Genome-Wide Identification and Analysis of the WRKY Transcription Factor Family Associated with Leaf Senescence in Alfalfa

Abstract

Leaves are the most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar ’XinjiangDaye’ using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources.

1. Introduction

WRKY transcription factors (TFs) are a type of sequence-specific DNA-binding proteins predominantly identified in plants [1,2]. The WRKY TF family members are characterized by a highly conserved WRKYGQK amino acid sequence and a C2H2 or C2HC zinc finger-like motif [2]. WRKY TFs are categorized into three groups depending on the number of WRKY sequences (two in group I, one in groups II and III) and the structure of the zinc finger-like motif (group III WRKY TFs have modified C2HC motifs) [1]. Group II WRKY proteins can be further classified into five subgroups (II-a, II-b, II-c, II-d, and II-e) based on their primary amino acid sequences [2]. In 1994, the first member of the WRKY gene family, SPF I, was identified in Ipomoea batatas and was shown to be triggered by sucrose and polygalacturonic acid [3]. Subsequently, an increasing number of WRKY gene families have been identified and studied in the genomes of model plants, including Arabidopsis thaliana [1], Medicago truncatula [4], maize [5], wheat [6], and Trifolium pratense L. [7].

Plants may encounter a variety of biotic and abiotic stresses during their growth and development. With the increase in climate change and extreme weather events, the proportion of abiotic stresses affecting crop production is growing. According to statistics, global yield loss of major crops due to various abiotic stresses is estimated to be around 50% [8]. Many transcription factors (TFs) contribute to plant resistance and tolerance to unfavorable environmental conditions, making them potential candidate genes for breeding. Among these TFs, WRKY transcription factors stand out. They regulate the expression of abiotic stress-responsive genes or directly control the expression of these genes by activating or repressing the transcription of downstream genes. WRKYs play key roles in abiotic and biotic growth and development [9]. Leaf senescence is the final stage of leaf development, which is a highly orchestrated process regulated by endogenous and environmental signals [10]. Leaf senescence is characterized by three main features: degradation of chlorophyll and chloroplasts, disruption of protein homeostasis, and induction of genes related to senescence [11]. Senescence-related traits are closely associated with yield and stress tolerance [12]. Expression profiling indicates that numerous WRKY genes are highly activated during senescence and they make up the second-largest category of transcription factor genes in the senescence transcriptome, indicating the key role of WRKY TFs in leaf senescence [13].

To date, several WRKY genes have been identified as key regulatory factors in Arabidopsis leaf senescence: Arabidopsis WRKY47 actively controls age-triggered leaf senescence by activating the programmed cell death-related genes BFN1 and MC6 [14], WRKY1 positively regulates leaf senescence by inducing SA biosynthesis genes [15], and WRKY45 interacts with the DELLA protein RGL1 [16]. Additionally, WRKY6 [17], WRKY22 [18], WRKY26 [19], WRKY42 [20], WRKY53 [21], WRKY55 [22], WRKY71 [23], and WRKY75 [24] positively regulate leaf senescence, whereas WRKY54 and WRKY70 [25] negatively regulate leaf senescence by delaying it. WRKY activation in crops against abiotic stresses also helps improve plant resilience and ensures crop yields even in challenging conditions [26,27]. WRKY TFs have also been documented to be involved in leaf senescence in several different plant species. In tobacco, NtWRKY70b triggers the expression of the ROS-synthesis-related gene NtRbohD and the chlorophyll degradation gene NtPPH, contributing to the positive regulation of leaf senescence. Additionally, in wheat, if the transcription of TaWRKY13-A is impaired, leaf senescence is delayed [28]. In sorghum, SbWRKY50 directly binds to the promoters of several chlorophyll catabolic genes and negatively regulates chlorophyll degradation, thus delaying leaf senescence [29].

Premature leaf senescence caused by abiotic stresses can significantly impact the yield and quality of alfalfa. However, the WRKY family is not well studied in Medicago Sativa. Alfalfa (M. sativa L.), known as the “queen of forages”, is considered one of the most important forage crops in the world [30] and is widely used as a high-quality legume forage due to its high yield, good palatability, and high nutritional value [31]. Leaves, as a primary harvesting organ, are a limiting factor affecting the yield and quality of M. sativa. Premature induction of leaf senescence can severely limit plant biomass [32]. Therefore, preventing or delaying premature senescence to increase biomass accumulation could improve the quality and economic benefits of alfalfa [33].

In this study, we first characterized the WRKY family members in the M. sativa genome through bioinformatics analysis. We then identified candidate WRKY genes involved in leaf senescence by analyzing the transcriptomes of M. sativa leaves subjected to darkness and salt stress. Similar approaches have been used in M. truncatula [34] to screen senescence-related genes and lay the foundation for subsequent studies on the regulatory mechanisms of M. sativa leaf senescence, as well as for breeding and selecting high-quality M. sativa germplasm resources.

2. Results

2.1. Gene Identification and Phylogenetic Tree Construction

The protein sequences of the WRKY TFs family from the Arabidopsis database were used to query the Pfam database to obtain the conserved domain (PF03106) file for WRKY proteins. The ‘hmmsearch’ command was executed with parameters set to E valuae ≤ 1 × 10^-10 and bits ≥ 85. A total of 198 MsWRKYs were identified in the M. sativa genome. Based on their chromosomal positions, they were named MsWRKY1 through MsWRKY198 (Table S1). Next, MEGA11.0 was used to conduct clustering analysis of the 72 Arabidopsis proteins and 198 putative MsWRKY proteins, using 1000 bootstrap replicates for statistical reliability (Figure 1). The 198 WRKY TFs were categorized into three groups, Group I, Group II, and Group III, (Figure 1), with 66, 114, and 18 WRKY members, respectively. Group II was further divided into five subclasses: Group II-a, -b, -c, -d, and -e. Group II-a, with only 4 MsWRKYs, had the least members among all subclasses. Groups II-b and II-d had 20 MsWRKYs each, Group II-c had 45, and Group II-e had 25. Since the functions of numerous Arabidopsis WRKYs have been investigated, it is possible to make inferences about the functions of MsWRKYs that are grouped with Arabidopsis WRKYs by considering previous research.

2.2. Sequence and Structural Analysis of MsWRKYs

A phylogenetic tree was created using MEGA11.0 to examine the structure and evolutionary connections of the MsWRKY genes. The conserved motifs were identified by utilizing the Multiple Em for MEME online tool, and the MsWRKY protein sequence as a reference “https://meme-suite.org/meme/ (accessed on 26 August 2024)” with the parameters set to zero or one motif per sequence, a maximum of 10 motifs, and an optimum motif width ranging from 4 to 14. Motifs with an e-value < 1 × 10⁻¹⁰ were retained for further analysis. Based on the genome Gff3 annotation file, the structure of the MsWRKY genes was determined. Gene Structure View (Advanced) of TBtools (v2.038) software was utilized to generate phylogenetic trees, motifs, and gene structure diagrams (Figure 2). It can be seen that MsWRKYs in group I have 7–9 types of motifs, MsWRKYs in group II have 2–6 types of motifs, and MsWRKYs in group III have 3 types of motifs. Correspondingly, MsWRKYs in group I contain 3–7 exons, MsWRKYs in group II contain 2–8 exons, and MsWRKYs in group III contain 3 exons. In brief, within the same group or subgroup, the distribution of conserved motifs as well as exon-intron organization were similar, indicating that genes within the same group or subgroup may have similar biological functions.

2.3. Cis-Acting Elements of MsWRKYs

Using the GXF Sequences Program in TBtools, the promoter sequences were obtained by determining the 2000 bp upstream sequences of the start codons of the MsWRKY genes. Using the PlantCARE database to predict potential cis-acting elements, we identified 11 types of cis-acting elements in 198 gene promoter regions, including two stress response elements (MBS, and TC-rich repeats), seven hormone response elements (ABRE, CGTCA motif, GARE motif, TATC box, TCA element, TGACG motif, and TGA element), the G-box element known as transcription factors (NAC, BHLH and bZIP) binding motif, and the W-box element of the WRKY transcription factor-binding site (Figure 3). 164 MsWRKYs contain light-responsive cis-acting elements (G-box), 152 MsWRKYs contain abscisic acid-responsive elements (ABRE), 143 MsWRKYs contain methyl jasmonate acid-responsive elements (CGTCA motif and TGACG motif), 120 MsWRKYs contain salicylic acid-responsive cis-acting elements (TCA-elements), 85 MsWRKYs contain MYB Binding Site (MBS) and 81 have TC-rich repeats. Each of the 198 MsWRKYs has at least two elements in the promoter region, with MsWRKY57containing 48 elements, the highest number among all MsWRKY genes. The majority of MsWRKY genes contain one or more W-box elements in the promoter region.

2.4. Verification of Screened Leaf Senescence-Related WRKY Genes

Expression data of 198 MsWRKYs were downloaded from the laboratory’s previously determined leaf senescent transcriptome (Table S2) and used to create a heat map (Figure 4). We examined the genes that were highly expressed in X3, X4 relative to X0, and the genes highly expressed in D4, D6, S4, and S6 relative to D0. We selected top five genes from each of these six comparisons based on the fold upregulation of their expression levels for subsequent experiments. A BLAST search on the alfalfa website was performed and the top-ranked genes without 100% homology were retained, resulting in a final selection of 14 MsWRKY genes for further analysis (Figure 4; Table S3).

The expression levels of these 14 genes at different leaf developmental stages and under different treatment conditions were verified by RT-qPCR, and the results were presented as column charts (Figure 5). With the exception of MsWRKY165, the other 13 genes showed trends consistent with that in the RNA-seq (Figure 4 and Figure 5). The qRT-PCR results revealed that MsWRKY5, chosen from the X4vsX0 comparison, was also induced by dark treatment and salt treatment at D1, D2 and S4 (Figure 5a–c). MsWRKY66, selected from the X3vsX0 comparison, was also up-regulated when exposed to salt treatment and showed significant up-regulation at the S6 period (Figure 5c). Similarly, MsWRKY92 and 141 exhibited significant changes in expression when subjected to dark and salt treatments.

2.5. Verification of Gene Function Using Transient Expression

Agrobacterium-mediated transient gene expression was performed in Nicotiana benthaminana leaves to quickly screen for M. sativa senescence-associated TFs (SA-TFs). The coding regions of the selected SA-TFs were amplified from cDNA derived from senescing M. sativa leaves using gene-specific primers, the PCR products were cloned into the pFGC-eYFP vector at the Bam HI site, and the sequence-verified constructs were then transformed competent Agrobacterium EHA105 cells. When the Agrobacterium cultures reached OD600 = 0.6 to 0.8, 3–4 week-old N. benthaminana plants were infiltrated. As MsSGR promotes leaf senescence [35], MsSGR was used as a positive control, and an empty vector was used as a negative control. After infiltration, the leaves were kept in the dark for one day, followed by normal light conditions. Phenotype observations were conducted 2–3 days later. The transient expression of MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 in N. benthaminana resulted in a significant premature aging phenotype, similar to the phenotype induced by infiltration of the MsSGR gene in alfalfa (Figure 6). These results suggest that these four MsWRKY genes play important roles in regulating leaf senescence in M. sativa.

3. Discussion

The WRKY transcription factor family is an important regulator of leaf senescence and one of the most extensively studied transcription factors [36]. Although it has been observed and researched in various species, it has not been documented in M. sativa. In this study, we conducted a bioinformatics analysis of 198 WRKY gene family members found in the alfalfa cultivar “Xinjiang Daye”. To determine the evolutionary relationships between the MsWRKY gene family, we constructed a phylogenetic tree of the WRKY gene family and investigated the gene structure and motif distribution. We also predicted the cis-acting elements of the WRKY gene family.

We identified 198 WRKY genes from the M. sativa genome, and quantitatively the number of WRKY genes in M. sativa was similar to that of tetraploid soybean [37], as well as significantly higher than that of diploid M. truncatula [4]. A total of 93 WRKY transcription factors were identified in M. truncatula, and the number identified in M. sativa was nearly twice that of M. truncatula. These indicate that the MsWRKY gene family is evolutionarily conserved in M. sativa in the context of gene number. The construction of a phylogenetic tree of the MsWRKY gene family revealed their evolutionary relationship; subsequently, the genetic structure and base distribution of the 198 MsWRKYs were analyzed. The phylogenetic analysis showed that the 198 MsWRKYs could be divided into three subgroups according to their evolutionary relationships. MsWRKY5, MsWRKY66, and MsWRKY92 are members of subclasses 2-d, 2-c, and 2-e, respectively, whereas MsWRKY141 is a member of Group 1. AtWRKY06 [17], AtWRKY42 [20], AtWRKY45 [16], AtWRKY47 [14], AtWRKY71 [23], AtWRKY75 [24], and AtWRKY22 [18] in Group 2, and AtWRKY01 [15] and AtWRKY26 [19] in Group 1 play a role in regulating leaf senescence. It can be inferred that MsWRKYs in the same subgroup with AtWRKYs are likely involved in the regulation of leaf senescence, laying the foundation for further research on the functions of MsWRKYs and the mechanisms of leaf senescence regulation. Conserved motif analysis revealed that all of these MsWRKYs contained motif 1, which was identified as WRKYGQK. This explains why members of the same subgroup share similar gene structures and motifs, supporting the phylogenetic results.

Cis-acting elements are DNA sequences present upstream of a gene, and they are involved in regulating its expression. They do not encode proteins but provide binding sites for transcription factors and other regulatory factors [38]. In this study, we used PlantCARE to predict cis-acting elements 2000 bp upstream of the start codon of the 198 MsWRKY genes. The promoter regions of the 198 MsWRKY genes were investigated for 11 types of elements, including two stress-responsive elements, seven hormone-responsive elements, one light-responsive elements G-box and one WRKY transcription factor-binding site W-box element. As the core sequence of the W-box is “TGAC”, the W-box can be used for predicting the target genes of WRKY TFs [39]. We found that 143 out of 198 MsWRKY genes contain two or more W-box elements in the promoter region, while 164 out of 198 MsWRKY genes have the important light-responsive cis-acting element known as G-box in their promoter. In plants, many genes related to photosynthesis and photomorphogenesis contain G-box element. G-box is able to respond to the environmental changes in light, temperature, and moisture leading to changes in the expression of related genes. This enables plants to adapt to different environmental conditions. Furthermore, at least 2 cis-acting elements were found in the promoter region of each MsWRKY gene. The cis-acting elements in the promoter region of the gene suggest that the gene may perform different biological functions under varying stress conditions. Studies on cis-regulatory elements are important for further understanding plant defense responses to abiotic and biotic stresses [40].

WRKYs play a central role in growth and development [9], including leaf senescence, the final stage of leaf growth and development [41]. They are the main players in mediating defense responses in plants [42]. In this study, we verified the expression patterns of 14 MsWRKYs by RT-qPCR and subsequently determined their function by overexpressing them in tobacco. The results showed that four MsWRKYs, namely MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141, exhibited the same leaf yellowing phenotype as the positive control MsSGR. We hypothesized that these four MsWRKYs may be involved in the regulation of leaf senescence in M. sativa.

Leaf senescence is the final stage of leaf development and is crucial for the fitness of the plant [43]. Inhibiting the expression of MsSGR in M. sativa can increase the crude protein content in hay by 2.3–5.5% and retain more than 50% of chlorophyll, greatly improving the quality of alfalfa hay [35]. In nature, plants often experience various stresses, such as salinity stress and insufficient light. These stresses can lead to premature leaf senescence. This is why we are studying WRKY genes, which are regulated by darkness and salt stress and are related to leaf senescence. In the future, silencing or mutating these WRKY genes may also improve forage quality, similar to silencing SGR.

The results of this study establish a foundation for future research into the roles and regulatory mechanisms of the WRKY gene family in M. sativa leaf senescence. These studies suggest that targeting specific WRKY transcription factors may offer a novel approach to manipulate leaf senescence in M. sativa, potentially enhancing yield and stress tolerance. In the future, more deep understanding of the interplay between WRKY transcription factors and other hormonal or signaling pathways may lead to biotechnological strategies that fine-tune senescence for better crop management.

4. Materials and Methods

4.1. Plant Material and Treatment

The plant materials of M. sativa ‘Xinjiang Daye’ and Nicotiana benthamiana were grown as described previously [44]. Briefly, M. sativa seeds were placed in petri dishes and covered with wet filter paper, once the radicle grew to 3–4 cm, the seedlings were then transferred to Hoagland’s nutrient solution for hydroponic cultivation; Nicotiana benthamiana seeds germinated in pots. All plants were grown in an artificial climate chamber (16 h light/8 h dark, temperature 22 °C; 65% relative humidity; 150 µmol·m⁻²·s⁻¹ light intensity).

When M. sativa ‘Xinjiang Daye’ plants were 4 weeks old, the first leaf that has not fully unfolded was collected and regarded as the first stage of leaf development (X0). Subsequent leaves were collected and identified as X1, X2, and X3 according to leaf position, with X4 representing the leaves with senescent symptom in the lower part of the plant. The compound leaves of the third leaf position from apex were collected and treated with darkness or salt for 0, 1, 2, 4, and 6 days in a culture dish. The darkness treatment solution was half-strength Murashige–Skoog containing 3 mM MES buffer, pH of 5.8; while the salt treatment solution is further supplemented with 150 mM NaCl.

Leaves with three biological replicates at different stages of development (X0, X1, X2, X3, X4) and at different treatment times (0, 2, 4, and 6d) were collected for RNAseq and RT-qPCR analysis.

4.2. Differential Expression Analysis

A differential expression analysis of genes was performed to identify differentially expressed genes between samples and to study their functions. To identify genes associated with leaf senescence, the expression levels of each transcript were determined using the transcripts per million reads method. The significantly differentially expressed genes were identified using DESeq2 [45] according to the following criteria: FDR < 0.05 and |log2FC| ≥ 1.

4.3. Sequence and Phylogenetic Analysis

In this study, we looked up the protein sequences of the WRKY gene family according to the Arabidopsis thaliana database and searched the Pfam database “https://pfam.xfam.org/ (accessed on 26 August 2024)” using their protein sequences as templates, downloaded the file of conserved structural domains of WRKY proteins (PF03106), and executed the hmmsearch command, in which E value ≤ 1 × 10⁻¹⁰, while bits ≥ 85, and finally a total of 198 MsWRKYs were identified in M. sativa. TBtools (v2.038) software was used to visualize the gene structure and motif distribution [46]. Conserved motifs were analysed for MsWRKYs using the MEME online website, with parameters set to zero or one motif per sequence, up to a maximum of 10 motifs. A total of 72 Arabidopsis WRKY sequences from The Arabidopsis Information Resource (TAIR) and 198 MsWRKY sequences from M. sativa were used to construct a phylogenetic tree. MEGA 11.0 was used to build phylogenetic trees using the neighbor-joining method, through applying the Poisson model, pairwise deletion, and 1000 bootstrap replications [47]. PlantCARE was used to predict the cis-acting elements of 2000 bp sequences upstream of the start codon of 198 MsWRKYs [48], and TBtools was used to visualize cis-acting elements.

4.4. Quantitative Polymerase Chain Reaction (RT-qPCR)

To identify the expression patterns of MsWRKYs, RNA was extracted from samples at each time point and analyzed using RNA-seq. RT-qPCR was performed for verification [49]. Total RNA was extracted from leaf samples at each stage using the Takara MiniBEST Plant RNA Extraction Kit (Plant RNA Purification Reagent for plant tissue; Invitrogen Corp., Carlsbad, CA, USA.) and reverse transcribed into cDNA using HiScript III^® RT SuperMix for qRT-PCR (+gDNA wiper) from Vazyme Biotech Co., Ltd. in Nanjing, China. RT-qPCR was conducted using the ChamQ SYBR Color qRT-PCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). MsUBC was used as the reference gene. Relative expression was calculated using the 2^–ΔCT method. The gene expression data is presented as the mean ± SD and was analyzed to detect significant differences using ANOVA in GraphPad Prism 8 (NS: not significant; * p < 0.05; ** p < 0.01). Each experiment was performed in three biological replicates, with each biological replicate being run in triplicate. Primers were designed using the NCBI database, and the primer sequences are listed in Table S3.

4.5. Verification of Gene Function through Transient Expression

Selected genes were cloned for transient expression in Nicotiana benthamiana, with MsSGR as a positive control. Using cDNA as a template, selected target genes were cloned and sequenced to verify the sequence accuracy. Target genes were ligated into the pFGC-eYFP vector using seamless DNA cloning (ClonExpress R Ultra One Step Cloning Kit; Vazyme Biotech Co., Ltd.). The recombinant plasmid was transformed into Escherichia coli DH5α (Vazyme Biotech Co., Ltd.) for amplification and detection, and positive colonies were selected and verified by sequencing. The plasmid was then extracted and transformed into Agrobacterium tumefaciens strain GV3101. A single positive A. tumefaciens colony was selected for infiltration into N. benthamiana using a previously described method [50]. Specific amplification primers were designed using CE Design software “http://www.vazyme.com (accessed on 26 August 2024)”, and the sequences are listed in Table S4.

5. Conclusions

In this study, we characterized MsWRKY transcription factors through genome-wide identification of the WRKY gene family in M. sativa, utilizing sequence homology and conserved WRKY domains to classify them into various groups based on their structure and phylogenetic relationships. An expression analysis revealed differential expression of several WRKY genes during M. sativa leaf senescence. Functional studies in N. benthaminana suggested the involvement of specific WRKY proteins in regulating senescence-associated phenotypes. Moreover, some senescence-associated MsWRKY genes responded to various abiotic stresses, such as salinity, indicating a dual role in stress resilience and senescence regulation. Collectively, these findings emphasize the complex roles of WRKY transcription factors in M. sativa leaf senescence and their potential as targets for enhancing crop resilience and productivity. Further investigations in this area will continue to elucidate the molecular mechanisms underlying senescence, offering a new path for the genetic enhancement of alfalfa and other forage crops.

Author Contributions

H.X. and X.P. designed and planned the feasibility of the experiment. X.P., J.H., X.D. and H.X. performed the experiments. X.P., J.H. and H.X. analyzed the data and wrote the manuscript. M.C., H.X., J.W. and Z.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures

Figure 1. A neighbor-joining phylogenetic tree was constructed using MEGA11.0 software with 1000 boot-strap replications, by comparing WRKY TFs from M. sativa L. (Ms) and Arabidopsis thaliana (At). Various highlighted colors correspond to the different subgroups.

Figure 2. Analysis of phylogenetic relationships, motifs, and gene structure of WRKY TFs from M. sativa. (a) Phylogenetic tree of 198 MsWRKYs in M. sativa. The colors highlighted the different subgroups are same as that in Figure 1. (b) Conserved motif arrangements of MsWRKYs. The motifs are highlighted in various colored boxes. Motif 1 represents the WRKY domain. (c) Exon-intron organizations of MsWRKYs. Green boxes indicate exons; black lines indicate introns.

Figure 3. The 2 kb promoter sequences of the MsWRKY gene contain various cis-acting elements. Different colored rectangles indicate different cis-elements, positioned according to their locations within the promoters.

Figure 4. A heatmap displaying the RNA−Seq data for 198 MsWRKYs, with expression levels normalized by row using the Z−Scores algorithm. The color scale on the right of the heatmap shows relative expression, with the color gradient from blue to red indicating increased expression levels. X0 (top not fully unfolded leaf), X1 (top fully unfolded first leaf), X2 (top fully unfolded second leaf), X3 (top fully unfolded third leaf), X4 (bottom leaf with senescent symptom); D0, D1, D2, D4, D6 (leaves treated in the dark for 0, 1, 2, 4 and 6 days); S1, S2, S4, S6 (leaves treated with salt for 1, 2, 4 and 6 days). Fourteen genes selected for RT-qPCR were labeled with an asterisk.

Figure 5. RT-qPCR results of 14 MsWRKYs in the process of leaf senescence under (a) natural condition (X0, X1, X2, X3, X4 represent different stages of leaf development), (b) dark stress (D0, D1, D2, D4, D6 represent 0, 1, 2, 4, and 6 days of dark treatment), (c) salt stress (S1, S2, S4, S6 represent 1, 2, 4, and 6 days of the 150 mM NaCl treatment). The error bars indicate the standard deviation of three biological replicates. Relative expression was calculated using the 2–ΔCT method. The data for gene expression are presented as the mean ± SD and were analyzed to detect significant differences by ANOVA using GraphPad Prism 8 (NS: not significant; * p [less than] 0.05; ** p [less than] 0.01) against D0 or X0.

Figure 6. Functional validation of selected MsWRKYs was conducted using an Agrobacterium-mediated transient expression assay. Symptoms of leaf senescence in representative Nicotiana benthamiana leaves appeared after infiltration with various constructs encoding MsWRKY5, MsWRKY66, MsWRKY92, MsWRKY141, MsSGR and an empty vector with YFP. Positive control: SGR. Negative control: empty vector with YFP. Bar = 1 cm.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13192725/s1. Table S1. Basic information of MsWRKYs gene. Table S2. Expression levels for the 198 MsWRKYs screened genes. Table S3. Sequences of primers used in qRT-PCR. Table S4. Sequences of primers used in PCR.

References

1. Eulgem, T.; Rushton, P.J.; Robatzek, S.; Somssich, I.E. The WRKY superfamily of plant transcription factors. Trends Plant Sci.; 2000; 5, pp. 199-206. [DOI: https://dx.doi.org/10.1016/S1360-1385(00)01600-9] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10785665]

2. Rushton, P.J.; Somssich, I.E.; Ringler, P.; Shen, Q.J. WRKY transcription factors. Trends Plant Sci.; 2010; 15, pp. 247-258. [DOI: https://dx.doi.org/10.1016/j.tplants.2010.02.006] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20304701]

3. Ishiguro, S.; Nakamura, K. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato. Mol. Gen. Genet. MGG; 1994; 244, pp. 563-571. [DOI: https://dx.doi.org/10.1007/BF00282746] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/7969025]

4. Song, H.; Nan, Z. Genome-wide identification and analysis of WRKY transcription factors in Medicago truncatula. Hereditas; 2014; 36, pp. 152-168. [DOI: https://dx.doi.org/10.3724/sp.j.1005.2014.00152]

5. Hu, W.; Ren, Q.; Chen, Y.; Xu, G.; Qian, Y. Genome-wide identification and analysis of WRKY gene family in maize provide insights into regulatory network in response to abiotic stresses. BMC Plant Biol.; 2021; 21, 427. [DOI: https://dx.doi.org/10.1186/s12870-021-03206-z]

6. Ye, H.; Qiao, L.; Guo, H.; Guo, L.; Ren, F.; Bai, J.; Wang, Y. Genome-Wide Identification of Wheat WRKY Gene Family Reveals That TaWRKY75-A Is Referred to Drought and Salt Resistances. Front. Plant Sci.; 2021; 12, 663118. [DOI: https://dx.doi.org/10.3389/fpls.2021.663118]

7. Yuan, G.; Zhang, N.; Zou, Y.; Hao, Y.; Pan, J.; Liu, Y.; Zhang, W.; Li, B. Genome-wide identification and expression analysis of WRKY gene family members in red clover (Trifolium pratense L.). Front. Plant Sci.; 2023; 14, 1289507. [DOI: https://dx.doi.org/10.3389/fpls.2023.1289507]

8. Kosakivska, I.V.; Vedenicheva, N.P.; Babenko, L.M.; Voytenko, L.V.; Romanenko, K.O.; Vasyuk, V.A. Exogenous phytohormones in the regulation of growth and development of cereals under abiotic stresses. Mol. Biol. Rep.; 2022; 49, pp. 617-628. [DOI: https://dx.doi.org/10.1007/s11033-021-06802-2]

9. Wani, S.H.; Anand, S.; Singh, B.; Bohra, A.; Joshi, R. WRKY transcription factors and plant defense responses: Latest discoveries and future prospects. Plant Cell Rep.; 2021; 40, pp. 1071-1085. [DOI: https://dx.doi.org/10.1007/s00299-021-02691-8]

10. Gan, S.; Amasino, R.M. Inhibition of Leaf Senescence by Autoregulated Production of Cytokinin. Science; 1995; 270, pp. 1986-1988. [DOI: https://dx.doi.org/10.1126/science.270.5244.1986]

11. Lei, P.; Yu, F.; Liu, X. Recent advances in cellular degradation and nuclear control of leaf senescence. J. Exp. Bot.; 2023; 74, pp. 5472-5486. [DOI: https://dx.doi.org/10.1093/jxb/erad273] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37453102]

12. Song, Y.; Li, C.; Zhu, Y.; Guo, P.; Wang, Q.; Zhang, L.; Wang, Z.; Di, H. Overexpression of ZmIPT2 gene delays leaf senescence and improves grain yield in maize. Front Plant Sci; 2022; 13, 963873. [DOI: https://dx.doi.org/10.3389/fpls.2022.963873] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35928712]

13. Guo, Y.; Cai, Z.; Gan, S. Transcriptome of Arabidopsis leaf senescence. Plant Cell Environ.; 2004; 27, pp. 521-549. [DOI: https://dx.doi.org/10.1111/j.1365-3040.2003.01158.x]

14. Cui, X.; Fan, X.; Xu, S.; Wang, S.; Niu, F.; Zhao, P.; Yang, B.; Liu, W.; Guo, X.; Jiang, Y.-Q. WRKY47 transcription factor modulates leaf senescence through regulating PCD-associated genes in Arabidopsis. Plant Physiol. Biochem.; 2024; 213, 108805. [DOI: https://dx.doi.org/10.1016/j.plaphy.2024.108805]

15. Zhang, W.; Tang, S.; Li, X.; Chen, Y.; Li, J.; Wang, Y.; Bian, R.; Jin, Y.; Zhu, X.; Zhang, K. Arabidopsis WRKY1 promotes monocarpic senescence by integrative regulation of flowering, leaf senescence and nitrogen remobilization. Mol. Plant; 2024; 17, pp. 1289-1306. [DOI: https://dx.doi.org/10.1016/j.molp.2024.07.005]

16. Chen, L.; Xiang, S.; Chen, Y.; Li, D.; Yu, D. Arabidopsis WRKY45 Interacts with the DELLA Protein RGL1 to Positively Regulate Age-Triggered Leaf Senescence. Mol. Plant; 2017; 10, pp. 1174-1189. [DOI: https://dx.doi.org/10.1016/j.molp.2017.07.008]

17. Robatzek, S.; Somssich, I.E. Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev.; 2002; 16, pp. 1139-1149. [DOI: https://dx.doi.org/10.1101/gad.222702]

18. Zhou, X.; Jiang, Y.; Yu, D. WRKY22 Transcription Factor Mediates Dark-Induced Leaf Senescence in Arabidopsis. Mol. Cells; 2011; 31, pp. 303-314. [DOI: https://dx.doi.org/10.1007/s10059-011-0047-1]

19. Cao, J.; Zhang, Y.; Tan, S.; Yang, Q.; Wang, H.-L.; Xia, X.; Luo, J.; Guo, H.; Zhang, Z.; Li, Z. LSD 4.0: An improved database for comparative studies of leaf senescence. Mol. Hortic.; 2022; 2, 24. [DOI: https://dx.doi.org/10.1186/s43897-022-00045-w]

20. Niu, F.; Cui, X.; Zhao, P.; Sun, M.; Yang, B.; Deyholos, M.K.; Li, Y.; Zhao, X.; Jiang, Y.Q. WRKY42 transcription factor positively regulates leaf senescence through modulating SA and ROS synthesis in Arabidopsis thaliana. Plant J.; 2020; 104, pp. 171-184. [DOI: https://dx.doi.org/10.1111/tpj.14914]

21. Zentgraf, U.; Laun, T.; Miao, Y. The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana. Eur. J. Cell Biol.; 2010; 89, pp. 133-137. [DOI: https://dx.doi.org/10.1016/j.ejcb.2009.10.014] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20004496]

22. Wang, Y.; Cui, X.; Yang, B.; Xu, S.; Wei, X.; Zhao, P.; Niu, F.; Sun, M.; Wang, C.; Cheng, H. et al. WRKY55 transcription factor positively regulates leaf senescence and defense response through modulating the transcription of genes implicated in ROS and SA biosynthesis in Arabidopsis. Development; 2020; 147, dev189647. [DOI: https://dx.doi.org/10.1242/dev.189647] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32680933]

23. Yu, Y.; Qi, Y.; Xu, J.; Dai, X.; Chen, J.; Dong, C.H.; Xiang, F. Arabidopsis WRKY71 regulates ethylene-mediated leaf senescence by directly activating EIN2, ORE1 and ACS2 genes. Plant J.; 2021; 107, pp. 1819-1836. [DOI: https://dx.doi.org/10.1111/tpj.15433] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34296474]

24. Guo, P.; Li, Z.; Huang, P.; Li, B.; Fang, S.; Chu, J.; Guo, H. A Tripartite Amplification Loop Involving the Transcription Factor WRKY75, Salicylic Acid, and Reactive Oxygen Species Accelerates Leaf Senescence. Plant Cell; 2017; 29, pp. 2854-2870. [DOI: https://dx.doi.org/10.1105/tpc.17.00438]

25. Besseau, S.; Li, J.; Palva, E.T. WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana. J. Exp. Bot.; 2012; 63, pp. 2667-2679. [DOI: https://dx.doi.org/10.1093/jxb/err450]

26. Wang, H.; Cheng, X.; Yin, D.; Chen, D.; Luo, C.; Liu, H.; Huang, C. Advances in the Research on Plant WRKY Transcription Factors Responsive to External Stresses. Curr. Issues Mol. Biol.; 2023; 45, pp. 2861-2880. [DOI: https://dx.doi.org/10.3390/cimb45040187]

27. Krupinska, K.; Desel, C.; Frank, S.; Hensel, G. WHIRLIES Are Multifunctional DNA-Binding Proteins With Impact on Plant Development and Stress Resistance. Front. Plant Sci.; 2022; 13, 880423. [DOI: https://dx.doi.org/10.3389/fpls.2022.880423]

28. Qiao, H.; Liu, Y.; Cheng, L.; Gu, X.; Yin, P.; Li, K.; Zhou, S.; Wang, G.; Zhou, C. TaWRKY13-A Serves as a Mediator of Jasmonic Acid-Related Leaf Senescence by Modulating Jasmonic Acid Biosynthesis. Front. Plant Sci.; 2021; 12, 717233. [DOI: https://dx.doi.org/10.3389/fpls.2021.717233]

29. Chen, W.; Zheng, Y.; Wang, J.; Wang, Z.; Yang, Z.; Chi, X.; Dai, L.; Lu, G.; Yang, Y.; Sun, B. Ethylene-responsive suppresses leaf senescence by inhibition of chlorophyll degradation in sorghum. New Phytol.; 2023; 238, pp. 1129-1145. [DOI: https://dx.doi.org/10.1111/nph.18757]

30. Du, J.; Lu, S.; Chai, M.; Zhou, C.; Sun, L.; Tang, Y.; Nakashima, J.; Kolape, J.; Wen, Z.; Behzadirad, M. et al. Functional characterization of PETIOLULE-LIKE PULVINUS (PLP) gene in abscission zone development in Medicago truncatula and its application to genetic improvement of alfalfa. Plant Biotechnol. J.; 2021; 19, pp. 351-364. [DOI: https://dx.doi.org/10.1111/pbi.13469]

31. Zhang, Y.; Wang, Z.; Zhang, F.; Wang, X.; Li, Y.; Long, R.; Li, M.; Li, X.; Wang, Q.; Yang, Q. et al. Overexpression of MsDREB1C Modulates Growth and Improves Forage Quality in Tetraploid Alfalfa (Medicago sativa L.). Plants; 2024; 13, 1237. [DOI: https://dx.doi.org/10.3390/plants13091237] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38732451]

32. Gregersen, P.L.; Culetic, A.; Boschian, L.; Krupinska, K. Plant senescence and crop productivity. Plant Mol. Biol.; 2013; 82, pp. 603-622. [DOI: https://dx.doi.org/10.1007/s11103-013-0013-8] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23354836]

33. Qiang, Y.; He, X.; Li, Z.; Li, S.; Zhang, J.; Liu, T.; Tursunniyaz, M.; Wang, X.; Liu, Z.; Fang, L. Genome-wide identification and expression analysis of the response regulator gene family in alfalfa (Medicago sativa L.) reveals their multifarious roles in stress response. Front. Plant Sci.; 2023; 14, 1149880. [DOI: https://dx.doi.org/10.3389/fpls.2023.1149880] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36998691]

34. Mahmood, K.; Torres-Jerez, I.; Krom, N.; Liu, W.; Udvardi, M.K. Transcriptional Programs and Regulators Underlying Age-Dependent and Dark-Induced Senescence in Medicago truncatula. Cells; 2022; 11, 1570. [DOI: https://dx.doi.org/10.3390/cells11091570] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35563875]

35. Zhou, C.; Han, L.; Pislariu, C.; Nakashima, J.; Fu, C.; Jiang, Q.; Quan, L.; Blancaflor, E.B.; Tang, Y.; Bouton, J.H. et al. From model to crop: Functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement. Plant Physiol.; 2011; 157, pp. 1483-1496. [DOI: https://dx.doi.org/10.1104/pp.111.185140]

36. Cao, J.; Liu, H.; Tan, S.; Li, Z. Transcription Factors-Regulated Leaf Senescence: Current Knowledge, Challenges and Approaches. Int. J. Mol. Sci.; 2023; 24, 9245. [DOI: https://dx.doi.org/10.3390/ijms24119245]

37. Bencke-Malato, M.; Cabreira, C.; Wiebke-Strohm, B.; Bücker-Neto, L.; Mancini, E.; Osorio, M.B.; Homrich, M.S.; Turchetto-Zolet, A.C.; De Carvalho, M.C.C.G.; Stolf, R. et al. Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhiziinfection. BMC Plant Biol.; 2014; 14, 236. [DOI: https://dx.doi.org/10.1186/s12870-014-0236-0]

38. Hernandez-Garcia, C.M.; Finer, J.J. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci.; 2014; 217–218, pp. 109-119. [DOI: https://dx.doi.org/10.1016/j.plantsci.2013.12.007]

39. Ishihama, N.; Yoshioka, H. Post-translational regulation of WRKY transcription factors in plant immunity. Curr. Opin. Plant Biol.; 2012; 15, pp. 431-437. [DOI: https://dx.doi.org/10.1016/j.pbi.2012.02.003]

40. Kaur, A.; Pati, P.K.; Pati, A.M.; Nagpal, A.K. In-silico analysis of cis-acting regulatory elements of pathogenesis-related proteins of Arabidopsis thaliana and Oryza sativa. PLoS ONE; 2017; 12, e0184523. [DOI: https://dx.doi.org/10.1371/journal.pone.0184523]

41. Wang, H.; Chen, W.; Xu, Z.; Chen, M.; Yu, D. Functions of WRKYs in plant growth and development. Trends Plant Sci.; 2023; 28, pp. 630-645. [DOI: https://dx.doi.org/10.1016/j.tplants.2022.12.012]

42. Jiang, J.; Ma, S.; Ye, N.; Jiang, M.; Cao, J.; Zhang, J. WRKY transcription factors in plant responses to stresses. J. Integr. Plant Biol.; 2017; 59, pp. 86-101. [DOI: https://dx.doi.org/10.1111/jipb.12513] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27995748]

43. Guo, Y.; Ren, G.; Zhang, K.; Li, Z.; Miao, Y.; Guo, H. Leaf senescence: Progression, regulation, and application. Mol. Hortic.; 2021; 1, 5. [DOI: https://dx.doi.org/10.1186/s43897-021-00006-9] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37789484]

44. Duan, X.; Tian, D.; Gao, P.; Sun, Y.; Peng, X.; Wen, J.; Xie, H.; Wang, Z.Y.; Chai, M. Transcriptome-Wide Identification of Dark- and Salt-Induced Senescence-Related NAC Gene Family Members in Alfalfa. Int. J. Mol. Sci.; 2024; 25, 8908. [DOI: https://dx.doi.org/10.3390/ijms25168908] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/39201594]

45. Tan, S.; Sha, Y.; Sun, L.; Li, Z. Abiotic Stress-Induced Leaf Senescence: Regulatory Mechanisms and Application. Int. J. Mol. Sci.; 2023; 24, 11996. [DOI: https://dx.doi.org/10.3390/ijms241511996] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37569371]

46. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant; 2020; 13, pp. 1194-1202. [DOI: https://dx.doi.org/10.1016/j.molp.2020.06.009]

47. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol.; 2016; 33, pp. 1870-1874. [DOI: https://dx.doi.org/10.1093/molbev/msw054]

48. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res.; 2002; 30, pp. 325-327. [DOI: https://dx.doi.org/10.1093/nar/30.1.325]

49. Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc.; 2008; 3, pp. 1101-1108. [DOI: https://dx.doi.org/10.1038/nprot.2008.73]

50. Li, X.; Chanroj, S.; Wu, Z.; Romanowsky, S.M.; Harper, J.F.; Sze, H. A distinct endosomal Ca²⁺/Mn²⁺ pump affects root growth through the secretory process. Plant Physiol.; 2008; 147, pp. 1675-1689. [DOI: https://dx.doi.org/10.1104/pp.108.119909]