| Summary: | <p>Cellulose is an essential structural component of the plant cell wall. Its biosynthesis involves genes encoding cellulose synthase enzymes and a complex transcriptional regulatory network. Three cellulose synthases have been identified in conifers as being potentially involved in secondary cell wall biosynthesis because of their preferential expression in xylem tissues; however, no direct functional association has been made to date. In the present work, we characterized the white spruce [<em>Picea glauca</em> (Moench) Voss] cellulose synthase <em>PgCesA3</em> gene and 5′ regulatory elements. Phylogenetic analysis showed that <em>PgCesA1-3</em> genes grouped with secondary cell wall-associated Arabidopsis cellulose synthase genes, such as <em>AtCesA8, AtCesA4</em>, and <em>AtCesA7</em>. We produced transgenic spruce expressing the GUS reporter gene driven by the <em>PgCesA3</em> promoter. We observed blue staining in differentiating xylem cells from stem and roots, and in foliar guard cells indicating that <em>PgCesA3</em> is clearly involved in secondary cell wall biosynthesis. The promoter region sequence of <em>PgCesA3</em> contained several putative MYB cis-regulatory elements including AC-I like motifs and secondary wall MYB-responsive element (SMRE); however, it lacked SMRE4, 7 and 8 that correspond to the sequences of AC-I, II, and III. Based on these findings and results of previous transient trans-activation assays that identified interactions between the <em>PgCesA3</em> promoter and different MYB transcription factors, we performed electrophoretic mobility shift assays with MYB recombinant proteins and cis-regulatory elements present in the <em>PgCesA3</em> promoter. We found that PgMYB12 bound to a canonical AC-I element identified in the <em>Pinus taeda</em> PAL promoter and two AC-I like elements. We hypothesized that the PgMYB12 could regulate <em>PgCesA3</em> in roots based on previous expression results. This functional study of <em>PgCesA3</em> sequences and promoter opens the door for future studies on the interaction between PgMYBs and the <em>PgCesA3</em> regulatory elements.</p>
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