Secondary Wall Regulating NACs Differentially Bind at the Promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL

Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating CESA4(Sha) and CESA4(Bay). In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of CESA4(Bay), CESA4(Sha), and the reference genome CESA4(Col). The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4(Sha) promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element.