Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection

Abstract

The intrusive growth, a type of plant cell elongation occurring in the depths of plant tissues, is characterized by the invasion of a growing cell between its neighbours due to a higher rate of elongation. In order to reveal the largely unknown molecular mechanisms of intrusive growth, we isolated primary flax phloem fibers specifically at the stage of intrusive growth by laser microdissection. The comparison of the RNA-Seq data from several flax stem parts enabled the characterization of those processes occurring specifically during the fiber intrusive elongation. The revealed molecular players are summarized as those involved in the supply of assimilates and support of turgor pressure, cell wall enlargement and modification, regulation by transcription factors and hormones, and responses to abiotic stress factors. The data obtained in this study provide a solid basis for developing approaches to manipulate fiber intrusive elongation, which is of importance both for plant biology and the yield of fiber crops.

The Toolbox for Fiber Flax Breeding: A Pipeline From Gene Expression to Fiber Quality

“…For example, genes for putative transcription factor LusBZIP5 (orthologous to AT3G49760) and transcriptional activator LusASML2 [orthologous to AT3G12890 encoding activator of spomin::LUC2 that regulates the expression of at least a subset of sugar-inducible genes (Masaki et al, 2005)], and auxin-responsive GH3 family protein Lus10003598 (orthologous to AT5G54510 and DFL1) were among the selected genes. Also, a number of O-Glycosyl hydrolases belonging to family 17 (four genes) that can be involved in callose metabolism were selected, because of their specific activation during intrusive growth (Gorshkova et al, 2018b).…”

“…The average length of individual fibers obtained from mature plants ranged from 14.7 ± 2.0 mm in L. angustifolium to 21.5 ± 1.2 mm in Drakkar. The proportion of fibers longer SAM, shoot apical meristem (Zhang and Deyholos, 2016); cPAR, cortical parenchyma; iFIB, intrusively growing fibers (Gorshkova et al, 2018b); tFIBa, fibers depositing tertiary cell wall, earlier stage (Gorshkov et al, 2019); tFIBb, fibers depositing tertiary cell wall, later stage (Gorshkov et al, 2017); sXYLa, xylem part, the same segment of the stem as for tFIBa (Gorshkov et al, 2019); sXYLb, xylem part, the same segment of the stem as for tFIBb (Mokshina et al, 2020); LEAF, upper leaves from individual plants under normal conditions (Dmitriev et al, 2016); ROOT, root tips, average for control samples (Dmitriev et al, 2017). When calculating the FC, to avoid division by zero, +1 was added to all the TGR values.…”

“…Transcript abundance of the selected genes was measured by RT-qPCR using the reverse transcription method in stem portions containing fibers at the intrusive growth stage and in isolated fibers at the advanced stage of fiber specialization. We did not isolate fiber cells at the intrusive growth stage and extracted RNA from whole stem parts because previously we have analyzed the transcriptome of fibers isolated by laser microdissection at this stage of development and revealed genes especially abounded in fiber cell at the intrusive growth stage as compared to other tissues (Gorshkova et al, 2018b); only such genes were analyzed in the current study to characterize expression at fiber elongation (Supplementary Table S1).…”

“…The current paper presents a more targeted functional approach based on the analysis of gene expression during specific stages of bast fiber development. The whole pipeline includes the identification of temporal and spatial localization of fiber intrusive elongation and TCW deposition in the stems of developing flax plants (Gorshkova et al, 2003(Gorshkova et al, , 2004, transcriptomic analysis of fibers at certain developmental stage (Gorshkov et al, 2017;Gorshkova et al, 2018b), identification of genes with fiber-specific expression by determination of tau score (Mokshina et al, 2020), and the relation of fiber quality parameters to the expression of genes important at key stages of bast fiber development (current paper). The identification of genes contributing to fiber intrusive elongation and TCW deposition is an important step in this pipeline because it reduces information noise in the analysis and determines the effectiveness of the subsequent steps.…”

“…These approaches considerably advanced the earlier important studies to large-scale molecular characterization of the flax genome and transcriptome (Day et al, 2005;Roach and Deyholos, 2007;Fenart et al, 2010). Further RNA-Seq experiments revealed those genes that are highly upregulated during intrusive growth (Gorshkova et al, 2018b) and TCW deposition (Gorshkov et al, 2017), and described genes with fiber-specific expression that are barely transcribed in other tissues (Mokshina et al, 2020). These studies opened the possibility of relating the structure and expression of genes important for fiber development to fiber quality.…”