Functional Analysis of the PgCesA3 White Spruce Cellulose Synthase Gene Promoter in Secondary Xylem.

Juliana Stival Sena,Denis Lachance,Armand Séguin,John Mackay,Thi Thuy An Nguyen,Isabelle Duval,Don Stewart

doi:10.3389/fpls.2019.00626

Abstract

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 [Picea glauca (Moench) Voss] cellulose synthase PgCesA3 gene and 5′ regulatory elements. Phylogenetic analysis showed that PgCesA1-3 genes grouped with secondary cell wall-associated Arabidopsis cellulose synthase genes, such as AtCesA8, AtCesA4, and AtCesA7. We produced transgenic spruce expressing the GUS reporter gene driven by the PgCesA3 promoter. We observed blue staining in differentiating xylem cells from stem and roots, and in foliar guard cells indicating that PgCesA3 is clearly involved in secondary cell wall biosynthesis. The promoter region sequence of PgCesA3 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 PgCesA3 promoter and different MYB transcription factors, we performed electrophoretic mobility shift assays with MYB recombinant proteins and cis-regulatory elements present in the PgCesA3 promoter. We found that PgMYB12 bound to a canonical AC-I element identified in the Pinus taeda PAL promoter and two AC-I like elements. We hypothesized that the PgMYB12 could regulate PgCesA3 in roots based on previous expression results. This functional study of PgCesA3 sequences and promoter opens the door for future studies on the interaction between PgMYBs and the PgCesA3 regulatory elements.

Highlights

Cellulose is a fundamental constituent of the plant cell wall
The white spruce cellulose synthases grouped in clades with angiosperm cellulose synthases previously linked to secondary cell wall formation including AtCesA8, AtCesA4, and AtCesA7 from Arabidopsis, PtiCesA8, PtiCesA4, and PtiCesA7-A from poplar, and EgCesA1, EgCesA2, and EgCesA3 from Eucalyptus (Holland et al, 2000; Tanaka et al, 2003; Xi et al, 2017)
We analyzed three white spruce cellulose synthases, PgCesA1, PgCesA2, and PgCesA3, that clustered in clades 4, 5, and 6 (Figure 1) with proteins known to be involved in secondary cell wall formation in angiosperms

Summary

Introduction

Cellulose is a fundamental constituent of the plant cell wall. It represents half of the mass of wood and is a crucial resource for the production of pulp, biomaterials and biofuels (Keijsers et al, 2013). Plants and trees accumulate large amounts of crystalline cellulose during the formation of secondary cell walls, which provide mechanical strength and are the main hallmark of secondary xylem and woody tissues. A protein complex formed of cellulose synthase enzymes that are specific to the secondary cell wall is responsible for building the cellulose microfibrils that make up the extracellular matrix (Zhong and Ye, 2015). Three cellulose synthases have been identified that are putatively involved in secondary cell wall formation but no direct functional association has been made to date. The regulation of cellulose synthase genes remains only partially understood in plants and trees in spite of their central role in development

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Conclusion