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

Abstract

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.

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation

“…These include the VNS, W-box, and Dof binding motif. The VNS motif featured heavily in secondary cell wall development, and is a consensus sequence bound by the VND, NST, and SND NAC transcription factors (Olins et al, 2018). Consistent with our observation of rhythmic secondary cell wall genes, this motif was enriched in both diurnal and constant conditions.…”

“…We identified 291 (out of 477 total DAP-seq motifs) significantly enriched motifs, including the core diurnal elements EE, GATA, TBX and G-box (Figs 9, S13). In contrast to ELEMENT, this approach enabled us to identify larger, split elements like VNS (VND/NST/SND: CTTNNNNNNAAG) (Olins et al, 2018), which was enriched in constant conditions and thermocycles, suggesting that it is very likely part of circadian clock regulation. In addition, we observed enrichment for the CACT motif in the morning in LDHC and LDHH.…”

Grass secondary cell walls, Brachypodium distachyon as a model for discovery

“…The same promoters that interact with the secondary cell wall regulating MYB transcription factors often interact with NAC transcription factors, collectively referred to as the SECONDARY WALL NACs (SWN) or the VND, NST/SND, SMB related (VNS) (Ohtani et al, 2011;Zhong et al, 2011). This group of proteins is generally classified into four clades, all binding the similarly named VNS element in in vitro assays (O'Malley et al, 2016;Olins et al, 2018), which is consistent with independently identified TERE and SNBE binding sites for the same proteins (Pyo et al, 2007;Valdivia et al, 2013). In A. thaliana, three of the clades that include the VASCULAR-RELATED NAC-DOMAINs (VNDs), activate cell wall thickening directly and by activating the previously described downstream MYBs (Kubo et al, 2005).…”

Changes in ambient temperature are the prevailing cue in determiningBrachypodium distachyondiurnal gene regulation

“…10) . In contrast to ELEMENT, This approach enabled us to identify larger, split elements like VNS (VND/NST/SND, CTTNNNNNNAAG, (Olins et al , 2018) ), which was enriched in constant conditions and thermocycles, suggesting that it is very likely part of circadian clock regulation. In addition, we observed enrichment for the CACT motif in the morning in LDHC and LDHH.…”

“…Light-dependent photoreceptor activity, in turn, influences the function of core clock proteins. In the dark, ZTL targets TOC1, PRR5, and CCA1 HIKING EXPEDITION (CHE) proteins for degradation, releasing the repression of the morning-expressed genes, CCA1 and LHY (Más et al , 2003;Lee et al , 2018) . Both cryptochrome and phytochrome activities have been implicated in controlling the morning loop function of PRR7 and PRR9 (Farré et al , 2005) .…”