Protoplast isolation and transcriptome analysis of developing xylem in Pinus massoniana (Pinaceae).

Abstract

With active physiological and biochemical activities, tissue-specific protoplasts from cambial derivatives, could serve as a specific source for information on xylogenesis for softwood species resistant to stable genetic transformation and lacking available mutants. In this study, protoplasts were isolated from developing xylem of the Chinese red pine, Pinus massoniana, by enzymolysis. High-quality RNAs were extracted from developing xylem and their protoplasts for constructing transcriptome libraries. Using Illumina HiSeq 2500 PE150 platform, a total of 362,328,426 clean paired-end reads (54.35G) were generated from multiple cDNA libraries and assembled into 146,422 unigenes. The transcriptome data were further analysed to identify 1567 differentially expressed genes (DEGs) between the isolated protoplasts and developing xylem of P. massoniana (Masson pine), 1126 DEGs were upregulated in protoplasts relative to developing xylem cells and 441 were downregulated. Most of the differentially expressed genes in biological process terms are related to plant response, which may be due to the response to cell wall removal. Further, the expression pattern of 71 unigenes involved in lignin biosynthesis was verified by RNA-seq. This study is the first to report the transcriptome profiles of the developing xylem and its protoplasts of coniferous trees, which provide a new perspective and valuable resource for tracking transcriptional regulatory events in wood formation of Masson pine.