Identification of Differentially Expressed Genes Related to Dehydration Resistance in a Highly Drought-Tolerant Pear, Pyrus betulaefolia, as through RNA-Seq

Abstract

Drought is a major abiotic stress that affects plant growth, development and productivity. Pear is one of the most important deciduous fruit trees in the world, but the mechanisms of drought tolerance in this plant are still unclear. To better understand the molecular basis regarding drought stress response, RNA-seq was performed on samples collected before and after dehydration in Pyrus betulaefolia. In total, 19,532 differentially expressed genes (DEGs) were identified. These genes were annotated into 144 Gene Ontology (GO) terms and 18 clusters of orthologous groups (COG) involved in 129 Kyoto Encyclopedia of Genes and Genomes (KEGG) defined pathways. These DEGs comprised 49 (26 up-regulated, 23 down-regulated), 248 (166 up-regulated, 82 down-regulated), 3483 (1295 up-regulated, 2188 down-regulated), 1455 (1065 up-regulated, 390 down-regulated) genes from the 1 h, 3 h and 6 h dehydration-treated samples and a 24 h recovery samples, respectively. RNA-seq was validated by analyzing the expresson patterns of randomly selected 16 DEGs by quantitative real-time PCR. Photosynthesis, signal transduction, innate immune response, protein phosphorylation, response to water, response to biotic stimulus, and plant hormone signal transduction were the most significantly enriched GO categories amongst the DEGs. A total of 637 transcription factors were shown to be dehydration responsive. In addition, a number of genes involved in the metabolism and signaling of hormones were significantly affected by the dehydration stress. This dataset provides valuable information regarding the Pyrus betulaefolia transcriptome changes in response to dehydration and may promote identification and functional analysis of potential genes that could be used for improving drought tolerance via genetic engineering of non-model, but economically-important, perennial species.

Fig 1. Time-course fresh water loss of Pyrus betulaefolia seedling during dehydration for 0, 1, 3 and 6 h in an ambient environment at 26°C, followed by recovery in water at 26°C for 24 h.

(A-E). Electrolyte leakage and relative water content (RWC) analysis of Pyrus betulaefolia leaves. (F). Content of photosynthetic rate (Pn) (G); content of stomatal conductance (Gs) (H); content of transpiration rate (Tr) (I).

Fig 2. Differentially expressed genes between different libraries.

Up-regulated (red) and down-regulated (green) genes were quantified (A). The ten comparison results are shown. Venn diagram showing the differentially expressed genes at each of the five time points associated with drought stress. (B) Up-regulated genes. (C) Down-regulated genes.

Fig 3. GO categories for the genes identified. Y-axis (left) represents the percentage of genes identified in this study; Y-axis (right) represents the actual gene number.

The genes were annotated into three main categories: biological process, cellular component and molecular function (X-axis).

Fig 4. Clustering and gene ontology enrichment of regulated genes.

Genes displaying some degree of regulation were clustered using Hierarchical Clustering. A description of the expression pattern and the number of transcripts belonging to the cluster form the title of each chart. Expression values were normalized and scaled to between –1.0 and 1.0 (Y-axis). Enriched “Biological Process” GO terms, generated in BinGO are listed at the bottom of each cluster. For Clusters 12, 13, 14, 16 and 18, no over-represented “Biological Process” terms were detected. The number of genes associated with an over-represented GO term in the cluster and the number of genes associated with the same GO term in the Pyrus transcriptome are shown in parentheses. Specific genes belonging to each cluster can be found in S3 File. 2 (D1), 3 (D3), 4, (D6), 5 (DH24).

Fig 5. Expression patterns of genes involved in ABA and GA metabolism.

Fig 6. Quantitative RT-PCR validation of differential gene expression during dehydration.

The left Y-axis indicates the gene expression levels calculated by the RPKM method. The right Y-axis indicates the relative gene expression levels.