SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

Abstract

Wood is an essential renewable raw material for industrial products and energy. However, knowledge of the genetic regulation of wood formation is limited. We developed a genome-wide high-throughput system for the discovery and validation of specific transcription factor (TF)-directed hierarchical gene regulatory networks (hGRNs) in wood formation. This system depends on a new robust procedure for isolation and transfection of Populus trichocarpa stem differentiating xylem protoplasts. We overexpressed Secondary Wall-Associated NAC Domain 1s (Ptr-SND1-B1), a TF gene affecting wood formation, in these protoplasts and identified differentially expressed genes by RNA sequencing. Direct Ptr-SND1-B1-DNA interactions were then inferred by integration of timecourse RNA sequencing data and top-down Graphical Gaussian Modeling-based algorithms. These Ptr-SND1-B1-DNA interactions were verified to function in differentiating xylem by anti-PtrSND1-B1 antibody-based chromatin immunoprecipitation (97% accuracy) and in stable transgenic P. trichocarpa (90% accuracy). In this way, we established a Ptr-SND1-B1-directed quantitative hGRN involving 76 direct targets, including eight TF and 61 enzyme-coding genes previously unidentified as targets. The network can be extended to the third layer from the second-layer TFs by computation or by overexpression of a second-layer TF to identify a new group of direct targets (third layer). This approach would allow the sequential establishment, one two-layered hGRN at a time, of all layers involved in a more comprehensive hGRN. Our approach may be particularly useful to study hGRNs in complex processes in plant species resistant to stable genetic transformation and where mutants are unavailable.

Genetic architecture of wood properties based on association analysis and co‐expression networks in white spruce

“…PgNAC‐7 and these three MYBs were proposed by Duval et al . (2014) to form a regulatory network similar to the SND1 network defined in Arabidopsis (Zhong et al ., 2006) that is conserved in poplar (Lin et al ., 2013; Wang et al ., 2014). The regulation of lignin biosynthesis by members of the conserved SND1 transcriptional network has been well documented in conifer trees (Patzlaff et al ., 2003a,b; Bomal et al ., 2008, 2014).…”

Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O -methyltransferase activity in poplar monolignol biosynthesis

“…PtrAldOMT2 has the most abundant monolignol biosynthetic gene transcript in stem-differentiating xylem (SDX) of P. trichocarpa and is the third highest transcribed gene in the whole SDX transcriptome (7). PtrAldOMT2 protein abundance is also the highest of all monolignol biosynthetic enzymes and accounts for 5.9% of the SDX proteome (22).…”

“…An expression vector containing a cauliflower mosaic virus 35S promoter and the PtrAldOMT2 gene fused at its C terminus with an sGFP coding sequence was overexpressed in P. trichocarpa SDX protoplasts, using our protocol (7,29). The transformed SDX protoplasts exhibited uniform green fluorescence consistent with the expression of a cytosolic enzyme (Fig.…”

“…Lignin is formed by the free-radical polymerization of monolignols, products of the highly regulated monolignol biosynthetic pathway. Monolignol biosynthesis is regulated by transcriptional activation and suppression by wood-associated NAC (NAM, ATAF1/2, and CUC2) domain factors and alternative splicing (5)(6)(7). At the metabolic level, monolignol biosynthesis is regulated by protein interactions (8,9) and a complex network of feed-forward and -back enzyme inhibitions (10)(11)(12)(13).…”

AspWood: High-Spatial-Resolution Transcriptome Profiles Reveal Uncharacterized Modularity of Wood Formation in Populus tremula

“…We explored whether 110 direct targets of the four SND1-clade genes were coexpression neighbors in our network, including 76 genes identified as targets using transactivation assays and chromatin immunoprecipitation (ChIP) in protoplast-derived xylary tissue with a low degree of differentiation (Lin et al, 2013) and 34 genes (hereafter, non-ChIP targets) identified as transactivation targets based on different lines of experimental evidence (Ye and Zhong, 2015) (see Supplemental Data Set 9 for all target genes). Seventy-six of these 110 putative direct targets of (Potri.001G248700 and Potri.016G112100), LAC11 (Potri.004G156400), and LAC17 (Potri.001G184300, Potri.001G401300, and Potri.006G087100).…”