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 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.

Keywords: CELLULOSE SYNTHASE A4; NAC transcription factor; VNS element; expression QTL; tracheary element-regulating cis-element.

FIGURE 1

(A) Histograms of relative expression of CESA4/7/8 within the Bay-0 × Sha recombinant inbred line population (RIL). (B) Genetic mapping identified a CESA4 expression quantitative trait locus coincident with the physical position of the CESA4 locus on chromosome five. (C) Relative expression of CESA4/7/8 from RIL parents carrying Bay-0 or Sha allele. Lines carrying Bay-0 allele had significantly higher expression of CesA4, while no differences were observed between RILS for the expression of CESA7 or CESA8. ^∗P < 0.01.

FIGURE 2

Sequence analysis of promoter of CESA4 for Col-0, Bay-0, and Sha accessions. Sequencing and alignment of CESA4 promoter contains only one single nucleotide polymorphism between CESA4^Bay and CESA4^Col and sixteen SNPs between CESA4^Sha and CESA4^Col. Colored lines and letter above the sequence indicate different fragments tested for affinity with NAC proteins. Boxes indicate sequence polymorphisms.

FIGURE 3

Yeast one-hybrid and protein–DNA interactions within CESA4 promoter. (A) Nine overlapping fragments (A–I) of CESA4^Bay and CESA4^Sha promoters were screened by yeast one-hybrid with CESA4^Col promoter interacting proteins. SND1 and NST2 interacted with two CESA4^Bay fragments (C and E) but only a single CESA4^Sha fragment (E). (B) A single nucleotide polymorphism disrupts the second position of a perfect TERE motif in CESA4^Sha fragment C. (C) Electrophoretic mobility shift assay for interactions between CESA4^Bay and CESA4^Sha promoters and NST2 and SND1. This polymorphic fragment CESA4^Bay fragment C failed to interact with SND1 and NST2.

FIGURE 4

Normalized gene expression results from qPCR between heterogeneous inbred families (HIFs) segregating for parental Bay-0 and Sha alleles in CESA4 promoter region demonstrated differential binding. Transcript abundance in developing stems containing CESA4^Bay was twofold greater than that of CESA4^Sha in both pairs of NILs. ^∗P < 0.01.

FIGURE 5

(A) Phloroglucinol stained cross sections with representative focus areas for measurements. (B) Cell wall thickness variation between Bay-0 and Sha NILS. CESA4^Bay NIL93 was the only line to exhibit slightly thicker cell walls, while the other three lines were similar. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means with surrounding gray box indicating 95% confidence interval.

FIGURE 6

Polarized light microscopy on stem cross sections revealed no differences in crystallinity. Representative images of (A) Sha 93, (B) Bay-0 93, (C) Sha 350, and (D) Bay-0 350.

FIGURE 7

Average probability matrix motif for the VND, NST, and SND secondary cell wall NAC protein DAP-seq showing the probability of a nucleotide at each position.