY transcription factor in P. grandiflorus. Identification and analysis of the YABBY gene family of P. grandiflorus using bioinformatics means. Six YABBY genes were identified and divided into five subgroups. Transcriptome data and qRT-PCR were used to analyze the expression patterns of YABBY. YABBY genes exhibited organ-specific patterns in expression in P grandiflorus. Upon salt stress and drought induction, P. grandiflorus presented different morphological and physiological changes with some dynamic changes. Under salt treatment, the YABBY gene family was down-regulated; PgYABBY5 was up-regulated in leaves at 24 h. In drought treatment, PgYABBY1, PgYABBY2, and PgYABBY3 were down-regulated to varying degrees, but PgYABBY3 was significantly up-regulated in the roots. PgYABBY5 was up-regulated gradually after being down-regulated. PgYABBY5 was significantly up-regulated in stem and leaf at 48 h. PgYABBY6 was down-regulated at first and then significantly up-regulated. The dynamic changes of salt stress and drought stress can be regarded as the responses of plants to resist damage. During the whole process of salt and drought stress treatment, the protein content of each tissue part of P grandiflorus changed continuously. At the same time, we found that the promoter region of the PgYABBY gene contains stress-resistant elements, and the regulatory role of YABBY transcription factor in the anti-stress mechanism of P grandiflorus remains to be studied. PgYABBY1, PgYABBY2, and PgYABBY5 may be involved in the regulation of saponins in P. grandiflorus. PgYABBY5 may be involved in the drought resistance mechanism in P. grandiflorus stems and leaves. This study may provide a theoretical basis for studying the regulation of terpenoids by the YABBY transcription factor and its resistance to abiotic stress.

Genome-wide identification and expression profiles analysis of the authentic response regulator gene family in licorice.

Introduction: As one of the traditional Chinese medicinal herbs that were most generally used, licorice attracts lots of interest due to its therapeutic potential. Authentic response regulators (ARRs) are key factors in cytokinin signal transduction and crucial for plant growth and stress response processes. Nevertheless, the characteristics and functions of the licorice ARR genes are still unknown. Results: In present study, a systematic genome-wide identification and expression analysis of the licorice ARR gene family were conducted and 51 ARR members were identified. Collinearity analysis revealed the significant roles of segmental duplications in the expansion of licorice ARR genes. The cis-acting elements associated with development, stress and phytohormone responses were identified, implying their pivotal roles in diverse regulatory processes. RNA-seq and qRT-PCR results suggested that A-type, but not B-type ARRs were induced by zeatin. Additionally, ARRs participated in diverse abiotic stresses and phytohormones responses. Yeast one-hybrid assay demonstrated that GuARR1, GuARR2, GuARR11, GuARR12, GuARR10-1, GuARR10-2 and GuARR14 were able to bind to the promoter of GuARR8-3, and GuARR1, GuARR12 bound to the GuARR8-1 promoter. GuARR1, GuARR2, GuARR11 and GuARR10-2 bound to the GuARR6-2 promoter as well as GuARR12 and GuARR10-2 bound to the GuARR6-1 promoter. Discussion: Collectively, these findings provide a basis for future ARR genes function investigations, shedding light on the potential medicinal properties and agricultural applications of licorice.

The LEA gene family in tomato and its wild relatives: genome-wide identification, structural characterization, expression profiling, and role of SlLEA6 in drought stress.

BACKGROUND: Late embryogenesis abundant (LEA) proteins are widely distributed in higher plants and play crucial roles in regulating plant growth and development processes and resisting abiotic stress. Cultivated tomato (Solanum lycopersicum) is an important vegetable crop worldwide; however, its growth, development, yield, and quality are currently severely constrained by abiotic stressors. In contrast, wild tomato species are more tolerant to abiotic stress and can grow normally in extreme environments. The main objective of this study was to identify, characterize, and perform gene expression analysis of LEA protein families from cultivated and wild tomato species to mine candidate genes and determine their potential role in abiotic stress tolerance in tomatoes. RESULTS: Total 60, 69, 65, and 60 LEA genes were identified in S. lycopersicum, Solanum pimpinellifolium, Solanum pennellii, and Solanum lycopersicoides, respectively. Characterization results showed that these genes could be divided into eight clusters, with the LEA_2 cluster having the most members. Most LEA genes had few introns and were non-randomly distributed on chromosomes; the promoter regions contained numerous cis-acting regulatory elements related to abiotic stress tolerance and phytohormone responses. Evolutionary analysis showed that LEA genes were highly conserved and that the segmental duplication event played an important role in evolution of the LEA gene family. Transcription and expression pattern analyses revealed different regulatory patterns of LEA genes between cultivated and wild tomato species under normal conditions. Certain S. lycopersicum LEA (SlLEA) genes showed similar expression patterns and played specific roles under different abiotic stress and phytohormone treatments. Gene ontology and protein interaction analyses showed that most LEA genes acted in response to abiotic stimuli and water deficit. Five SlLEA proteins were found to interact with 11 S. lycopersicum WRKY proteins involved in development or resistance to stress. Virus-induced gene silencing of SlLEA6 affected the antioxidant and reactive oxygen species defense systems, increased the degree of cellular damage, and reduced drought resistance in S. lycopersicum. CONCLUSION: These findings provide comprehensive information on LEA proteins in cultivated and wild tomato species and their possible functions under different abiotic and phytohormone stresses. The study systematically broadens our current understanding of LEA proteins and candidate genes and provides a theoretical basis for future functional studies aimed at improving stress resistance in tomato.

Genome-Wide Identification, Evolution, and Expression Pattern Analysis of the GATA Gene Family in Tartary Buckwheat (Fagopyrum tataricum).

GATA is a transcription factor that exerts a vital function in plant growth and development, physiological metabolism, and environmental responses. However, the GATA gene family has rarely been studied in Tartary buckwheat since the completion of its genome. This study used bioinformatics methods to identify GATA genes of Tartary buckwheat and to analyze their subfamily classification, structural composition, and developmental evolution, as well as to discuss the expression patterns of FtGATA genes in different subfamilies. The twenty-eight identified FtGATA genes in the Tartary buckwheat genome were divided into four subfamilies and distributed on eight chromosomes. One pair of tandem repeat genes and eight pairs of fragments were found in chromosome mapping. Spatiotemporal expression patterns of eight FtGATA genes in different subfamilies indicated that the FtGATA gene family has regulatory roles in tissue specificity, fruit development, abiotic stress, and hormonal responses. This study creates a theoretical and scientific foundation for further research on the evolutionary relationship and biological function of FtGATA.

Full-Length Transcriptomic Sequencing and Temporal Transcriptome Expression Profiling Analyses Offer Insights into Terpenoid Biosynthesis in Artemisia argyi.

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC-MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila).

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

Transcriptome-wide identification of WRKY transcription factors and their expression profiles in response to methyl jasmonate in Platycodon grandiflorus.

Platycodon grandiflorus, a perennial flowering plant widely distributed in China and South Korea, is an excellent resource for both food and medicine. The main active compounds of P. grandiflorus are triterpenoid saponins. WRKY transcription factors (TFs) are among the largest gene families in plants and play an important role in regulating plant terpenoid accumulation, physiological metabolism, and stress response. Numerous studies have been reported on other medicinal plants; however, little is known about WRKY genes in P. grandiflorus. In this study, 27 PgWRKYs were identified in the P. grandiflorus transcriptome. Phylogenetic analysis showed that PgWRKY genes were clustered into three main groups and five subgroups. Transcriptome analysis showed that the PgWRKY gene expression patterns in different tissues differed between those in Tongcheng City (Southern Anhui) and Taihe County (Northern Anhui). Gene expression analysis based on RNA sequencing and qRT-PCR analysis showed that most PgWRKY genes were expressed after induction with methyl jasmonate (MeJA). Co-expressing PgWRKY genes with triterpenoid biosynthesis pathway genes revealed four PgWRKY genes that may have functions in triterpenoid biosynthesis. Additionally, functional annotation and protein-protein interaction analysis of PgWRKY proteins were performed to predict their roles in potential regulatory networks. Thus, we systematically analyzed the structure, evolution, and expression patterns of PgWRKY genes to provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in triterpenoid biosynthesis.

Molecular cloning, characterization, and functional analysis of the uncharacterized C11orf96 gene.

BACKGROUND: The mammalian genome encodes millions of proteins. Although many proteins have been discovered and identified, a large part of proteins encoded by genes are yet to be discovered or fully characterized. In the present study, we successfully identified a host protein C11orf96 that was significantly upregulated after viral infection. RESULTS: First, we successfully cloned the coding sequence (CDS) region of the cat, human, and mouse C11orf96 gene. The CDS region of the C11orf96 gene is 372 bp long, encodes 124 amino acids, and is relatively conserved in different mammals. From bioinformatics analysis, we found that C11orf96 is rich in Ser and has multiple predicted phosphorylation sites. Moreover, protein interaction prediction analysis revealed that the protein is associated with several transmembrane family proteins and zinc finger proteins. Subsequently, we found that C11orf96 is strictly distributed in the cytoplasm. According to the tissue distribution characteristics, C11orf96 is distributed in all tissues and organs, with the highest expression levels in the kidney. These results indicate that C11orf96 may play a specific biological role in the kidney. CONCLUSIONS: Summarizing, these data lay the foundation for studying the biological functions of C11orf96 and for exploring its role in viral replication.

Genome-Wide Characterization and Expression Analysis of GATA Transcription Factors in Response to Methyl Jasmonate in Salvia miltiorrhiza.

Salvia miltiorrhiza is an important medicinal plant, which is mainly used for treatment of cardiovascular and cerebrovascular diseases. GATA transcription factors are evolutionarily conser-ved proteins that play essential roles in biological process of plants. In this study, we systematically characterized the GATA transcription factors in S. miltiorrhiza. A total 28 SmGATA genes were identified and divided into four subfamilies based on phylogenetic analysis and domain. SmGATA genes being clustered into a subfamily have similar conserved motifs and exon-intron patterns, and unevenly distribute on eight chromosomes of S. miltiorrhiza. Tissue-specific expression analysis based on transcriptome datasets showed that the majority of SmGATA genes were preferentially expressed in roots. Under methyl jasmonate (MeJA) treatment, the quantitative real-time PCR (qRT-PCR) analysis indicated that several SmGATA genes in roots showed distinct upregulation post-MeJA treatment, especially SmGATA08, which was highly responsive to MeJA, and might be involved in the jasmonate signal, thereby affecting root growth, development, tolerance to various stresses, or secondary metabolites biosynthesis. The study found that several SmGATAs, like SmGATA08, are highly responsive to MeJA, indicating that these SmGATAs might be vital in the biosynthesis of tanshinones and phenolic acids by regulating the response to MeJA in S. miltiorrhiza. Our results laid the foundation for understanding their biological roles and quality improvement in S. miltiorrhiza.

SWEET Gene Family in Sugar Beet (Beta vulgaris): Genome-Wide Survey, Phylogeny and Expression Analysis.

<b>Background and Objective:</b> The SWEET (Sugars Will Eventually be Exported Transporter) proteins play important roles in modulating the growth and development processes in plants. However, little information is available on the SWEET family in sugar beet (<i>Beta vulgaris</i>). The objectives of this present study were to genome-wide identify and characterize the BvSWEET family in sugar beet. <b>Materials and Methods:</b> Based on the available genome, proteome and transcriptome databases of sugar beet, various computational tools have been used to analyze the nucleotide and full-length protein sequences of members of the BvSWEET family. <b>Results:</b> A total of 16 members of the BvSWEET family has been identified in sugar beet at the genome-wide scale. Structural analysis indicated that the BvSWEET family exhibited variable characteristics. Furthermore, the BvSWEET family in sugar beet could be categorized into four distinct groups like in other plant species. Of our interest, we found that some <i>BvSWEET</i> genes exhibited strongly preferential expression in major organs/tissues under adverse environmental stimuli. <b>Conclusion:</b> The results provided a comprehensive foundation for further functional characterization of the <i>BvSWEET </i>gene family.

Genome-Wide Identification and Characterization of the WRKY Gene Family in Scutellaria baicalensis Georgi under Diverse Abiotic Stress.

The WRKY gene family is an important inducible regulatory factor in plants, which has been extensively studied in many model plants. It has progressively become the focus of investigation for the secondary metabolites of medicinal plants. Currently, there is no systematic analysis of the WRKY gene family in Scutellaria baicalensis Georgi. For this study, a systematic and comprehensive bioinformatics analysis of the WRKY gene family was conducted based on the genomic data of S. baicalensis. A total of 77 WRKY members were identified and 75 were mapped onto nine chromosomes, respectively. Their encoded WRKY proteins could be classified into three subfamilies: Group I, Group II (II-a, II-b, II-c, II-d, II-e), and Group III, based on the characteristics of the amino acid sequences of the WRKY domain and genetic structure. Syntenic analysis revealed that there were 35 pairs of repetitive fragments. Furthermore, the transcriptome data of roots, stems, leaves, and flowers showed that the spatial expression profiles of WRKYs were different. qRT-PCR analysis revealed that 11 stress-related WRKYs exhibited specific expression patterns under diverse treatments. In addition, sub cellular localization analysis indicated that SbWRKY26 and SbWRKY41 were localized in nucleus. This study is the first to report the identification and characterization of the WRKY gene family in S. baicalensis, which is valuable for the further exploration of the biological function of SbWRKYs. It also provides valuable bioinformatics data for S. baicalensis and provides a reference for assessing the medicinal properties of the genus.

Genome-wide identification of PEBP gene family members in potato, their phylogenetic relationships, and expression patterns under heat stress.

BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP) gene family is involved in regulating many plant traits. Genome-wide identification of PEPB members and knowledge of their responses to heat stress may assist genetic improvement of potato (Solanum tuberosum). METHODS AND RESULTS: We identified PEBP gene family members from both the recently-updated, long-reads-based reference genome (DM v6.1) and the previous short-reads-based annotation (PGSC DM v3.4) of the potato reference genome and characterized their heat-induced gene expression using RT-PCR and RNA-Seq. Fifteen PEBP family genes were identified from DM v6.1 and named as StPEBP1 to StPEBP15 based on their locations on 6 chromosomes and were classified into FT, TFL, MFT, and PEBP-like subfamilies. Most of the StPEBP genes were found to have conserved motifs 1 to 5. Tandem or segmental duplications were found between StPEBP genes in seven pairs. Heat stress induced opposite expression patterns of certain FT and TFL members but involving different members in leaves, roots and tubers. CONCLUSION: The long-reads-based genome assembly and annotation provides a better genomic resource for identification of PEBP family genes. Heat stress tends to decrease FT gene activities but increases TFL gene activities, but this opposite expression involves different FT/TFL pairs in leaves, roots, and tubers. This tissue-specific expression pattern of PEBP members may partly explain why different potato organs differ in their sensitivities to heat stress. Our study provides candidate PEBP family genes and relevant information for genetic improvement of heat tolerance in potato and may help understand heat-induced responses in other plants.

Genome-wide identification of MAPK gene family members in Fagopyrum tataricum and their expression during development and stress responses.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) plays essential roles in the development, hormone regulation and abiotic stress response of plants. Nevertheless, a comprehensive study on MAPK family members has thus far not been performed in Tartary buckwheat. RESULTS: Here, we identified 16 FtMAPKs in the Fagopyrum tataricum genome. Phylogenetic analysis showed that the FtMAPK family members could be classified into Groups A, B, C and D, in which A, B and C members contain a Thr-Glu-Tyr (TEY) signature motif and Group D members contain a Thr-Asp-Tyr (TDY) signature motif. Promoter cis-acting elements showed that most ProFtMAPks contain light response elements, hormone response elements and abiotic stress response elements, and several ProFtMAPks have MYB-binding sites, which may be involved in the regulation of flavonoid biosynthesis-related enzyme gene expression. Synteny analysis indicated that FtMAPKs have a variety of biological functions. Protein interaction prediction suggested that MAPKs can interact with proteins involved in development and stress resistance. Correlation analysis further confirmed that most of the FtMAPK genes and transcription factors involved in the stress response have the same expression pattern. The transient transformation of FtMAPK1 significantly increased the antioxidant enzymes activity in Tartary buckwheat leaves. In addition, we also found that FtMAPK1 can respond to salt stress by up-regulating the transcription abundance of downstream genes. CONCLUSIONS: A total of 16 MAPKs were identified in Tartary buckwheat, and the members of the MAPK family containing the TDY motif were found to have expanded. The same subfamily members have relatively conserved gene structures and similar protein motifs. Tissue-specific expression indicated that the expression of all FtMAPK genes varied widely in the roots, stems, leaves and flowers. Most FtMAPKs can regulate the expression of other transcription factors and participate in the abiotic stress response. Our findings comprehensively revealed the FtMAPK gene family and laid a theoretical foundation for the functional characterization of FtMAPKs.

General gene expression patterns and stemness of the gingiva and dental pulp.

BACKGROUND/PURPOSE: Due to the unique properties of healing processes and cellular differentiation, the gingiva and dental pulp have attracted attention as a potential source of mesenchymal stem cells (MSCs). The purpose of this study was to obtain molecular-level information on these tissues in terms of their function and differentiation processes and investigate stemness. MATERIALS AND METHODS: Healthy gingival tissues were collected from patients (n = 9; aged 7-12 years) who underwent simple surgical procedures, and normal dental pulp tissues were obtained from patients (n = 25; aged 11-25 years) undergoing tooth extraction for orthodontic reasons. Complementary DNA microarray, qRT-qPCR, and immunohistochemical staining were performed to assess general and MSC gene expression patterns. RESULTS: In the gingival tissue, genes related to keratinization, the formation of epithelial cells and ectoderm, and immune and/or inflammatory responses were highly expressed. Meanwhile, in the dental pulp tissue, genes related to ion transport, neuronal development and axon guidance, bone and enamel mineralization, extracellular matrix organization, and angiogenesis were highly expressed. When focusing on the expression of MSC genes, induced pluripotent stem (iPS) cell genes, such as Sox2, c-Myc, and KLF4, were expressed at higher levels in the gingival tissue, whereas dental stem cell genes, such as NT5E and VCAM1, were expressed in dental pulp tissue. CONCLUSION: We found different general and MSC gene expression patterns between the gingival and dental pulp tissue. These results have implications for future regenerative medicine, considering the application of gingival tissue as a potential source of iPS cells.

Identification and expression analysis of the sucrose synthase gene family in pomegranate (Punica granatum L.).

BACKGROUND: Sucrose synthase (SUS, EC 2.4.1.13) is one of the major enzymes of sucrose metabolism in higher plants. It has been associated with C allocation, biomass accumulation, and sink strength. The SUS gene families have been broadly explored and characterized in a number of plants. The pomegranate (Punica granatum) genome is known, however, it lacks a comprehensive study on its SUS genes family. METHODS: PgSUS genes were identified from the pomegranate genome using a genome-wide search method. The PgSUS gene family was comprehensively analyzed by physicochemical properties, evolutionary relationship, gene structure, conserved motifs and domains, protein structure, syntenic relationships, and cis-acting elements using bioinformatics methods. The expression pattern of the PgSUS gene in different organs and fruit development stages were assayed with RNA-seq obtained from the NCBI SRA database as well as real-time quantitative polymerase chain reaction (qPCR). RESULTS: Five pomegranate SUS genes, located on four different chromosomes, were divided into three subgroupsaccording to the classification of other seven species. The PgSUS family was found to be highly conserved during evolution after studying the gene structure, motifs, and domain analysis. Furthermore, the predicted PgSUS proteins showed similar secondary and tertiary structures. Syntenic analysis demonstrated that four PgSUS genes showed syntenic relationships with four species, with the exception of PgSUS2. Predictive promoter analysis indicated that PgSUS genes may be responsive to light, hormone signaling, and stress stimulation. RNA-seq analysis revealed that PgSUS1/3/4 were highly expressed in sink organs, including the root, flower, and fruit, and particularly in the outer seed coats. qPCR analysis showed also that PgSUS1, PgSUS3, and PgSUS4 were remarkably expressed during fruit seed coat development. Our results provide a systematic overview of the PgSUS gene family in pomegranate, developing the framework for further research and use of functional PgSUS genes.

Genome-Wide Identification of the Hypericum perforatum WRKY Gene Family Implicates HpWRKY85 in Drought Resistance.

WRKY, named for its special heptapeptide conserved sequence WRKYGOK, is one of the largest transcription factor families in plants and is widely involved in plant responses to biotic, abiotic, and hormonal stresses, especially the important regulatory function in response to drought stress. However, there is no complete comprehensive analysis of this family in H. perforatum, which is one of the most extensively studied plants and is probably the best-known herbal medicine on the market today, serving as an antidepressant, neuroprotective, an antineuralgic, and an antiviral. Here, we identified 86 HpWRKY genes according to the whole genome database of H. perforatum, and classified them into three groups through phylogenetic analysis. Gene structure, conserved domain, motif, cis-elements, gene ontology, and expression profiling were performed. Furthermore, it was found that HpWRKY85, a homologous gene of AtWRKY75, showed obvious responses to drought treatment. Subcellular localization analysis indicated that this protein was localized in the nucleus by the Arabidopsis protoplasts transient transfection. Meanwhile, HpWRKY85-overexpressing Arabidopsis plants showed a stronger ability of root growth and scavenging endogenous reactive oxygen species. The results provide a reference for further understanding the role of HpWRKY85 in the molecular mechanism of drought resistance of H. perforatum.