Chitinase-Like (CTL) and Cellulose Synthase (CESA) Gene Expression in Gelatinous-Type Cellulosic Walls of Flax (Linum usitatissimum L.) Bast Fibers

Abstract

Plant chitinases (EC 3.2.1.14) and chitinase-like (CTL) proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs), belonging to glycoside hydrolase family 19 (GH19). Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21) that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA) family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8) was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2) that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls.

RNA-seq Transcriptome Response of Flax (Linum usitatissimum L.) to the Pathogenic Fungus Fusarium oxysporum f. sp. lini

“…As markers of the response to fungal infection, we used quantitative PCR to measure transcript abundance of four Glycosyl Hydrolase family 19 (GH19) chitinase genes of flax (Mokshina et al, 2014). These chitinases were selected based on homology to A. thaliana genes that had been previously characterized as responsive to pathogens or other related processes (Supplementary Figure S3).…”

“…Asterisks denote significant differences between the inoculation treatment and the respective water control (one-tailed t -test, p < 0.05). Names of chitinases correspond to those referenced in Mokshina et al (2014).…”

“…Four chitinases (chitinase-like CTL2, 4, 10, and 11) from Glycosyl Hydrolase family 19 (GH19) were selected to test the response to the pathogen (Mokshina et al, 2014). To select reference genes, we tested primers from six genes for stability upon our treatments from a list of 13 genes previously published as normalizers in qRT-PCR experiments in flax (Huis et al, 2010): Elongation factor 1-α (EF1A), Eukaryotic translation initiation factor 3E (ETIF3E), Eukaryotic translation factor 5A (ETIF5A), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Ubiquitin (UBI), and Ubiquitin extension protein (UBI2; Supplementary Table S1).…”

Studying Secondary Growth and Bast Fiber Development: The Hemp Hypocotyl Peeks behind the Wall

“…It was highly expressed in H9, H15, and H20. In flax, three isoforms of chitinase-like genes, most similar to chitinases of class IV, were highly expressed in the phloem fibers (Mokshina et al, 2014), where they may be involved in the development of the G-layer. A signal peptide was found in the deduced hemp chitinase protein sequence, a finding corroborating its action in the cell wall.…”

Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

“…We separated the bast fibres in a mortar, by repeatedly pressing the cortex with a pestle (Supplementary Materials Figure S1), as described in [11,12].…”