Transcriptomic changes during cell wall biogenesis and modifications during xylogenesis in stems and pioneer roots of Populus trichocarpa

We studied the changes that occur in gene transcription and the biosynthesis of cell-wall-related compounds during xylogenesis in Populus trichocarpa pioneer roots and stems. Transcriptional profiling was conducted with the use of microarrays to identify genes involved in xylem formation. Pioneer roots were examined in segments corresponding to their developmental stage: apical meristem (PR1), primary development (PR2), and secondary development (PR3). Comparative segments were used to analyze xylogenesis in stems: apical meristem with primary development (PS1), secondary development (PS2), and isolated secondary xylem (PS3). Results indicated that only approximately 10% of the genes that were identified to be differentially expressed during xylogenesis were common to both pioneer roots and stems. Despite the dissimilarity in gene expression, however, many fundamental mechanisms were similar, e.g. the pattern of expression of genes involved in the biosynthesis of cell wall proteins, polysaccharides and lignins. While hemicellulose degradation was typical for stems, possibly due to the intensive level of cell wall lignification. Our study is the first to conduct a comprehensive analysis, at the structural and molecular level, of xylogenesis, and clearly reveals the great complexity of molecular mechanisms underlying cell wall formation and modification during xylogenesis. All experiments were performed on seed-grown Populus trichocarpa (Torr. & Gray) growing in rhizotrons filled with natural soil with shoots extending from the top into the air. Waterlogging was avoided by installing a drainage hole in the bottom of each rhizotron that permitted soil aeration and drainage of excess water. At sampling, pioneer roots were divided into the following segments corresponding to their developmental stage: 0-2cm – root tips with apical meristem (PR1); 4-6cm – primary development (PR2); and 13-16cm – secondary development (PR3). Similarly, stems were also sampled based on developmental stages: 0-2cm – apical meristem with primary development (PS1); 20-25cm – secondary development (PS2) and 40-45cm – isolated secondary xylem (PS3). ). Root tips were treated as a negative control for the process of xylogenesis, while isolated secondary xylem served as a positive control. Total RNA was extracted from three biological replicates of each developmental stage of roots and stems using an RNeasy Plant Mini kit (Qiagen, USA). RNA quantity and quality were assessed using a NanoDrop1000 (Thermo Fisher Scientific Inc., USA). cRNA synthesis and microarray hybridization to an Affymetrix GeneChip Poplar Genome Array (A-AFFY-131) were performed according to the provided Affymetrix protocol.