Data_Sheet_1_Secondary Wall Regulating NACs Differentially Bind at the Promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL.docx

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 CESA4Sha and CESA4Bay. 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 CESA4Bay, CESA4Sha, and the reference genome CESA4Col. The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4Sha 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.

拟南芥（Arabidopsis thaliana）的纤维素合酶A亚基4/7/8（CELLULOSE SYNTHASE A4/7/8，CESA4/7/8）是次生细胞壁纤维素合酶复合物的三个非冗余亚基。这些基因的转录本丰度在不同基因型间存在差异，研究人员在Bay-0与Shahdara生态型的重组群体中鉴定到了表达数量性状位点（expression quantitative trait loci，eQTL）。通过两个独立近等基因系（near isogenic lines，NILs）间的遗传定位与CESAs转录水平分析，证实CESA4的表达变化在该区间内发生分离。我们对其启动子进行测序，鉴定出16个可区分CESA4Sha与CESA4Bay的多态性位点。为明确其中哪些单核苷酸多态性（single nucleotide polymorphism，SNP）可能介导该eQTL，我们针对CESA4Bay、CESA4Sha以及参考基因组CESA4Col的启动子片段，开展了转录因子蛋白亲和性筛选实验。结果显示，次生细胞壁增厚激活蛋白NAC次生细胞壁增厚促进因子2（NAC SECONDARY WALL THICKENING PROMOTING FACTOR2，NST2）与NST3，与携带导管细胞调控顺式元件（tracheary element-regulating cis-element，TERE）多态性的CESA4Sha启动子的结合能力出现下降。尽管携带该TERE多态性的近等基因系的CESA4表达存在显著差异，但其纤维素结晶度与细胞壁厚度并无显著区别。上述结果表明，TERE多态性可导致转录因子结合能力与CESA4表达的差异；但拟南芥能够耐受这种转录调控变异，且不会影响植株的结构特征，这为解析与细胞壁生物合成及调控相关的基因调控弹性提供了新的研究视角。此外，我们还利用已公开的DNA亲和纯化测序数据，确定了次生壁NAC类蛋白的核心结合基序C(G/T)TNNNNNNNA(A/C)G，该基序被称为VNS元件。