WRKY Transcription Factor Family Research Articles

Identification of WRKY transcription factor family genes in Pinus massoniana Lamb. and their expression patterns and functions in response to drought stress

BackgroundPinus massoniana Lamb. is the timber species with the widest distribution and the largest afforestation area in China, providing a large amount of timber, turpentine and ecological products. Seasonal drought caused by climate warming severely constrains the quality and growth of P. massoniana forests. WRKY transcription factors play an important role in plant responses to abiotic stress. In this study, the molecular mechanisms by which P. massoniana responds to drought stress were analysed based on the P. massoniana WRKY (PmWRKY) family of genes.ResultsForty-three PmWRKYs are divided into three major families, 7 sub-families, and the conserved motifs are essentially the same. Among these 43 PmWRKYs express under drought stress but with different expression patterns in response to stress. PmWRKYs respond to drought stress induced by exogenous hormones of SA, ABA, and MeJA. The expression of PmWRKY6, PmWRKY10, and PmWRKY30 up-regulate in different families and tissues under drought stress, while PmWRKY22 down-regulate. Transgenetic tobaccos of PmWRKY31 are with lower malondialdehyde (MDA) content and higher proline (Pro) content than wild type (WT) tobaccos. In transgenic tobaccos of PmWRKY31, expression levels of related genes significantly improve, and drought tolerance enhance.ConclusionsThis study analysed the molecular biological characteristics of PmWRKYs and investigated the expression patterns and functions of PmWRKYs in response to drought stress in P. massoniana. The results of this study provide a basis for in-depth research of the molecular functions of PmWRKYs in response to drought stress.

Transcriptome-Based Identification, Characterization, Evolutionary Analysis, and Expression Pattern Analysis of the WRKY Gene Family and Salt Stress Response in Panax ginseng

WRKY transcription factors are some of the most important transcription factors in planta, and they are involved in biological and abiotic stresses, growth and development, and biochemical processes. The WRKY gene family has been found in many higher plants, while little is known about WRKY-encoding genes in ginseng. As a traditional and important economic medicinal herb and health food, ginseng has been a model species for other related medicinal plants. Here, we analyze the WRKY transcript factor gene family in ginseng from a genetics and genomics perspective in the hope that this study can create a new avenue for understanding the role of PgWRKYs. By identifying and analyzing all candidate WRKY transcription factor family members based on the transcriptome of ginseng, a total of 118 PgWRKY genes was obtained following gene classification, the phylogenetic relationship, conserved domain structure, functional differentiation, and an expression activity analysis. A phylogenetic analysis revealed that the PgWRKYs were clustered into three groups: I, II, and III transcription factors. Members in group Ⅱ were further divided into five sub-groups: Ⅱa to Ⅱe. An expression analysis showed a trend of co-expression among most PgWRKY genes, forming an interaction network. The coding sequences were WRKYGQK; only two genes were WRKYGKK, and only one gene was WSKYGQK. Moreover, a salt stress treatment analysis of the response of PgWRKY39-01, PgWRKY062, and PgWRKY064 genes was investigated using RT-qPCR. After salt stress treatment, the expression of three PgWRKY genes was increased, indicating that PgWRKYs can participate in regulating the response to salt stresses in ginseng. These comprehensive data provide a reference for elucidating the functions of this transcription factor family in the growth, development, and salt stress response of ginseng.

Analysis of Floral Organ Development and Sex Determination in Schisandra chinensis by Scanning Electron Microscopy and RNA-Sequencing.

S. chinensis is a typical monoecious plant, and the number and development of female flowers determines the yield of S. chinensis. Due to a lack of genetic information, the molecular mechanism of sex differentiation in S. chinensis remains unclear. In this study, the combination of scanning electron microscopy (SEM) and RNA sequencing (RNA-seq) was used to understand the way of sex differentiation of S. chinensis and to mine the related genes of sex determination. The result shows the development of male and female S. chinensis flowers was completed at the same time, the unisexual S. chinensis flowers did not undergo a transition stage between sexes, and sex may have been determined at an early stage in flower development. The results of the gene function analysis of the plant hormone signaling pathway and sucrose metabolism pathway suggest that auxin and JA could be the key hormones for sex differentiation in S. chinensis, and sucrose may promote pollen maturation at the later stage of male flower development. Two AGAMOUS (GAG) genes, 10 AGAMOUS-like MADS-box (AGLs) genes, and the MYB, NAC, WRKY, bHLH, and Trihelix transcription factor families may play important roles in sex determination in S. chinensis. Taken together, the present findings provide valuable genetic information on flower development and sex determination in S. chinensis.

Systematic Analysis of the Grafting-Related Glucanase-Encoding GH9 Family Genes in Pepper, Tomato and Tobacco.

Grafting is an important agricultural practice to control soil-borne diseases, alleviate continuous cropping problems and improve stress tolerance in vegetable industry, but it is relatively less applied in pepper production. A recent study has revealed the key roles of β-1, 4-glucanase in graft survival. We speculated that the GH9 family gene encoding glucanase may be involved in the obstacles of pepper grafting. Therefore, we performed a systematic analysis of the GH9 family in pepper, tomato and tobacco. A total of 25, 24 and 42 GH9 genes were identified from these three species. Compared with the orthologues of other solanaceous crops, the deduced pepper GH9B3 protein lacks a conserved motif (Motif 5). Promoter cis-element analysis revealed that a wound-responsive element exists in the promoter of tobacco NbGH9B3, but it is absent in the GH9B3 promoter of most solanaceous crops. The auxin-responsive related element is absent in CaGH9B3 promoter, but it presents in the promoter of tobacco, tomato, potato and petunia GH9B3. Tissue and induction expression profiles indicated that GH9 family genes are functionally differentiated. Nine GH9 genes, including CaGH9B3, were detected expressing in pepper stem. The expression patterns of NbGH9B3 and CaGH9B3 in grafting were different in our test condition, with obvious induction in tobacco but repression in pepper. Furthermore, weighted correlation network analysis (WGCNA) revealed 58 transcription factor genes highly co-expressed with NbGH9B3. Eight WRKY binding sites were detected in the promoter of NbGH9B3, and several NbWRKYs were highly co-expressed with NbGH9B3. In conclusion, the missing of Motif 5 in CaGH9B3, and lacking of wound- and auxin-responsive elements in the gene promoter are the potential causes of grafting-related problems in pepper. WRKY family transcription factors could be important regulator of NbGH9B3 in tobacco grafting. Our analysis points out the putative regulators of NbGH9B3, which would be helpful to the functional validation and the study of signal pathways related to grafting in the future.

OsWRKY114 Negatively Regulates Drought Tolerance by Restricting Stomatal Closure in Rice.

The WRKY family of transcription factors plays a pivotal role in plant responses to biotic and abiotic stress. The WRKY Group III transcription factor OsWRKY114 is a positive regulator of innate immunity against Xanthomonas oryzae pv. oryzae; however, its role in abiotic stress responses is largely unknown. In this study, we showed that the abundant OsWRKY114 transcripts present in transgenic rice plants are reduced under drought conditions. The overexpression of OsWRKY114 significantly increased drought sensitivity in rice, which resulted in a lower survival rate after drought stress. Moreover, we showed that stomatal closure, which is a strategy to save water under drought, is restricted in OsWRKY114-overexpressing plants compared with wild-type plants. The expression levels of PYR/PYL/RCAR genes, such as OsPYL2 and OsPYL10 that confer drought tolerance through stomatal closure, were also markedly lower in the OsWRKY114-overexpressing plants. Taken together, these results suggest that OsWRKY114 negatively regulates plant tolerance of drought stress via inhibition of stomatal closure, which would otherwise prevent water loss in rice.

Overexpression of Myrothamnus flabellifolia MfWRKY41 confers drought and salinity tolerance by enhancing root system and antioxidation ability in Arabidopsis.

Myrothamnus flabellifolia is the only woody resurrection plant discovered so far and could recover from extreme desiccation condition. However, few genes related to its strong drought tolerance have been characterized, and the underlying molecular mechanisms remains mysterious. Members of WRKY transcription factor family are effective in regulating abiotic stress responses or tolerance in various plants. An early dehydration-induced gene encoding a WRKY transcription factor namely MfWRKY41 was isolated from M. flabellifolia, which is homologous to AtWRKY41 of Arabidopsis. It contains a typical WRKY domain and zinc finger motif, and is located in the nucleus. Comparing to wild type, the four transgenic lines overexpressing MfWRKY41 showed better growth performance under drought and salt treatments, and exhibited higher chlorophyll content, lower water loss rate and stomatal aperture and better osmotic adjustment capacity. These results indicated that MfWRKY41 of M. flabellifolia positively regulates drought as well as salinity responses. Interestingly, the root system architecture, including lateral root number and primary root length, of the transgenic lines was enhanced by MfWRKY41 under both normal and stressful conditions, and the antioxidation ability was also significantly improved. Therefore, MfWRKY41 may have potential application values in genetic improvement of plant tolerance to drought and salinity stresses. The molecular mechanism involving in the regulatory roles of MfWRKY41 is worthy being explored in the future.

Transcriptomic and metabolomic analyses reveal that exogenous strigolactones alleviate the response of melon root to cadmium stress

Strigolactones (SLs) are classified into plant hormones, playing a key role as a mediator of plant growth in response to several abiotic stresses. Cadmium (Cd), a common heavy metal and soil pollutant, can suppress plant growth and development. In this work, we explored the effects of exogenous SLs on root formation in response to Cd stress using melon seeds subjected to seven germination treatments: CK (control), Cd (300 μmol·L-1 CdCl2), and SL1–SL5 (CdCl2-stressed seeds pretreated with 0.1, 0.5, 1, 2, and 3 μmol·L-1 GR24 solutions). The results indicated that SLs increased the antioxidant enzyme activities and root vigor and decreased the malondialdehyde (MDA) contents in the roots of Cd-stressed melon seedlings. Then we used transcriptomic and metabolomic analyses to explore the mechanisms by which exogenous SLs protect against Cd stress. There were 242 significant differentially expressed genes (DEGs) (78 upregulated, 164 downregulated) and 247 significantly differentially expressed metabolites (DEMs) (222 upregulated, 25 downregulated) between the Cd and SL3 treatments. SLs altered the expression of genes related to redox formation processes, including peroxidase (POD), lipoxygenase (LOX), glutamate dehydrogenase (GDH), and glutathione S-transferase (GST). In addition, we found that SLs regulated the expression of the MYB, AP2/ERF, bHLH, and WRKY transcription factor families. The combined transcriptomic and metabolomic analyses revealed that the DEGs and DEMs involved in Cd stress alleviation were mainly related to the gene expression of jasmonic acid (JA) and flavonoid biosynthesis. SLs might induce LOX-related genes to regulate JA biosynthesis. Moreover, SLs might promote flavonoid biosynthesis by regulating eleven flavonoid-related genes and eight metabolites. The results provide a new perspective for studying the adaptation of plants to Cd stress.

Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis.

OscWRKY1 from Ocimum sanctum positively regulates phenylpropanoid pathway genes and rosmarinic acid content. OscWRKY1 overexpression promotes resistance against bacterial pathogen in Arabidopsis. WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TF's is not well characterized. In the present study, we have characterized an OscWRKY1 gene from Ocimum sanctum which shows induced expression by methyl jasmonate (MeJA), salicylic acid (SA), and wounding. The recombinant OscWRKY1 protein binds to the DIG-labeled (Digoxigenin) W-box cis-element TTGAC[C/T] and activates the LacZ reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have been identified to contain the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. Furthermore, the metabolite analysis of OscWRKY1 silenced plants showed reduced rosmarinic acid content. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes leading to the alteration of rosmarinic acid content and enhances the resistance against bacterial pathogen in Arabidopsis.

A Comprehensive Study of the WRKY Transcription Factor Family in Strawberry.

WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry.

MeWRKY IIas, Subfamily Genes of WRKY Transcription Factors From Cassava, Play an Important Role in Disease Resistance.

Cassava (Manihot esculenta Crantz) is an important tropical crop for food, fodder, and energy. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) occurs in all cassava growing regions and threatens global cassava production. WRKY transcription factor family plays the essential roles during plant growth, development, and abiotic or biotic stress. Particularly, previous studies have revealed the important role of the group IIa WRKY genes in plant disease resistance. However, a comprehensive analysis of group IIa subfamily in cassava is still missing. Here, we identified 102 WRKY members, which were classified into three groups, I, II, and III. Transient expression showed that six MeWRKY IIas were localized in the nucleus. MeWRKY IIas transcripts accumulated significantly in response to SA, JA, and Xam. Overexpression of MeWRKY27 and MeWRKY33 in Arabidopsis enhanced its resistance to Pst DC3000. In contrast, silencing of MeWRKY27 and MeWRKY33 in cassava enhanced its susceptibility to Xam. Co-expression network analysis showed that different downstream genes are regulated by different MeWRKY IIa members. The functional analysis of downstream genes will provide clues for clarifying molecular mechanism of cassava disease resistance. Collectively, our results suggest that MeWRKY IIas are regulated by SA, JA signaling, and coordinate response to Xam infection.

Genome-Wide Analysis and Exploration of WRKY Transcription Factor Family Involved in the Regulation of Shoot Branching in Petunia.

The WRKY transcription factors (TFs) participate in various physiological, growth and developmental processes of plants. In our study, a total of 79 WRKY family members were identified and classified into three groups (Group I, Group IIa–e, and Group III) based on phylogenetic and conservative domain analyses. Conserved motif analysis showed that seven WRKYGQK domains changed. The promoter sequence analysis suggested that there were multiple stress- and hormone-related cis-regulatory elements in the promoter regions of PhWRKY genes. Expression patterns of PhWRKYs based on RNA-seq data revealed their diverse expression profiles in five tissues and under different treatments. Subcellular localization analysis showed that PhWRKY71 was located in the nucleus. In addition, overexpression of PhWRKY71 caused a significant increase in branch number. This indicated that PhWRKY71 played a critical role in regulating the shoot branching of Petunia hybrida. The above results lay the foundation for further revealing the functions of PhWRKY genes.

Transcriptional cascades in the regulation of 2-AP biosynthesis under Zn supply in fragrant rice.

Transcription factors (TFs) regulate gene expression to control certain genetic programs, such as growth and development, phytohormone regulation, and environmental stresses. 2-acetyl-1-pyrroline (2-AP) is the key element involved in aroma biosynthesis pathway, and the application of micronutrients can increase the 2-AP levels. However, little is known about the micronutrient-induced TFs involved in 2-AP biosynthesis. Here, we identify a number of TF families in two fragrant rice varieties, "Meixiangzhan-2" (M) and "Xiangyaxiangzhan" (X), in response to Zinc (Zn) application through transcriptomic analysis. A total of ~678 TFs were identified and grouped into 26 TF families, each of which was found to be involved in numerous signaling pathways. The WRKY TF family was found to be the most abundant, followed by bHLH and MYB. Furthermore, members of the WRKY, bHLH, MYB, ERF, HSF, MADS-box, NFY, and AP2 TF families were significantly upregulated and may be involved in the transcriptional regulation of aroma biosynthesis. In brief, this study enhances our understanding of the molecular mechanism of 2-AP biosynthesis and highlights the key TFs potentially involved in the production of aroma in fragrant rice.

Mercury toxicity affects oxidative metabolism and induces stress responsive mechanisms in wheat (Triticum aestivum L.).

Mercury (Hg) toxicity is an increasing problem worldwide, with a negative impact on the environment and living organisms including both animals and plants. In this study, we analyzed molecular and biochemical changes related to Hg toxicity in wheat (Triticum aestivum L.) seedlings. Seven-day-old seedlings were exposed to various concentrations (5, 10, and 20 µM) of HgCl2 for 24 and 48h. Our results showed that HgCl2 treatments led to an increase in the Hg content of wheat leaves in a time- and concentration-dependent manner. Furthermore, significant increases were observed in hydrogen peroxide, malondialdehyde, and proline contents in response to Hg toxicity. While all HgCl2 treatments decreased the level of superoxide dismutase (SOD), the level of catalase (CAT) was reduced only in seedlings exposed to 5 µM of HgCl2. Mercury stress caused a decline in the expression of Cu/Zn-SOD, Fe-SOD, TaWRKY19, and TaDREB1 genes at both exposure times. On the other hand, 10 and 20 µM HgCl2 treatments caused significant induction (1.9 to 6.1-fold) in the expression of the CAT gene in wheat leaves. The mRNA level of the Mn-SOD and TaWRKY2 genes showed different patterns depending on the concentration and exposure period of HgCl2. In conclusion, the findings of this work demonstrate that Hg toxicity causes oxidative damage in wheat seedlings and changes the expression of some genes encoding WRKY and DREB transcription factor families, which have important functions in abiotic stress response.

Genome-Wide Analysis of the Banana WRKY Transcription Factor Gene Family Closely Related to Fruit Ripening and Stress.

WRKY transcription factors (TFs) play an important role in plant responses to biotic and abiotic stress as well as in plant growth and development. In the present study, bioinformatics methods were used to identify members of the WRKY transcription factor family in the Musa acuminata (DH-Pahang) genome (version 2). A total of 164 MaWRKYs were identified and phylogenetic analysis showed that MaWRKYs could be categorized into three subfamilies. Overall, the 162 MaWRKYs were distributed on 11 chromosomes, and 2 genes were not located on the chromosome. There were 31 collinear genes from segmental duplication and 7 pairs of genes from tandem duplication. RNA-sequencing was used to analyze the expression profiles of MaWRKYs in different fruit development, ripening stages, under various abiotic and biotic stressors. Most of the MaWRKYs showed a variety of expression patterns in the banana fruit development and ripening stages. Some MaWRKYs responded to abiotic stress, such as low temperature, drought, and salt stress. Most differentially expressed MaWRKYs were downregulated during banana’s response to Foc TR4 infection, which plays an important role in physiological regulation to stress. Our findings indicate that MaWRKY21 directly binds to the W-box of the MaICS promoter to decrease MaICS transcription and then reduce the enzyme activity. These studies have improved our understanding of the molecular basis for the development and stress resistance of an important banana variety.

The WRKY transcription factor family in cowpea: Genomic characterization and transcriptomic profiling under root dehydration

Cowpea [Vigna unguiculata (L.) Walp.] is one of the most tolerant legume crops to drought and salt stresses. WRKY transcription factor (TF) family members stand out among plant transcriptional regulators related to abiotic stress tolerance. However, little information is currently available on the expression of the cowpea WRKY gene family (VuWRKY) in response to water deficit. Thus, we analyzed genomic and transcriptomic data from cowpea to identify VuWRKY members and characterize their structure and transcriptional response under root dehydration stress. Ninety-two complete VuWRKY genes were found in the cowpea genome based on their domain characteristics. They were clustered into three groups: I (15 members), II (58), and III (16), while three genes were unclassified. Domain analysis of the encoded proteins identified four major variants of the conserved heptapeptide motif WRKYGQK. In silico analysis of VuWRKY gene promoters identified eight candidate binding motifs of cis-regulatory elements, regulated mainly by six TF families associated with abiotic stress responses. Ninety-seven VuWRKY modulated splicing variants associated with 55 VuWRKY genes were identified via RNA-Seq analysis available at the Cowpea Genomics Consortium (CpGC) database. qPCR analyses showed that 22 genes are induced under root dehydration, with VuWRKY18, 21, and 75 exhibiting the most significant induction levels. Given their central role in activating signal transduction cascades in abiotic stress response, the data provide a foundation for the targeted modification of specific VuWRKY family members to improve drought tolerance in this important climate-resilient legume in the developing world and beyond.

Single-Cell Transcriptome and Network Analyses Unveil Key Transcription Factors Regulating Mesophyll Cell Development in Maize.

Background: Maize mesophyll (M) cells play important roles in various biological processes such as photosynthesis II and secondary metabolism. Functional differentiation occurs during M-cell development, but the underlying mechanisms for regulating M-cell development are largely unknown. Results: We conducted single-cell RNA sequencing (scRNA-seq) to profile transcripts in maize leaves. We then identified coregulated modules by analyzing the resulting pseudo-time-series data through gene regulatory network analyses. WRKY, ERF, NAC, MYB and Heat stress transcription factor (HSF) families were highly expressed in the early stage, whereas CONSTANS (CO)-like (COL) and ERF families were highly expressed in the late stage of M-cell development. Construction of regulatory networks revealed that these transcript factor (TF) families, especially HSF and COL, were the major players in the early and later stages of M-cell development, respectively. Integration of scRNA expression matrix with TF ChIP-seq and Hi-C further revealed regulatory interactions between these TFs and their targets. HSF1 and COL8 were primarily expressed in the leaf bases and tips, respectively, and their targets were validated with protoplast-based ChIP-qPCR, with the binding sites of HSF1 being experimentally confirmed. Conclusions: Our study provides evidence that several TF families, with the involvement of epigenetic regulation, play vital roles in the regulation of M-cell development in maize.

Moss transcription factors regulating development and defense responses to stress.

Transcription factors control gene expression, leading to regulation of biological processes that determine plant development and adaptation to the environment. Land colonization by plants occurred 450-470 million years ago and was accompanied by an increase in the complexity of transcriptional regulation associated to transcription factor gene expansions. AP2/ERF, bHLH, MYB, NAC, GRAS, and WRKY transcription factor families increased in land plants compared with algae. In angiosperms, they play crucial roles in regulating plant growth and responses to environmental stressors. However, less information is available in bryophytes and only in a few cases is the functional role of moss transcription factors in stress mechanisms known. In this review, we discuss current knowledge of the transcription factor families involved in development and defense responses to stress in mosses and other bryophytes. By exploring and analysing the Physcomitrium patens public database and published transcriptional profiles, we show that a high number of AP2/ERF, bHLH, MYB, NAC, GRAS, and WRKY genes are differentially expressed in response to abiotic stresses and during biotic interactions. Expression profiles together with a comprehensive analysis provide insights into relevant transcription factors involved in moss defenses, and hint at distinct and conserved biological roles between bryophytes and angiosperms.

Identification of stress-related characteristics of the <i>WRKY</i> gene family: A case study of <i>Dendrobium catenatum</i>

As one of the largest families of transcription factors (TFs) in plants, the WRKY TF family plays a key role in regulating plant responses to various biotic and abiotic stresses. However, there is no confirmed method to quickly identify stress-responsive members from the <italic>WRKY</italic> gene family. In this study, all reported functional <italic>WRKY</italic> genes were first analyzed, and the amino acid patterns in response to stress were identified in group II-c (T-R/K-S/T-E/Q/D-V/I/L-E/D-I/V/H/N-L/M-D/E-D-G/E-F/Y-K/R-WRKYG-Q/K-K-A/T-VKN-S/N-P), group II-d (VPA-I/V-S-X-K-M/L/V/I-ADIP-P/A/V-D-D/E-Y/F-S-WRKYGQKPIKGSP-H/Y-PRGYYKCS-S/T-V/M-RGCPARKVER), and group II-e (PSD-S/A/L-WAWRKYGQKPIKGSPYPR-G/S-YYRCSSSKGC). <italic>WRKY</italic> genes in<italic> Dendrobium catenatum</italic> were used to validate the accuracy of these patterns. A total of 63 <italic>DcaWRKY</italic> genes were identified, their gene structures, conserved motifs, and gene expression patterns were analyzed, and a phylogenetic tree was constructed. Gene expression patterns were then analyzed under drought stress, and seven <italic>DcaWRKY</italic> genes (<italic>Dca002550</italic>, <italic>Dca002715</italic>, <italic>Dca005648</italic>, <italic>Dca007842</italic>, <italic>Dca010430</italic>, <italic>Dca016437</italic>, and <italic>Dca006787</italic>) were randomly selected to determine their expression levels and verify their expression patterns by quantitative real-time polymerase chain reaction analysis. The identified amino acid patterns were validated by drought-responsive <italic>WRKY</italic> genes in <italic>D. catenatum</italic>, confirming the accuracy of these amino acid patterns and providing valuable insights into further research of the <italic>WRKY</italic> family in <italic>D. catenatum</italic>.

Flavonoid biosynthesis in four Dendrobium species based on transcriptome sequencing and metabolite analysis.

Dendrobium is a genus of plants used as traditional Chinese herbal medicines, with high economic and medicinal value. To reveal the mechanism of flavonoid biosynthesis in Dendrobium, the metabolites and transcriptomes of four Dendrobium species (D. chrysotoxum, D. nobile, D. fimbriatum, and D. denneanum) were analyzed comprehensively. Ultra-high-performance liquid chromatography-tandem mass spectrometry analysis revealed ten flavonoid compounds in Dendrobium. In total, 100,096 unigenes were obtained from the transcript database of the four Dendrobium species. Among the identified differentially expressed genes, 51 were associated with flavonoid biosynthesis, and 670 differentially expressed transcription factors were predicted, including 194 MYB, 87 bHLH, and 100 WRKY family transcription factors, respectively. Transcriptome analysis showed that the expression levels of structural genes such as chalcone synthase (CHS), cinnamate-4-hydroxylase (C4H), and flavonoid 3'-hydroxylase (F3'H) were lower in D. chrysotoxum, D. nobile, and D. fimbriatum than those in D. denneanum, which may be the main reason for the low flavonoid contents in D. chrysotoxum, D. nobile, and D. fimbriatum. The expression level of structural genes corresponded to the accumulation level of flavonols in the different Dendrobium species. The results deepen the understanding of the molecular mechanism of flavonoid biosynthesis in Dendrobium and provide novel insights into the synthesis and accumulation of flavonoids in Dendrobium.

Composition of Flavonoids in the Petals of Freesia and Prediction of Four Novel Transcription Factors Involving in Freesia Flavonoid Pathway.

Freesia hybrida is rich in flower colors with beautiful flower shapes and pleasant aroma. Flavonoids are vital to the color formation of its flowers. In this study, five Freesia cultivars with different flower colors were used to study on the level of accumulation of their flavonoids and expression of flavonoid-related genes and further explore new novel transcription factor (TF). Ultra-high-performance liquid chromatography and VION ion mobility quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS) were used to determine the flavonoids. Combined with transcriptome sequencing technology, the molecular mechanism of the flavonoid metabolism difference in Freesia was revealed. A total of 10 anthoxanthin components and 12 anthocyanin components were detected using UPLC-Q-TOF-MS. All six common anthocyanin aglycones in high plants, including cyanidin, delphinidin, petunidin, peonidin, malvidin, and pelargonidin, were detected in Freesia at first time in this study. In orange, yellow, and white cultivars, anthoxanthins gradually decreased with the opening of the petals, while in red and purple cultivars, anthoxanthins first increased and then decreased. No anthocyanin was detected in yellow and white cultivars, while anthocyanins increased with the opening of the petals and reached their maximum at the flowering stage (S3) in other three cultivars. The correlation analysis revealed that the color of Freesia petals was closely related to the composition and content of anthoxanthins and anthocyanins. Petals of five cultivars at S3 were then selected for transcriptome sequencing by using the Illumina Hiseq 4000 platform, and a total of 100,539 unigenes were obtained. There were totally 5,162 differentially expressed genes (DEGs) when the four colored cultivars were compared with the white cultivar at S3. Comparing all DEGs with gene ontology (GO), KEGG, and Pfam databases, it was found that the genes involved in the flavonoid biosynthesis pathway were significantly different. In addition, AP2, WRKY, and bHLH TF families ranked the top three among all differently expressed TFs in all DEGs. Quantitative real-time PCR (qRT-PCR) technology was used to analyze the expression patterns of the structural genes of flavonoid biosynthesis pathway in Freesia. The results showed that metabolic process was affected significantly by structural genes in this pathway, such as CHS1, CHI2, DFR1, ANS1, 3GT1, and FLS1. Cluster analysis was performed by using all annotated WRKY and AP2 TFs and the above structural genes based on their relatively expression. Four novel candidate TFs of WRKY and AP2 family were screened. Their spatiotemporal expression patterns revealed that these four novel TFs may participate in the regulation of the flavonoid biosynthesis, thus controlling its color formation in Freesia petals.