Identification and sequence analysis of WRKY transcription factor in Dioscorea spp.
After manually removing redundant entries through screening (manually delete the genes without ORF/WRKY motif) and validation of the search results, a number of 22 WRKY transcription factors were characterized from Dioscorea spp. and named them from DoWRKY1 to DoWRKY22. The characteristics concerning each gene, including gene number, length of coding sequence and amino acid sequence, MW and PI of the proteins, are summarized in Table 1. Subsequent sequence analysis of these 22 DoWRKYs showed that the encoded DoWRKY proteins lengths are ranged from 167 amino acids (aa) (DoWRKY20) to 709 amino acids (DoWRKY21) (average length: 385 amino acids). The calculated molecular weight (MW) from 19.01 (DoWRKY20) to 76.91 (DoWRKY21) kDa and isoelectric points (PI) from 4.88 (DoWRKY18) to 9.93 (DoWRKY19). The subcellular localization prediction showed that 7 of the 22 DoWRKYs proteins were extracellular (DoWRKY4, 10, 11, 17, 18, 20, 22), and the rest were localized in the nucleus.
Multiple sequence alignment and phylogenetic analysis of DoWRKYs
To detect the evolutionary relationships and classification of the WRKY family in Dioscorea spp., unrooted phylogenetic maximum likelihood method trees were constructed with the 22 DoWRKY proteins and the known WRKY protein from Oryza (Table S2). The results indicated that the 22 DoWRKYs were divided into 6 clades (Ⅰ,Ⅱa,Ⅱc,Ⅱd,Ⅱe,Ⅲ) according to the groups in Oryza (Fig. 1). Group I contained the highest WRKY members with 7 DoWRKYs, with four proteins in IIa groups, one members in IIc, three in IId, two in IIe, and group five in Ⅲ (Table 1). These results suggest that WRKY members in these groups of Dioscorea spp. could have the similar functions with those members in Oryza. Group 1 WRKY family members all have two WRKY domains and a C2H2 type zinc-finger motif (CX4-CX22 − 23-HXH). Group 3 were characterized by a C2HC type zinc-finger motif, but only the N-terminal WRKY domain of DoWRKY20 was lost (Fig. 2).
Gene structure and conserved motifs analysis
To further analyze the characteristics of DoWRKYs, the MEME online tool was used to predict the potential conserved motifs. A total of 10 motifs were identified, with lengths ranging between 17 and 50 amino acids. Motif 1 contained the domain present in every WRKY member. The motif information and the multiple alignment information showed that Motif1 was the highly conserved WRKY domain (WRKYGQK) in WRKY genes in Dioscorea spp.. Excluding DoWRKY15, all genes contained at least 2 Motifs. Notably, the members with high similarity in the same group shared a common motif composition. For instance, DoWRKY7 and DoWRKY9 were found to contain seven motifs (Fig. 3). It indicates that these two genes may have a similar function.
Expression analysis of DoWRKYs in different developmental stages of tuber
By comprehensively considering the protein family classification, subcellular localization, and expression level, we selected 8 genes from six groups to analyze expression level of Dioscorea spp(Table S3). under different growth and development stages. DoWRKY7 was up-regulated in the whole process and highly expressed in 105d. DoWRKY4 and DoWRKY12 showed the same trend and reached the highest value at 120d. Two genes DoWRKY16 and 18, downregulation expressed in earlier stage and upregulated expression in mature stage(Fig. 4).
DoWRKYs gene expression following abiotic stress by qRT-PCR
To further investigate the role of DoWRKYs in abiotic stress responses, we randomly selected 8 genes to made sure their responses to the cold, ABA, and JA treatment.
Following cold treatment (Fig. 5), the expression levels of four genes (DoWRKY5, DoWRK Y7, DoWRKY14, DoWRKY16) increased by more than twofold, among which the expression of DoWRKY7 was 15 times and DoWRKY16 was 9 times higher than that of the control. The expression of two genes(DoWRKY4 and DoWRKY11) decreased significantly at 1h, but their expression were significantly increased and peaked at 24 h.
The expression levels of three genes (DoWRKY7, DoWRKY11, DoWRKY16) were increased by more than twofold following treatment with ABA(Fig. 5). The expression levels of DoWRKY7 and DoWRKY16 were 15 fold higher than those of the control, respectively. The expression levels of DoWRKY18 decreased significantly than control.
For the case of MeJA stress (Fig. 5), four genes (DoWRKY4, DoWRKY5, DoWRKY14, DoWRKY18) hava the highest level of expression at 24 h. DoWRKY14 were upregulated in the whole process, but DoWRKY16 rapidly rising at early time points but with subsequently decreased expression at 24h. In contrast, DoWRKY7, DoWRKY11, and DoWRKY12 showed a trend of downregulation(Table S4).
To sum up, DoWRKY16 was upregulated expression in mature stage and responded to abiotic stresses in cold, ABA, MeJA. we selected it for further study.
Subcellular localization of DoWRKY71
To determine the subcellular localization of the DoWRKY71, the recombinant vector CaMV35S::GFP-DoWRKY71 was transiently expressed in the tobacco leaves. The GFP fluorescence of the CaMV35S::GFP-DoWRKY71 vector showed a strong fluorescence signal only in the nucleus, while the CaMV35S::GFP vector was relatively uniformly distributed inside the tobacco cells (Fig. 6).
Physiological and biochemical changes and detection of reactive oxygen species in tobacco treated at 4℃
Investigating the cold stress tolerance of DoWRKY71 in transgenic tobacco. The growth of transgenic tobacco under control conditions was better than that of WT tobacco, and the cold tolerance of transgenic tobacco was better (Fig. 7, A). The chlorophyll content decreased in the whole treatment processes (Fig. 7, B); The soluble protein content decreased firstly and reached the highest level after treatment for 24h, and the transgenic plants was significantly higher than that of WT plants (Fig. 7, C); The activities of SOD and POD both tended to increase, the transgenic plants were significantly higher than WT plants (Fig. 7, D, E).
Low temperature usually induces membranous peroxidation and overproduction of reactive oxygen species, which eventually leads to oxidative stress reaction. NBT and DAB staining was performed on tobacco treated at 4℃ (Fig. 7, F), staining was seen in both control WT and transgenic plants, but the O2·- and The H2O2 level was lower than that of the WT line. In addition, MDA content of both transgenic and WT plants increased with time when low temperature stress occurred, but decreased slightly at 24h. MDA content accumulated by transgenic lines in control and experimental groups was significantly lower than that of WT plants (Fig. 7, G) (Table S5). These results indicated that overexpression of DoWRKY71 regulated reactive oxygen species homeostasis and reduced oxidative damage in tobacco.
Changes of physiological indexes, ABA and GA contents in tobacco under ABA treatment
With the extension of spraying time, the soluble protein content decreased firstly and then increased, the transgenic plants in control and treatment groups was significantly higher than that in WT tobacco (Fig. 8, A). The change trend of SOD and POD activity were basically the same as that under 4℃ treatment, and POD activity was significantly higher than that in WT tobacco(Fig. 8, B, C). The GA content showed an overall upward trend and reached the highest value at 12h, the ABA content increased firstly and then decreased. The GA content of the transgenic lines in the control and experimental groups was significantly lower than that of the WT plants (Fig. 8, D), and ABA content is significantly higher than that in WT plants (Fig. 8, E) ( Table S6).
Effects of ABA treatment on stomata of tobacco overexpressing DoWRKY71
The stomata of DoWRKY71 transgenic plants and WT plants under ABA treatment were observed under microscope (100×) (Fig. 9, A). Under normal conditions, the stomatal pore size index of T3 and T4 was significantly higher than that of WT (Fig. 9, A). Under 100 µmol/L ABA spraying treatment, the stomatal index of DoWRKY71 transgenic lines was significantly smaller than that of WT plants. The results indicated that overexpression of DoWRKY71 could promote ABA mediated stomatal closure in tobacco.
Expression analysis of DoWRKY71 gene in transgenic tobacco at 4℃ and ABA treatment
Under 4℃ and ABA treatment time, the expression level of this gene showed a trend of first increase and then decrease. The expression level of transgenic plants was higher than that of untreated plants, and there was a significant difference (Fig. 10) ( Table S7).