Secondary Metabolism and Hormone Response Reveal the Molecular Mechanism of Triploid Mulberry (Morus Alba L.) Trees Against Drought

Abstract

The improvement of a plant's tolerance to drought is a major endeavor in agriculture. Polyploid plants often exhibit enhanced stress tolerance relative to their diploid progenitor, but the matching stress tolerance is still little understood. Own-rooted stem cuttings of mulberry (Morus alba L.) cultivar Shinichinose (2n = 2x = 28) and Shaansang-305 (2n = 3x = 42) were used in this study, of which the latter (triploid) has more production and application purposes. The responses of triploid Shaansang-305 and diploid progenitor ShinIchinose under drought stress were compared through an investigation of their physiological traits, RNA-seq, and secondary metabolome analysis. The results showed that the triploid exhibited an augmented abscisic acid (ABA) content and a better stress tolerance phenotype under severe drought stress. Further, in the triploid plant some genes (TSPO, NCED3, and LOC21398866) and ATG gene related to ABA signaling showed significantly upregulated expression. Interestingly, the triploid accumulated higher levels of RWC and SOD activity, as well as more wax on the leaf surface, but with less reductive flavonoid than in diploid. Our results suggest triploid plants may better adapt to with drought events. Furthermore, the flavonoid metabolism involved in drought resistance identified here may be of great value to medicinal usage of mulberry. The findings presented here could have substantial implications for future studies of crop breeding.

Keywords: RNA-Seq; drought stress; mulberry; polyploidy; secondary metabolism.

Figure 1

Physiological parameters in diploid (2X) and autotriploid (3X) subjected to different days of drought stress. (A) Water content of soil; (B) Ci: Intercellular CO2 concentration; (C) Cleaf: stomatic conductance; (D) Leaf relative water content; (E) MDA: malonaldehyde; (F) SOD: superoxide dismutase. (G-I) Hormonal content in leaves of diploid and triploid with drought stress for 9 days for ABA (G), GA (H) and JA (I). All assays were carried out at least three times and statistical significance levels were calculated using Student's t-test (^*P ≤ 0.05; ^**P ≤ 0.01). CK, control; SD, severe drought.

Figure 2

Transcriptome analysis of diploid (2X) and triploid (3X) in response to drought stress. (A) GO classification analysis of DEGs in diploid (2X) response to drought stress. (B) GO classification analysis of DEGs in triploid (3X) in response to drought stress. (C) The top 20 enriched pathway in drought stress conditions in diploid (2X). (D) The top 20 enriched pathway in drought stress conditions in triploid (3X). The size and the color of solid circles represent the number of transcripts involved in certain pathways and the significant value (q-value) of the rich factor, respectively. CK, control; SD, severe drought.

Figure 3

KEGG enrichment analysis of the DEGs on the 9th day and Weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) identified in drought treated and control over three sampling time points (5, 9, and 15 days) during drought stress. (A,B) The 20 most significantly enriched KEGG pathways for DEGs in response to drought after nine days in diploids (2X) (A) and triploids (3X) (B). (C) Salmon module eigengene expression values across all the samples. The upper portions of (D) figure is heat maps of the expression values of each gene (row) in each sample (column): red—highly expressed; green—lowly expressed; black—neutral; Control: (a: CK-2X, e: CK-3X); Severe drought with 5 days: (b:SD-2X-5, f:SD-3X-5); Severe drought with 9 days: (c: SD-2X-9, g: SD-3X-9); Severe drought with 15 days: (d: SD-2X-15, h: SD-3X-15). CK: control; SD: severe drought. (D) Pathway and process enrichment analysis. Each term is represented by a circle node, where its size is proportional to the number of input genes that fall into that term, and its color represent its cluster identity.

Figure 4

Differentially accumulated metabolites identified from diploid (2X) and triploid (3X) involved in the flavonoid biosynthesis under drought stress and overview of the major metabolic and transcript changes response to drought stress. (A) Heatmap hierarchical clustering showing metabolite fold change in diploid (2X) and triploid (3X) in comparison with the control. The color scale for hierarchical clustering is labeled. The scale bar displays fold change values. (B) Box plot for the temporal variability of key flavonoid compound signal intensities in all samples. (C) Overview of the major metabolic and transcript changes response to drought stress in diploid (2X) and triploid (3X). Arrows show the metabolic stream. The genes and metabolites that are up-regulated and down-regulated are shown in red and blue, respectively. Circled in black is the metabolite data. The scale bar displays fold change values. Abbreviations: cinnamic acid-4-hydroxylase, C4H, chalcone synthase; CHS, UDP-glucose favonoid-3-O-glucosyltransferase; UFGT, O-methyltransferase; OMT. CK, control; SD, severe drought.

Figure 5

Surface wax load, surface permeability, and histological traits of leaf in diploid (2X) and triploid (3X). (A) Morphological display of surface wax in the pressure side of leaf of diploid (2X) and triploid (3X) under two water treatments observed using scanning electron microscopy (SEM). Ample water treatment: (a1) CK-2X; (a3) CK-3X; Severe drought stress: (a2) SD-2X; (a4) SD-3X. (B) The wax coverage changes of leaf during water deprivation treatment for 9 days. (C) Histological characterization of leaves of diploid (2X) and triploid (3X) in well-watered and drought stress for 9 days. Ample water treatment: (c1) CK-2X; (c3) CK-3X; Severe drought stress: (c2) SD-2X; (c4) SD-3X. Bar = 250 um. (D) Palisade tissue-spongy tissue ratio of two cultivars. Surface permeability of leaves with drought stress. (E) Water loss rates and (F) chlorophyll extraction yields. Values represent means of three replicates. Error bars = SD. (G,H) Quantitative real-time PCR analysis of LOC21411095, LOC21396807 expression between diploid (2X) and triploid (3X) with drought stress, which related to wax synthesis. All assays were carried out at least three times and statistical significance levels were calculated using Student's t-test (^*P ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001). CK, control; SD, severe drought.

Figure 6

Heatmap of transcription factor and autophagy related transcripts. (A) Heatmap of gene expression for TFs enriched. (B) Heatmap of gene expression for eight DEGs enriched in the ‘autophagy’ pathway. Heatmap color indicates fold change of expression under drought compared with that in well-watered of both diploid (2X) and triploid (3X). (C) Quantitative real-time PCR analysis of LOC21395145 gene of diploid (2X) and triploid (3X) identified in the drought treatment and control over two sampling time points (9 and 15 days). All assays were carried out at least three times and statistical significance levels were calculated using Student's t-test (^*,p ≤ 0.05;^**,p ≤ 0.01). CK, control; SD, severe drought.

Figure 7

A model for mechanisms underlying the enhanced drought tolerance of mulberry triploid (3X). Under drought stress, triploid plants exhibit enhanced drought tolerance in comparison with diploid progenitors due to the activation of multifaceted defense machinery in leaves. For the first, activation of ABA hormonal signaling and antioxidant enzyme (SOD) leads to efficient hormonal signaling and improved ROS scavenging ability, and ABA-induced stomatal closure concurrently. Secondly, some genes (TSPO, NCED3, and LOC21398866) and ATG gene related to ABA signaling showed significant upregulation in expression. Autophagy is regulated by autophagy-related (ATG)gene, meanwhile, wax load increased.