BdGT43B2 functions in xylan biosynthesis and is essential for seedling survival in Brachypodium distachyon

Abstract

Xylan is the predominant hemicellulose in the primary cell walls of grasses, but its synthesis and interactions with other wall polysaccharides are complex and incompletely understood. To probe xylan biosynthesis, we generated CRISPR/Cas9 knockout and amiRNA knockdown lines of BdGT43B2, an ortholog of the wheat TaGT43-4 xylan synthase scaffolding protein in the IRX14 clade, in Brachypodium distachyon.Knockout of BdGT43B2 caused stunting and premature death in Brachypodium seedlings. Immunofluorescence labeling of xylans was greatly reduced in homozygous knockout BdGT43B2 mutants, whereas cellulose labeling was unchanged or slightly increased. Biochemical analysis showed reductions in digestible xylan in knockout mutant walls, and cell size was smaller in knockout leaves. BdGT43B2 knockdown plants appeared morphologically normal as adults, but showed slight reductions in seedling growth and small decreases in xylose content in isolated cell walls. Immunofluorescence labeling of xylan and cellulose staining was both reduced inBdGT43B2 knockdown plants. Together, these data indicate that BdGT43B2 functions in the synthesis of a form of xylan that is required for seedling growth and survival in Brachypodium distachyon. K E Y W O R D S artificial microRNA (amiRNA), Brachypodium distachyon, CRISPR/Cas9, glycosyl transferase, polysaccharide analysis by carbohydrate electrophoresis, xylanBrachypodium distacyhon is a useful model species for the analysis of grass cell walls because it is diploid, has a small stature and an 8-10 week seed-to-seed life cycle, has a sequenced genome, and is relatively easy to transform (Vogel et al., 2010). The composition of its cell walls has been characterized (Rancour, Marita, & Hatfield, 2012). Phylogenetic analysis of Brachypodium distachyon GT43 members shows that there are eight members in Clade A and two members in Clade B (Whitehead et al., 2018). RNAi knockdown of one these GT43 genes, Bradi5g24290, results in modestly reduced

Identification and expression analysis of the glycosyltransferase GT43 family members in bamboo reveal their potential function in xylan biosynthesis during rapid growth

“…PeGT43–15/PeGT43–5 and PeGT43–4 were presumed to be involved in glucuronide xylan synthesis as they were homologous to OsGT43A (LOC Os05g03174) and OsGT43E (LOC Os05g48600), respectively [ 23 ]. PeGT43–17/PeGT43–2 were homologs of BdGT43B2 (Bradi5g24290), which is involved in arabinoxylan synthesis [ 21 ].

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“…The biomass of the RNA interference lines showed higher digestibility, decreased xylan, arabinose and ferulic acid, and increased coumaric acid, supporting a causative role of BdGT43A in Brachypodium distachyon [ 20 ]. The BdGT43B2 knockout lines showed that the immunofluorescence labeling of xylan was greatly reduced, whereas cellulose labeling was unchanged or slightly increased [ 21 ]. Overexpression of OsIRX9 , OsIRX9L and OsIRX14 in Arabidopsis mutants irx9 and irx14 resulted in xylosyltransferase (XylT) activity in the stems that was greater thandouble that in wild type plants, and the stem strength increased to 124% above that of wild-type plants, suggesting that OsIRX9 / OsIRX9L , and OsIRX14 , have similar functions to Arabidopsis IRX9 and IRX14 , respectively [ 22 ].…”

BdFAR4, a root‐specific fatty acyl‐coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon

“…(2019) and Petrik et al . (2020), to generate a loss‐of‐function knockout into the BdFAR4 gene in B. distachyon genotype Bd21. In the resulting T 1 transgenic knockout lines, the amounts of C20:0 and C22:0 fatty alcohols were significantly reduced while C16:0 and C18:0 fatty alcohols showed no significant changes (Figure 5), thus corroborating that BdFAR4 preferred C20:0 and C22:0 acyl‐CoA substrates in B. distachyon .…”

“…Cells expressing BdFAR4 accumulated mainly C20:0 fatty alcohol and a small amount of C22:0 fatty alcohol (Figure 3(b)), strongly suggesting that BdFAR4 possesses distinct substrate specificity for C20:0 and C22:0 acyl-CoAs. The substrate specificity of BdFAR4 was further tested by CRISPR/Cas9mediated gene editing, following the pioneering work of Qin et al (2019) and Petrik et al (2020), to generate a lossof-function knockout into the BdFAR4 gene in B. distachyon genotype Bd21. In the resulting T 1 transgenic knockout lines, the amounts of C20:0 and C22:0 fatty alcohols were significantly reduced while C16:0 and C18:0 fatty alcohols showed no significant changes (Figure 5), thus corroborating that BdFAR4 preferred C20:0 and C22:0 acyl-CoA substrates in B. distachyon.…”

RNAi suppression of xylan synthase genes in wheat starchy endosperm

“…IRX10 appears to be responsible for catalysis but IRX9 and IRX14 are both required for correct localisation of the complex in the Golgi [ 10 ]. The IRX14 component may play a key role in assembly of the complex in wheat [ 13 ] and its knock-out reduces xylan in primary cell walls of Brachypodium with profound consequences for growth [ 14 ].…”