Over-expression of WRKY2 in wheat enhanced the tolerance of transgenic wheat to drought stress,while over-expression of WRKY44 and WRKY46 enhanced the tolerance of both drought and salt stress,respectively.In addition,it is reported that WRKYs can also participate in the response of hormone signals.For example,salicylic acid can activate the expression of WRKY70 transcription factor,while jasmonic acid inhibits its expression.ABA can induce the WRKY gene to regulate the abscisic acid and gibberellin signaling pathway.Previous studies have proved that WRKY genes can regulate stress,respond to hormone signals to affect plant growth and development,and also plays an important role in fiber development.Gibberellins,as a type of important plant growth regulator,play a key role in fiber development and stem elongation.However,there is no report on the WRKY gene in seed hemp so far.It is imperative to identify and analyze WRKY transcription factors in seed hemp based on its whole genome and transcriptome data.The aims of this study were to identify CasWRKY genes at the whole genome-scale,analyze its expression patterns in different tissues,and analyze the effect of CasWRKYs on plant height according to GA3 stress in seed hemp.The diversity of gene structure can reflect the evolution of polygenic families.To gain the structural evolution,the exon–intron structures of the CasWRKY genes were determined by comparing the cDNA sequences of WRKYs with their corresponding genomic DNA sequences and then visualized by GSDS program..Gene structure analysis showed that the number of exons ranged from 2 to 6.More than half number of the CasWRKY genes had three exons.This indicates that exon loss and gain events occurred in the CasWRKY gene family during evolution,which may be the reason for the functional diversity among CasWRKY genes.
In addition,it was found that CasWRKY genes of the same group had similar exon/intron structures.Members of II-c and II-e had fewer exons and simple structures,while,members of I、II-a and II-b had many exons.To gain insights into the conservation and diversification,marijuana grow system motif structures on 39 CasWRKY proteins were constructed using the MEME program.A total of 10 conserved motifs were identified in the 39 CasWRKY protein sequences.The length of ten identified conserved motifs of CasWRKYs ranged from 6 to 50 amino acids.The number of motifs in CasWRKY proteins ranged from 5 to 15.As expected,most CasWRKYs that have been observed to be in the same group or subgroup usually shared highly similar motif compositions.Among them,motifs 1 and 3 contained the heptapeptide stretch WRKYGQK,with motif 3 being present only in group I.Motifs 1 and 2 were distributed all over the groups and motif 2 was immediately downstream of motif 1.In addition,five genes have similar protein lengths and same motif distributions.Motif 8 was present only in groups II-a and II-b,while Motif 6 was found mainly in subgroup II-B.Motif 4 was present in most genes in groups I and II-c.Furthermore,although CasWRKY27 belonged to subgroup II-d,it had similar conserved motifs with subgroup II-c.Totally,the analysis of CasWRKY motifs showed that every group or subgroup of CasWRKYs had similar motif compositions,which further validated the taxonomic and phylogenetic relationship ofCasWRKY.The cis-acting elements in the promoter region are essential for gene expression.The 2000 bp upstream promoter regions were searched using Plant CARE,and it was found that there are many types of cisacting elements.Among them,twenty cis-acting elements contain hormone-related elements,promoter-related elements,stressrelated elements,development-related elements.the 20P-box were distributed on the promoter region of 13 CasWRKY genes.The genes that contain P-box were concentrated in group I,group II-b,group II-c,III.Particularly,CasWRKY05 promoters had four gibberellin-responsive elements.In addition,cis-acting regulatory elements involved in light responsiveness were analyzed.17 CasWRKY promoters contain part of a light-responsive elements,which are cis-regulatory elements essential for light induction.It was noted that the three genes contain the common cisacting elements in promoter and enhancer regions.These results showed that most of CasWRKYs might play a crucial role in multiple stress responses in seed hemp.To investigate the plant in response to hormones at the growth boom in seed hemp,several phenotype responses induced by hormones were studied.
Changes in plant height after treatment with different exogenous hormones were shown in Fig.S.1.After the first exogenous hormone treatment,the plant height changes of different concentrations of GA3 treatments were significantly different.After 5 consecutive treatments,the plant height variation of high concentration GA3 treatment was significantly higher than that of the other two low concentration GA3 treatments and other exogenous hormones.No significant phenotypic differences were observed after treatment with exogenous hormones IAA and BR compared with the control.These results showed that GA3 at different concentrations could promote the plant height of “Yushema”,while BR and IAA had no significant effect.The above results indicated that “Yushema” was a gibberellin-sensitive variety.Since bio-active GA3 plays the key roles in the development of the stem,the RNA-seq data sampled from different time after GA3 treatment were used to study the differential expression pattern of CasWRKY genes under the GA3 stress.At 24 h of GA3 stress,most genes showed different expression levels and expression patterns,but some genes in the same subfamilies showed the same expression patterns.The expression levels of CasWRKY01,CasWRKY02,CasWRKY06,CasWRKY24,and CasWRKY29 were significantly up-regulated at 3 h after GA3 treatment.Moreover,thirteen genes exhibited the opposite trend with exposure to GA3 stress.In addition,among these 39 CasWRKY genes,the transcription levels of 4 CasWRKY genes s were continuously up-regulated in the stem barks at different time points under the GA3 stress.It should be noted that the expression of CasWRKY09 was significantly down-regulated after GA3 treatment,and then gradually up-regulated.Interestingly,nine out of the 39 CasWRKY genes were not expressed at all-time points,indicating that they might be pseudogenes or may not be involved in expressed in the stem barks.These results suggest that CasWRKY genes in seed hemp could respond to GA3 stress and form a complicated network to regulate hormone regulatory network.To verify the accuracy of RNA-seq data,9 CasWRKYs were randomly selected for RT-qPCR analysis.The results showed that the RT-qPCR assays of the expression patterns of the 9 CasWRKY genes were similar to the results of FPKM.As one of the largest transcriptional regulatory factor families in plants,WRKY gene family plays important role in plant growth and development and abiotic stress.To date,WRKY genes have been widely studied in a variety of plants,but similar studies have not yet been performed in seed hemp.we identified a total of 39 WRKY genes in the whole genome of seed hemp.The number of WRKY genes in seed hemp is significantly lower than that in A.thaliana ,soybean ,cabbage.hypogaea ,and cotton ,close to that in E.ulmoides ,C.capsularis ,sugar beet ,and canola.
By comparing with different species,it was found that the number of WRKY genes in different species is not directly related to their genome size.Previous studies believe that gene replication,fragment replication,and whole-genome replication play crucial roles in the large-scale expansion of gene families.With the continuous progress of high-throughput sequencing technology and the continuous optimization of data analysis methods,WRKY gene family members have been comprehensively studied.In this study,we conducted a comprehensive genome-wide analysis of WRKY gene family based on the seed hemp genome for the first time.Therefore,the results of this study will contribute to further functional analysis of WRKY genes in seed hemp and provide a reference for gene family analysis of other species.In Arabidopsis,a widely accepted WRKY gene family classification system was established by analyzing the genome data of WRKY gene family.According to the classical classification on the basis of sequence alignment and phylogenetic tree analysis,39 WRKY proteins identified in seed hemp were divided into three main groups,and group II of proteins was further clustering into five subgroups.Among the 39 CasWRKY proteins,there are five proteins in Group III,eight proteins are in Group I,and the remaining 26 CasWRKY proteins belong to group II.This classification results were similar to that in Eucommia ulmoides,Brassica napus and Brassica rapa,and the maximum numbers of group II proteins in this gene family,which means that this group may have experienced more gene duplications during the process of evolutionary.However,group III has the largest number of WRKY genes in rice,while group I has the largest number in Arabidopsis andpoplar.In this study,three domain loss events were found and concentrated in group III,which is a common phenomenon in the plant WRKY gene family and is considered to be a divergent force for the expansion of this gene family.In addition,it was also found that majority of the CasWRKY genes contain a highly conserved heptapeptide WRKYGQK motif followed by a C2H2 or C2HC type zinc finger motif except for several variants.WRKYGQK,a highly conserved domain,was replaced with WRKYGKK in CasWRKY20 and CasWRKY21.Both genes belong to subgroup II-c.Similar phenomena have been reported in Arabidopsis and cucumber.Previous reports have shown that the WRKYGKK domain failed to bind to W-box,and it needs further verification about whether CasWRKY20 and CasWRKY21 can bind to the W-box.
In addition,previous studies indicated that variations in the WRKYGQK motif of WRKY domain might influence normal interactions of WRKY genes with downstream target genes,cannabis vertical farming and therefore the molecular mechanism of these family members in plants still needs to be further explored.During the process of evolution,tandem and segmental duplication contribute to the expansion of the WRKY gene family.These replicated WRKY genes may form new gene functions to adapt to various conditions required in the process of plant growth and development.Chromosomal distribution and synteny analyses revealed that six tandem duplication events with 12 CasWRKY genes and five segmental duplication events with 10 CasWRKY genes were observed.The number of tandem duplications was more than segmental duplications,which increased the number of members in the WRKY gene family of seed hemp.Therefore,it could be deduced that tandem and segmental duplication were the main evolutionary driving force of the WRKY gene family in the evolution of seed hemp.In general,analyzing the distribution of introns and exons can help us better to understand the phylogenetic relationship,because they play crucial rolse in the evolution process of many gene families.Here,the comprehensive analysis of the distribution and length of exons and introns of CasWRKY genes were conducted.By analyzing the gene structures of CasWRKYs,it was found that exon loss and gain events occurred in the evolution of CasWRKY gene family,which may increase the functional diversity of genes in the gene family of CasWRKY.Cis-acting elements of promoters are essential for gene transcription and expression.Twenty cis-acting elements involved in hormone-related elements,promoter-related elements,stress-related elements,development-related elements,and cis-acting regulatory elements involved in light responsiveness were analyzed.It should be noted that 20P-box were distributed on the promoter region of 13 CasWRKY genes,the genes that contain these response elements are concentrated in group I,group II-b,group II-c,group III,and CasWRKY05 promoters had four gibberellin-responsive elements.These results suggest that these genes may produce corresponding expression responses under gibberellin stress,which will be conducive to the functional study of CasWRKYs.It is well known that gene expression pattern is closely related to gene biological function.In the present study,all 39 tested CasWRKY genes were found to be expressed in at least one of those tissues,except that CasWRKY22 and CasWRKY39 were not expressed in the three tissues examined and might be expressed in other tissues.
In addition,some CasWRKY genes showed preferential expression in all tissues tested.For example,six genes have high expression levels in stems and are mainly concentrated in group I.The CasWRKY genes which exhibited preferentially expression among different tissues are likely to have a unique role in organ development.In addition,CasWRKY15 was tissue-specific and is expressed only in stems.Its orthologous gene Atwrky12 in Arabidopsis has been reported to participate in the formation of secondary cell walls which could significantly increase plant biomass.These results suggest that the expression of WRKY transcription factor may be conducive to the regulation of fiber development,at least there is a certain relationship between them.WRKY transcription factors not only play vital roles in plant growth and development but also play key roles in response to various hormone regulations,which provides a favorable guarantee for crop genetic improvement.Phytohormones play an irreplaceable role in plant cellulose biosynthesis.As a key hormone,gibberellin participates in a variety of regulatory processes of plant life activities and almost accompanies entire plant life.Gibberellin can greatly affect fiber initiation,differentiation,and development,and promote fiber elongation by inducing more fiber initial cells.In this study,spraying gibberellin on plants of “Yushema” significantly promoted the elongation of plant heights,which proved that “Yushema” was the typical gibberellinsensitive germplasm.Therefore,the expression patterns of 39 CasWRKYs under GA3 stress were systematically analyzed.Expression pattern showed that most genes showed different expression levels at 24 h of GA3 stress,but some genes in the same subfamilies showed the same expression patterns.