In silico and functional characterization of the cellulose synthase 8 gene promoter of Eucalyptus trees

Abstract

Cellulose is a highly abundant biopolymer which forms the basis of several industrial applications including paper and textile products. Cellulose is deposited into the plant cell wall by a large membrane bound protein complex which is comprised of different cellulose synthase (CesA) subunits. Plants maintain several different CesA genes which have specific expression patterns depending on the cell wall type, tissue type, developmental stage and environment of the cell. While CesA genes and proteins have been the focus of many studies, the upstream regulatory regions which govern their complex expression patterns have remained largely unexplored. The aim of this study was to use the previously identified CesA promoter regulatory modules and putative cis-elements to identify conserved cis-element clusters in the Eucalyptus CesA and the transcription factors which interact with the regulatory regions of the EgrCesA8 promoter. The promoters of six cellulose synthase genes (CesA1, CesA3, CesA6, CesA4, CesA7 and CesA8) were isolated from 13 Eucalyptus species of different sections in the Symphyomyrtus subgenus of Eucalyptus. Species-level nucleotide diversity was calculated for the promoters of each gene. The promoters each contained a highly conserved region at the transcriptional start site (TSS), possibly marking the core promoter. The Eucalyptus promoters appeared to be TATA-less and cis-elements which resembled alternate core plant promoter elements were found clustered close to the TSS. Other localised regions of low species-level nucleotide diversity were identified upstream of the TSS in each promoter set and could indicate the location of cis-regulatory modules (CRMs). The conserved promoter regions and cis-element maps of the SCW-associated EgrCesA8 promoter were used to direct promoter truncation for reporter gene analysis in Arabidopsis, Eucalyptus and Populus. Comparative analysis of the cis-element maps and GUS expression data revealed that two main conserved regions of the CesA8 promoter harboured clusters of cis-elements and modulated GUS expression. The CT(11)-microsatellite in the conserved TSS-associated cis-element cluster produced strong non-specific GUS expression in Eucalyptus and Arabidopsis when appended to the 5’UTR which suggests a role in the EgrCesA8 core promoter. Further upstream in the promoter a second conserved promoter region coincided with a cluster of SCW-associated cis-elements and caused a loss of expression in leaf vasculature, suggesting a role for this CRM in modulating tissue-specific expression of EgrCesA8. The conserved EgrCesA8 promoter regions which coincided with cis-element clusters and GUS regulatory modules were used as baits in a yeast-1-hybrid screen against the a panel of 14 Eucalyptus SCW transcription factors. EgrMYB31 (AtMYB46) and EgrZincFinger-A were found to interact with the EgrCesA8 5’UTR. The interaction of these transcription factors with the 5’UTR were blocked by the presence of the CT(11)-microsatellite and could explain the loss of tissue-specific expression. EgrNAC170 (AtSND2) directly interacted with the CRM containing a cluster of SCW-related cis-elements. A dual Y1H assay revealed that EgrKNAT7 in the presence of EgrMYB80 (AtMYB52) or EgrMYB87 (AtMYB54) could also interact with the ErgCesA8 CRM. Together the results of the dissertation indicate that EgrCesA8 regulation is modulated by different protein-DNA and protein-protein interactions acting at highly conserved regions of the promoter.