Genome-wide identification of long non-coding RNAs and their potential functions in radish response to salt stress.

Long non-coding RNAs (lncRNAs) are increasingly recognized as cis- and trans-acting regulators of protein-coding genes in plants, particularly in response to abiotic stressors. Among these stressors, high soil salinity poses a significant challenge to crop productivity. Radish (Raphanus sativus L.) is a prominent root vegetable crop that exhibits moderate susceptibility to salt stress, particularly during the seedling stage. Nevertheless, the precise regulatory mechanisms through which lncRNAs contribute to salt response in radish remain largely unexplored. In this study, we performed genome-wide identification of lncRNAs using strand-specific RNA sequencing on radish fleshy root samples subjected to varying time points of salinity treatment. A total of 7,709 novel lncRNAs were identified, with 363 of them displaying significant differential expression in response to salt application. Furthermore, through target gene prediction, 5,006 cis- and 5,983 trans-target genes were obtained for the differentially expressed lncRNAs. The predicted target genes of these salt-responsive lncRNAs exhibited strong associations with various plant defense mechanisms, including signal perception and transduction, transcription regulation, ion homeostasis, osmoregulation, reactive oxygen species scavenging, photosynthesis, phytohormone regulation, and kinase activity. Notably, this study represents the first comprehensive genome-wide analysis of salt-responsive lncRNAs in radish, to the best of our knowledge. These findings provide a basis for future functional analysis of lncRNAs implicated in the defense response of radish against high salinity, which will aid in further understanding the regulatory mechanisms underlying radish response to salt stress.

Genome-Wide Identification and Expression Profiling of CONSTANS-Like Genes in Pepper (Capsicum annuum): Gaining an Insight to Their Phylogenetic Evolution and Stress-Specific Roles.

CONSTANS-like (COL) genes play important regulatory roles in multiple growth and development processes of plants but have rarely been studied in Capsicum annuum. This study explored the evolutionary relationship and expression patterns of COL genes from C. annuum. A total of 10 COL genes were identified in the genome of the cultivated pepper Zunla-1 and were named CaCOL01-10. These genes were unequally distributed among five chromosomes and could be divided into three groups based on differences in gene structure characteristics. During evolutionary history, duplications and retentions were divergent among different groups of COL genes. Tandem duplication caused amplification of group I genes. Genetic distance among COL genes was the largest in group III, suggesting that group III genes undergo more relaxed selection pressure compared with the other groups. Expression patterns of CaCOLs in tissues were significantly different, with CaCOL08 exhibiting the highest expression in stem and leaf. Some COL orthologous genes showed markedly different expression patterns in pepper compared with tomato, such as COL_1 orthologs, which may be involved in fruit development in pepper. In addition, CaCOLs participated in the regulation of abiotic stresses to varying degrees. Five CaCOL genes were induced by cold, and CaCOL02 and CaCOL03 were specifically upregulated by cold and downregulated by heat. This study provides a theoretical basis for the in-depth understanding of the functions of COL genes in pepper and their molecular mechanisms involved in growth and development and responses to abiotic stresses.

Chromosome-level pepino genome provides insights into genome evolution and anthocyanin biosynthesis in Solanaceae.

Pepino (Solanum muricatum, 2n = 2x = 24), a member of the Solanaceae family, is an important globally grown fruit. Herein, we report high-quality, chromosome-level pepino genomes. The 91.67% genome sequence is anchored to 12 chromosomes, with a total length of 1.20 Gb and scaffold N50 of 87.03 Mb. More than half the genome comprises repetitive sequences. In addition to the shared ancient whole-genome triplication (WGT) event in eudicots, an additional new WGT event was present in the pepino. Our findings suggest that pepinos experienced chromosome rearrangements, fusions, and gene loss after a WGT event. The large number of gene removals indicated the instability of Solanaceae genomes, providing opportunities for species divergence and natural selection. The paucity of disease-resistance genes (NBS) in pepino and eggplant has been explained by extensive loss and limited generation of genes after WGT events in Solanaceae. The outbreak of NBS genes was not synchronized in Solanaceae species, which occurred before the Solanaceae WGT event in pepino, tomato, and tobacco, whereas it was almost synchronized with WGT events in the other four Solanaceae species. Transcriptome and comparative genomic analyses revealed several key genes involved in anthocyanin biosynthesis. Although an extra WGT event occurred in Solanaceae, CHS genes related to anthocyanin biosynthesis in grapes were still significantly expanded compared with those in Solanaceae species. Proximal and tandem duplications contributed to the expansion of CHS genes. In conclusion, the pepino genome and annotation facilitate further research into important gene functions and comparative genomic analysis in Solanaceae.

Comprehensive analysis of SSRs and database construction using all complete gene-coding sequences in major horticultural and representative plants.

Simple sequence repeats (SSRs) are one of the most important genetic markers and widely exist in most species. Here, we identified 249,822 SSRs from 3,951,919 genes in 112 plants. Then, we conducted a comprehensive analysis of these SSRs and constructed a plant SSR database (PSSRD). Interestingly, more SSRs were found in lower plants than in higher plants, showing that lower plants needed to adapt to early extreme environments. Four specific enriched functional terms in the lower plant Chlamydomonas reinhardtii were detected when it was compared with seven other higher plants. In addition, Guanylate_cyc existed in more genes of lower plants than of higher plants. In our PSSRD, we constructed an interactive plotting function in the chart interface, and users can easily view the detailed information of SSRs. All SSR information, including sequences, primers, and annotations, can be downloaded from our database. Moreover, we developed Web SSR Finder and Batch SSR Finder tools, which can be easily used for identifying SSRs. Our database was developed using PHP, HTML, JavaScript, and MySQL, which are freely available at http://www.pssrd.info/ . We conducted an analysis of the Myb gene families and flowering genes as two applications of the PSSRD. Further analysis indicated that whole-genome duplication and whole-genome triplication played a major role in the expansion of the Myb gene families. These SSR markers in our database will greatly facilitate comparative genomics and functional genomics studies in the future.

Isolation, Identification, and Anti-Inflammatory Activity of Polysaccharides of Typha angustifolia.

The present study aimed to purify, structurally characterize, and evaluate the anti-inflammatory activity of the polysaccharide extracted from Typha angustifolia. Two purified polysaccharides (PTA-1 and PTA-2) were obtained via DEAE-52 cellulose chromatography. Their structural characterizations and antioxidant activity were in vitro analyzed. To evaluate the anti-inflammatory activity of PTA-2, the levels of inflammatory cytokines, intracellular ROS production, and the inhibitory effects of the transcriptional activation of the nuclear factor kappa B (NF-κB) signaling pathway were determined. PTA-1 comprises glucose (100%) with α-(1 → 3) glycosidic bonds, and PTA-2 comprises glucose (66.7%) and rhamnose (33.3%) formed by ß-(1 → 3) glycosidic bonds. PTA-1 and PTA-2 showed strong antioxidant activity in vitro. Moreover, PTA-2 intervention (50, 100, and 200 µg/mL) suppressed the production of inflammatory cytokines, the activation of NF-κB signaling, and reactive oxygen species production significantly. The results identified PTA-2 as a natural product that could be applied in anti-inflammatory drugs.

Molecular Evolutionary and Expression Pattern Analysis of AKR Genes Shed New Light on GalUR Functional Characteristics in Brassica rapa.

The aldo-keto reductase (AKR) superfamily plays a major role in oxidation-reduction in plants. D-galacturonic acid reductase (GalUR), an ascorbic acid (AsA) biosynthetic enzyme, belongs to this superfamily. However, the phylogenetic relationship and evolutionary history of the AKR gene family in plants has not yet been clarified. In this study, a total of 1268 AKR genes identified in 36 plant species were used to determine this phylogenetic relationship. The retention, structural characteristics, and expression patterns of AKR homologous genes in Brassica rapa and Arabidopsis thaliana were analyzed to further explore their evolutionary history. We found that the AKRs originated in algae and could be divided into A and B groups according to the bootstrap value; GalURs belonged to group A. Group A AKR genes expanded significantly before the origin of angiosperms. Two groups of AKR genes demonstrated functional divergence due to environmental adaptability, while group A genes were more conservative than those in group B. All 12 candidate GalUR genes were cloned, and their expression patterns under stress were analyzed, in Pak-choi. These genes showed an obvious expression divergence under multiple stresses, and BrcAKR22 exhibited a positive correlation between its expression trend and AsA content. Our findings provide new insights into the evolution of the AKR superfamily and help build a foundation for further investigations of GalUR's functional characteristics.

Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa.

Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-Mannose/L-Galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), B. oleracea (C genome) and B. napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. (i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. (ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. (iii) Under NaCl, Cu(2+), MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments.

Comprehensive analysis of the polygalacturonase and pectin methylesterase genes in Brassica rapa shed light on their different evolutionary patterns.

Pectins are fundamental polysaccharides in the plant primary cell wall. Polygalacturonases (PGs) and pectin methylesterases (PMEs), major components of the pectin remodeling and disassembly network, are involved in cell separation processes during many stages of plant development. A comprehensive study of these genes in plants could shed light on the evolution patterns of their structural development. In this study, we conducted whole-genome annotation, molecular evolution and gene expression analyses of PGs and PMEs in Brassica rapa and 8 other plant species. A total of 100 PGs and 110 PMEs were identified in B. rapa; they primarily diverged from 12-18 MYA and PMEs were retained more than PGs. Along with another 305 PGs and 348 PMEs in the 8 species, two different expansion or evolution types were discovered: a new branch of class A PGs appeared after the split of gymnosperms and angiosperms, which led to the rapid expansion of PGs; the pro domain was obtained or lost in the proPMEs through comprehensive analyses among PME genes. In addition, the PGs and PMEs exhibit diverged expression patterns. These findings will lead to novel insight regarding functional divergence and conservation in the gene families and provide more support for molecular evolution analyses.

Temperature expression patterns of genes and their coexpression with LncRNAs revealed by RNA-Seq in non-heading Chinese cabbage.

BACKGROUND: Non-heading Chinese cabbage (NHCC, Brassica rapa ssp. chinensis) is an important leaf vegetable grown worldwide. However, little is known about the molecular mechanisms underlying tolerance for extreme temperature in NHCC. The limited availability of NHCC genomic information has greatly hindered functional analysis and molecular breeding. RESULTS: Here, we conduct comprehensive analyses of cold and heat treatments in NHCC using RNA-seq. Approximately 790 million paired-end reads representing 136,189 unigenes with N50 length of 1705 bp were obtained. Totally, 14,329 differentially expressed genes (DEGs) were detected. Among which, 10 DEGs were detected in all treatments, including 7 up-regulated and 3 down-regulated. The enrichment analyses showed 25 and 33 genes were enriched under cold and heat treatments, respectively. Additionally, 10,001 LncRNAs were identified, and 9,687 belonged to novel LncRNAs. The expression of miRNAs were more than that of pri-miRNAs and LncRNAs. Furthermore, we constructed a coexpression network for LncRNAs and miRNAs. It showed 67 and 192 genes were regulated by LncRNAs under cold and heat treatments, respectively. We constructed the flowchart for identifying LncRNAs of NHCC using transcriptome. Except conducting the de novo transcriptome analyses, we also compared these unigenes with the Chinese cabbage proteins. We identified several most important genes, and discussed their regulatory networks and crosstalk in cold and heat stresses. CONCLUSIONS: We presented the first comprehensive characterization for NHCC crops and constructed the flowchart for identifying LncRNAs using transcriptome. Therefore, this study represents a fully characterized NHCC transcriptome, and provides a valuable resource for genetic and genomic studies under abiotic stress.

Molecular evolution, characterization, and expression analysis of SnRK2 gene family in Pak-choi (Brassica rapa ssp. chinensis).

The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) family members are plant-specific serine/threonine kinases that are involved in the plant response to abiotic stress and abscisic acid (ABA)-dependent plant development. Further understanding of the evolutionary history and expression characteristics of these genes will help to elucidate the mechanisms of the stress tolerance in Pak-choi, an important green leafy vegetable in China. Thus, we investigated the evolutionary patterns, footprints and conservation of SnRK2 genes in selected plants and later cloned and analyzed SnRK2 genes in Pak-choi. We found that this gene family was preferentially retained in Brassicas after the Brassica-Arabidopsis thaliana split. Next, we cloned and sequenced 13 SnRK2 from both cDNA and DNA libraries of stress-induced Pak-choi, which were under conditions of ABA, salinity, cold, heat, and osmotic treatments. Most of the BcSnRK2s have eight exons and could be divided into three groups. The subcellular localization predictions suggested that the putative BcSnRK2 proteins were enriched in the nucleus. The results of an analysis of the expression patterns of the BcSnRK2 genes showed that BcSnRK2 group III genes were robustly induced by ABA treatments. Most of the BcSnRK2 genes were activated by low temperature, and the BcSnRK2.6 genes responded to both ABA and low temperature. In fact, most of the BcSnRK2 genes showed positive or negative regulation under ABA and low temperature treatments, suggesting that they may be global regulators that function at the intersection of multiple signaling pathways to play important roles in Pak-choi stress responses.

Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of CO-like genes in plant kingdom.

In plants, flowering is the most important transition from vegetative to reproductive growth. The flowering patterns of monocots and eudicots are distinctly different, but few studies have described the evolutionary patterns of the flowering genes in them. In this study, we analysed the evolutionary pattern, duplication and expression level of these genes. The main results were as follows: (i) characterization of flowering genes in monocots and eudicots, including the identification of family-specific, orthologous and collinear genes; (ii) full characterization of CONSTANS-like genes in Brassica rapa (BraCOL genes), the key flowering genes; (iii) exploration of the evolution of COL genes in plant kingdom and construction of the evolutionary pattern of COL genes; (iv) comparative analysis of CO and FT genes between Brassicaceae and Grass, which identified several family-specific amino acids, and revealed that CO and FT protein structures were similar in B. rapa and Arabidopsis but different in rice; and (v) expression analysis of photoperiod pathway-related genes in B. rapa under different photoperiod treatments by RT-qPCR. This analysis will provide resources for understanding the flowering mechanisms and evolutionary pattern of COL genes. In addition, this genome-wide comparative study of COL genes may also provide clues for evolution of other flowering genes.

Retention, Molecular Evolution, and Expression Divergence of the Auxin/Indole Acetic Acid and Auxin Response Factor Gene Families in Brassica Rapa Shed Light on Their Evolution Patterns in Plants.

Auxin/indole acetic acids (Aux/IAAs) and auxin response factors (ARFs), major components of the Aux signaling network, are involved in many developmental processes in plants. Investigating their evolution will provide new sight on the relationship between the molecular evolution of these genes and the increasing morphotypes of plants. We constructed comparative analyses of the retention, structure, expansion, and expression patterns of Aux/IAAs and ARFs in Brassica rapa and their evolution in eight other plant species, including algae, bryophytes, lycophytes, and angiosperms. All 33 of the ARFs, including 1 ARF-like (AL) (a type of ARF-like protein) and 53 Aux/IAAs, were identified in the B. rapa genome. The genes mainly diverged approximately 13 Ma. After the split, no Aux/IAA was completely lost, and they were more preferentially retained than ARFs. In land plants, compared with ARFs, which increased in stability, Aux/IAAs expanded more rapidly and were under more relaxed selective pressure. Moreover, BraIAAs were expressed in a more tissue-specific fashion than BraARFs and demonstrated functional diversification during gene duplication under different treatments, which enhanced the cooperative interaction of homologs to help plants adapt to complex environments. In addition, ALs existed widely and had a closer relationship with ARFs, suggesting that ALs might be the initial structure of ARFs. Our results suggest that the rapid expansion and preferential retention of Aux/IAAs are likely paralleled by the increasingly complex morphotypes in Brassicas and even in land plants. Meanwhile, the data support the hypothesis that the PB1 domain plays a key role in the origin of both Aux/IAAs and ARFs.

Genome-wide analysis of the MADS-box gene family in Brassica rapa (Chinese cabbage).

The MADS-box gene family is an ancient and well-studied transcription factor family that functions in almost every developmental process in plants. There are a number of reports about the MADS-box family in different plant species, but systematic analysis of the MADS-box transcription factor family in Brassica rapa (Chinese cabbage) is still lacking. In this study, 160 MADS-box transcription factors were identified from the entire Chinese cabbage genome and compared with the MADS-box factors from 21 other representative plant species. A detailed list of MADS proteins from these 22 species was sorted. Phylogenetic analysis of the BrMADS genes, together with their Arabidopsis and rice counterparts, showed that the BrMADS genes were categorised into type I (Mα, Mß, Mγ) and type II (MIKC(C), MIKC*) groups, and the MIKC(C) proteins were further divided into 13 subfamilies. The Chinese cabbage type II group has 95 members, which is twice as much as the Arabidopsis type II group, indicating that the Chinese cabbage type II genes have been retained more frequently than the type I genes. Finally, RNA-seq transcriptome data and quantitative real-time PCR analysis revealed that BrMADS genes are expressed in a tissue-specific manner similar to Arabidopsis. Interestingly, a number of BrMIKC genes showed responses to different abiotic stress treatments, suggesting a function for some of the genes in these processes as well. Taken together, the characterization of the B. rapa MADS-box family presented here, will certainly help in the selection of appropriate candidate genes and further facilitate functional studies in Chinese cabbage.

High concentrate:forage ratio diet inhibiting omasal epithelium growth is associated with decreased cyclin D1 and CDK4 expression in growing goats.

The hypothesis that different concentrate : forage ratio diets alter omasal epithelium proliferation of growing goats via cyclins and regulation of the cell cycle was tested. Growing goats were fed with a high concentrate (HC, n = 8) or a low concentrate (LC, n = 8) diet for 42 days. The concentrate : forage ratio was 40:60 in the HC group and 0:100 in the LC group. In the HC group, the relative weight and DNA content of the omasal epithelium were lower, but the protein : DNA ratio was higher. Flow cytometry revealed that HC omasal cell numbers were smaller in S- and G2 /M-phases of the cell cycle and higher in the G0 /G1 -phases and were accompanied by reduced expression of cyclin D1 and CDK4 mRNA and protein. These data are consistent with morphologic observations in the HC that cell density decreased in the stratum spinosum (SS) plus stratum granulosum (SG) and stratum basale, and that cell density was lower in the SS plus SG. Thus, high-concentrate : forage ratio diet retards omasal epithelial growth by slowing the G1 to S phase transition of the cell cycle and is associated with decreased cyclin D1 and CDK4 expression in growing goats.

Genome-wide identification, classification and expression analysis of the heat shock transcription factor family in Chinese cabbage.

The Hsf gene family, one of the most important transcription factor families, plays crucial roles in regulating heat resistance. However, a systematic and comprehensive analysis of this gene family has not been reported in Chinese cabbage. Therefore, systematic analysis of the Hsf gene family in Chinese cabbage has profound significance. In this study, 35 BrHsf genes were identified from Chinese cabbage, which could be classified into three groups according to their structural characteristics and phylogenetic comparisons with Arabidopsis and rice. Thirty-three BrHsf genes mapped on chromosomes were further assigned to three subgenomes and eight ancestral karyotypes. Distribution mapping showed that BrHsf genes were non-randomly localized on chromosomes. Chinese cabbage and Arabidopsis shared 22 orthologous gene pairs. The expansion of BrHsf genes mainly resulted from genome triplication. Comparative analysis showed that the most Hsf genes were in Chinese cabbage among the five species analyzed. Interestingly, the number of Hsf genes of heat-resistant plants (Theobroma cacao and Musa acuminata) was fewer than that in Chinese cabbage. The expression patterns of BrHsf genes were different in six tissues, based on RNA-seq. Quantitative real-time-PCR analysis showed that the expression level of BrHsf genes varied under various abiotic stresses. In conclusion, this comprehensive analysis of BrHsf genes will provide rich resources, aiding the determination of Hsfs functions in plant heat resistance. Furthermore, the comparative genomics analysis deepened our understanding of Hsf genes' evolution accompanied by the polyploidy event of Chinese cabbage.

Genes associated with agronomic traits in non-heading Chinese cabbage identified by expression profiling.

BACKGROUND: The genomes of non-heading Chinese cabbage (Brassica rapa ssp. chinensis), heading Chinese cabbage (Brassica rapa ssp. pekinensis) and their close relative Arabidopsis thaliana have provided important resources for studying the evolution and genetic improvement of cruciferous plants. Natural growing conditions present these plants with a variety of physiological challenges for which they have a repertoire of genes that ensure adaptability and normal growth. We investigated the differential expressions of genes that control adaptability and development in plants growing in the natural environment to study underlying mechanisms of their expression. RESULTS: Using digital gene expression tag profiling, we constructed an expression profile to identify genes related to important agronomic traits under natural growing conditions. Among three non-heading Chinese cabbage cultivars, we found thousands of genes that exhibited significant differences in expression levels at five developmental stages. Through comparative analysis and previous reports, we identified several candidate genes associated with late flowering, cold tolerance, self-incompatibility, and leaf color. Two genes related to cold tolerance were verified using quantitative real-time PCR. CONCLUSIONS: We identified a large number of genes associated with important agronomic traits of non-heading Chinese cabbage. This analysis will provide a wealth of resources for molecular-assisted breeding of cabbage. The raw data and detailed results of this analysis are available at the website http://nhccdata.njau.edu.cn.

Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapa.

Ascorbic acid (AsA) is an important antioxidant in plants and an essential vitamin for humans. Extending the study of AsA-related genes from Arabidopsis thaliana to Brassica rapa could shed light on the evolution of AsA in plants and inform crop breeding. In this study, we conducted whole-genome annotation, molecular-evolution and gene-expression analyses of all known AsA-related genes in B. rapa. The nucleobase-ascorbate transporter (NAT) gene family and AsA l-galactose pathway genes were also compared among plant species. Four important insights gained are that: 1) 102 AsA-related gene were identified in B. rapa and they mainly diverged 12-18 Ma accompanied by the Brassica-specific genome triplication event; 2) during their evolution, these AsA-related genes were preferentially retained, consistent with the gene dosage hypothesis; 3) the putative proteins were highly conserved, but their expression patterns varied; and 4) although the number of AsA-related genes is higher in B. rapa than in A. thaliana, the AsA contents and the numbers of expressed genes in leaves of both species are similar, the genes that are not generally expressed may serve as substitutes during emergencies. In summary, this study provides genome-wide insights into evolutionary history and mechanisms of AsA-related genes following whole-genome triplication in B. rapa.

Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis).

Basic helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotic organisms and are thought to be one of the largest families of regulatory proteins. This important family of transcriptional regulators plays crucial roles in plant development. However, a systematic analysis of the bHLH transcription factor family has not been reported in Chinese cabbage. In this study, 230 bHLH transcription factors were identified from the whole Chinese cabbage genome and compared with proteins from other representative plants, fungi and metazoans. The Chinese cabbage bHLH (BrabHLH) gene family could be classified into 24 subfamilies. Phylogenetic analysis of BrabHLHs along with bHLHs from Arabidopsis and rice indicated 26 subfamilies. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns and interaction networks of BrabHLHs were analyzed. Distribution mapping showed that BrabHLHs were non-randomly located on the ten Chinese cabbage chromosomes. One hundred and twenty-four orthologous bHLH genes were identified between Chinese cabbage and Arabidopsis, and the interaction networks of the orthologous genes were constructed in Chinese cabbage. Quantitative RT-PCR analysis showed that expressions of BrabHLH genes varied widely under different abiotic stress treatments for different times. Thus, this comprehensive analysis of BrabHLHs represents a rich resource, aiding the elucidation of the roles of bHLH family members in plant growth and development. Furthermore, the comparative genomics analysis deepened our understanding of the evolution of this gene family after a polyploidy event.

Characterization and co-expression analysis of WRKY orthologs involved in responses to multiple abiotic stresses in Pak-choi (Brassica campestris ssp. chinensis).

BACKGROUND: The WRKY transcription factor is an important member of the stress-related transcription factors, which mediate diverse abiotic stresses in many plants. However, up until now, the number of WRKY members, and the regulatory mechanisms involved in abiotic stress responses in Pak-choi (Brassica campestris ssp. chinensis), remained unknown. RESULTS: We isolated and identified 56 full-length WRKY cDNAs from a Pak-choi stress-induced cDNA library. The 56 putative BcWRKY proteins were divided into three groups based on structural and phylogenetic analyses. A subcellular localization prediction indicated that the putative BcWRKY proteins were enriched in the nuclear region. Experiments involving BcWRKY25 and BcWRKY40 confirmed the prediction. A total of 22 BcWRKYs were differentially expressed in response to at least one stress condition (abscisic acid, cold, salinity, heat, or osmosis) tested on Pak-choi leaves, and a co-expression analysis indicated stress-inducible BcWRKYs co-regulated multiple abiotic stresses. BcWRKY33, BcWRKY40, BcWRKY53, and BcWRKY70 acted as key regulators and played dominant roles within co-regulatory networks of stress-inducible BcWRKYs. CONCLUSIONS: We first isolated and characterized the 56 stress-inducible WRKY transcription factor family members. A total of 22 stress-inducible BcWRKYs found in leaves can co-regulate multiple environmental stresses by integrating the potential mutual interactions of WRKYs in Pak-choi. This information will be valuable when exploring the molecular mechanisms of WRKYs in response to abiotic stresses in plants.