Transcriptome analysis of genes involved in defense against alkaline stress in roots of wild jujube (Ziziphus acidojujuba)

Abstract

Wild jujube (Ziziphus acidojujuba Mill.) is highly tolerant to alkaline, saline and drought stress; however, no studies have performed transcriptome profiling to study the response of wild jujube to these and other abiotic stresses. In this study, we examined the tolerance of wild jujube to NaHCO3-NaOH solution and analyzed gene expression profiles in response to alkaline stress. Physiological experiments revealed that H2O2 content in leaves increased significantly and root activity decreased quickly during alkaline of pH 9.5 treatment. For transcriptome analysis, wild jujube plants grown hydroponically were treated with NaHCO3-NaOH solution for 0, 1, and 12 h and six transcriptomes from roots were built. In total, 32,758 genes were generated, and 3,604 differentially expressed genes (DEGs) were identified. After 1 h, 853 genes showed significantly different expression between control and treated plants; after 12 h, expression of 2,856 genes was significantly different. The expression pattern of nine genes was validated by quantitative real-time PCR. After gene annotation and gene ontology enrichment analysis, the genes encoding transcriptional factors, serine/threonine-protein kinases, heat shock proteins, cysteine-like kinases, calmodulin-like proteins, and reactive oxygen species (ROS) scavengers were found to be closely involved in alkaline stress response. These results will provide useful insights for elucidating the mechanisms underlying alkaline tolerance in wild jujube.

Fig 1. Physiologic characteristics of wild jujube under salt and alkaline-salt treatment.

Seedlings at the six-leaf stage were subjected to an alkaline treatment (pH 9.5). (A) Time-course observation for tolerance phenotype at 3, and 5 d after the start of treatment. Bars = 2 cm. (B) Quantitative measurement of H₂O₂ in wild jujube leaves during pH 9.5 treatment. Values are means± SD (n = 3). (C) Quantitative measurement of root activity in wild jujube during pH 9.5 treatment. Values are means± SD (n = 3).

Fig 2. The overlap of differentially expressed genes (DEGs) in wild jujube (Ziziphus acidojujuba) roots under alkaline treatment.

The three data sets compared 0 h vs. 1 h (yellow), 1 h vs. 12 h (red), and 0 h vs. 12 h (blue).

Fig 3. Gene ontology (GO) categories assigned to the DEGs in response to alkaline stress.

The X- axis represents the gene numbers, and the Y- axis shows the GO subcategories.

Fig 4. Expression analysis of selected genes.

(A) Quantitative real-time PCR was conducted on the roots of hydroponically grown seedlings of wild jujube at the six-leaf stage. ZaActin was used as an internal control. Data shown are mean values of three biological replicates with SD. (B) The heat map was constructed based on log₂ FPKM values at different time points. The color scale is shown at the top.

Fig 5. Expression profile of the different family genes in response to alkaline stress at different time points.

The heat map was constructed based on log₂ FPKM values. The color scales are shown at the top of each analysis.