Integrated analysis of the physiology, RNA, and microRNA involved in black locust (Robinia pseudoacacia) rejuvenation

Abstract

Abstract Background Rejuvenation is a key process that enables perennial woody plants to regain growth potential. In Robinia pseudoacacia plantations, natural root sprouting individuals provide good material for studying the rejuvenation of woody plants. However, the physiological differences and molecular mechanisms underlying black locust rejuvenation remain unclear. In this study, we compared the physiological conditions and molecular responses of rejuvenated individuals and mother trees. Results Our analysis of leaf structures and physiological indices showed that the epidermis thickness, leaf thickness and leaf-tissue tightness of rejuvenated individuals were less than those of mother trees. The soluble-sugar content and total SOD activity of rejuvenated individuals were also lower than those of mother trees. The younger the rejuvenated individuals were, the lower the ABA content, ABA/ZT and GA3/ZT in the leaves. The ZT content increased with decreasing age of rejuvenated individuals. Using high-throughput sequencing strategies, the mRNA and miRNA involved in the rejuvenation of black locust were identified. RNA-seq identified 175,862 unigenes by de novo transcript assembly. Of those, 4,727 differentially expressed genes were identified based on clean reads mapped to the assembled transcriptome for gene expression analysis(fold change≥2 or ≤0.5 and q-value≤0.05). These genes were enriched to 53 gene ontology(GO) terms and 20 KEGG pathways (FDR≤0.01). Among these were a major pathway related to flavone and flavonol biosynthesis. High-throughput miRNA sequencing identified a total of 991 miRNAs, including 671 novel miRNAs. Furthermore, 262 known and 625 novel differentially expressed miRNAs were identified(fold change≥1.5 or ≤0.67 and p≤0.05). The main functions identified in the GO analysis of the target predictions overlapped with differentially expressed genes derived from RNA-seq. KEGG pathway enrichment showed that circadian rhythm-fly and signaling pathways regulating pluripotency of stem cells attracted considerable attention during rejuvenation. Conclusion Our study revealed physiological differences between rejuvenated individuals and mother trees of R. pseudoacacia. Differential genes between mother trees and rejuvenated individuals may vary according to the tree ages, but miRNAs may play a key regulatory role in rejuvenation. The same genotype system composed of root germinating individuals and mother-tree individuals provides a solid starting point for further elucidation of the rejuvenation of woody plants.