Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing

Abstract

Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to osmotic stress and ABA treatment

“…r-project.org/) as Wang et al (2014) reported. Single variable analysis was used to compare the overall gene expression of the 13 genes at each of the time points (with Plant Growth Regul PEG-6000 or ABA treatment) to the control (without treatment), and to compare the overall gene expression between the tissues.…”

“…The difference values of CT (dCT) between a sample and the average of the the three house-keeping genes (ACT, EF1 and UBQ) as the expression level of the sample. Therefore, the higher the dCT value, the lower expression of mRNA in the sample, and vice versa (Wang et al 2014). …”

“…Reproduction twigs of the plant materials were harvested from the same clone of di-haploid P. simonii 9 P. nigra as Wang reported (Wang et al 2014). In order to monitor gene expression changes by different treatments, we designed a time course of experiment.…”

“…Total RNAs were extracted as previously described, and ACT, EF1 and UBQ were used as house-keeping genes (Wang et al 2014). The samples from three seedlings per time point per tissue were mixed, and then RNA was isolated for qRT-PCR analysis.…”

“…In our previous parallel paper (Wang et al 2014), we focused on expression of the 13 genes and their functions in salinity tolerance. In this study, we focus on the spatio-temporal expression patterns of the13 genes in response to osmotic stress and ABA treatment.…”