CkWRKY33 cloning and sequence analysis
In our previous study, we used one month old seedling of C. korshinskii Kom to do drought treatment, and then we did RNA-seq and de novo assembly (BioSample: SAMN03121496). The results showed that there were 440 differentially expressed genes (DEGs) between drought and control plants, and among the DEGs, 39 unigenes showed up-regulated expression after drought treatment .After comparing with the database, we named one unigene, com66203 as CkWRKY33.
The full-length cDNA of CkWRKY33 was obtained from total RNA extracted from drought-stressed C. korshinskii Kom leaves using RT-PCR. The nucleotide sequence of the CkWRKY33 gene is 2,075 bp in length, consisting of a 23-bp 5’ untranslated region, an 1,614 bp open reading frame (ORF) and a 345-bp 3’ UTR. The ORF encodes a putative 537-amino acid protein. Sequence alignments between CkWRKY33 and other plant WRKY proteins indicated that the amino acid sequences of these proteins share a high similarity. The sequence identity between CkWRKY33 and the other proteins in the analysis ranged from 39% to 85% (Fig. 1). A multiple sequence alignment analysis revealed that CkWRKY33 contains two putative WRKY domains followed by a C2H2-type zinc-finger motif, a putative nuclear localization signal and a short conserved structural motif (C-motif), indicating that CkWRKY33 belongs to Group-I of the WRKY family (Fig. 1).
A phylogenetic tree was constructed to investigate the evolutionary relationships among CkWRKY33 and other WRKY proteins. As shown in Fig. 2, CkWRKY33 showed a close relationship with AtWRKY33 in Arabidopsis, WRKY24 in rice and WRKY115 in maize. These proteins participate in plant response to abiotic stresses [26, 27]. Thus, these proteins having high homology levels among different species, may share some similar functions.
Subcellular localization of CkWRKY33
To determine the subcellular localization of CkWRKY33, the ORF of CkWRKY33 without the termination codon was fused to the 5’ end of the GFP reporter gene under the control of the CaMV35S promoter. The recombinant construct and the GFP vector were independently introduced into tobacco epidermal cells. Confocal imaging showed that the 35s-CkWRKY33-GFP fusion protein was exclusively localized in the nuclear. By contrast, tobacco epidermal cells transformed with the 35s-GFP vector alone displayed fluorescence throughout the entire cell, demonstrating that CkWRKY33 is a nuclear localized protein (Fig. 3).
Overexpression of CkWRKY33 enhances tolerance to mannitol stress
The full-length cDNA of CkWRKY33 under the control of the CaMV35S promoter was transformed into Arabidopsis. After positive transformants were screened and self-crossed, the seeds of T2 transgenic homozygous and wild-type (WT) lines were sown on normal 1/2MS medium and 1/2MS medium supplemented with mannitol. On 1/2MS solid medium, the growth of transgenic lines was generally similar to that of WT, with no obvious change in root length. Under mannitol-treatment conditions, the seedlings of both the transgenic plants and WT plants grew weakly, the rosette leaves turned yellow, and the root lengths became shorter as the mannitol concentration increased. With both 50mM and 100mM mannitol treatments, the root lengths of transgenic plants were longer than those of WT (Fig. 4). Thus, the root length of Arabidopsis was changed by mannitol stress, and the CkWRKY33 gene may have effect on the mannitol resistance of the plant.
Overexpression of CkWRKY33 enhances the tolerance to drought stresses
In addition to the mannitol-stress treatment, the seeds of three transgenic and WT lines were sown in soil. After these plants had grown for 21 d under normal conditions, watering was stopped. After 15 d of the natural drought treatment, most leaves of WT had lost their green color and turned yellow, or even died. The transgenic plants showed slight yellowing and curling at the leaf tips and dehydration; however, they grew well and had a normal phenotype. After rehydration, WT plants showed complete wilting and dehydration, while the leaves of transgenic lines showed a low degree of atrophy and good growth. Compared with under drought-stress conditions, the leaves of transgenic plants became tender after 3 d of rehydration, indicating that transgenic plants were strongly resilient after rehydration (Fig. 5A).
The survival rate of WT was 8.33%, which was lower than any of the three transgenic lines. The survival rate of the three lines was 80% on average. Thus, the expression of the transformed CkWRKY33 increased the drought resistance of transgenic plants (Fig. 5B).
A qRT-PCR experiment was used to analyze the expression patterns of CkWRKY33 in transgenic plants before and after drought treatment. The relative expression level revealed that compared with before drought treatment, the CkWRKY33 was highly induced expression by drought treatment in transgenic plants (Fig. 5C). After the drought treatment, the gene was expressed 3 to 5 times more in transgenic plants than before dought treatment. After rehydration, the gene’s expression level in the transgenic plants decreased. Thus, that expression of the WRKY TF may improve the tolerance of transgenic Arabidopsis to drought stress.
Changes in physiological traits under stress conditions
The leaf water loss rates of WT and transgenic plants were detected. The water loss rate increased as the processing time increased for all the plants. It was greater in WT than in transgenic plants (Fig. 6A). After 1h, the water loss rate of the WT was 27.8% and the rates of the transgenic lines ranged from 11.8% to 20.5%. After a 12h treatment, the water loss rates of all the transgenic lines were less than 86%, while that of the WT was as high as 92.3% (Fig. 6A). This indicated that the transgenic lines had lower water loss rates and stronger drought- tolerance levels.
The values of four physiological traits, soluble sugar content, malondialdehyde (MDA) content, proline content and peroxidase (POD) activity, were determined for the drought-treated transgenic plants. The soluble sugar content of each transgenic plant line was higher than that of the WT. The highest value for the transgenic plants was 23.70 mg/g and the lowest value was 16.08 mg/g. The MDA content was lower in transgenic plants than in WT, and the lowest value among the transgenic lines was 2.32 nmol/g. Thus, as the MDA content in the transgenic plants decreased, the damage to plant cell membranes decreased and the drought resistance increased. The proline content was higher in transgenic plants than in WT after drought stress, and the POD content followed the same trend (Fig. 6B-E).