Functional genetic analysis of the Eucalyptus grandis cellulose synthase 1 (EgCesA1) gene in Arabidopsis thaliana

Abstract

Cellulose is the most important component of paper and pulp products and increased cellulose biosynthesis in commercially important trees like Eucalyptus spp. could greatly benefit paper and pulp industries. Cellulose in plants occurs mostly in the secondary cell walls together with lignin and hemicellulose. It is biosynthesised by membrane-bound rosette-shaped protein complexes. The rosette complexes are believed to be comprised of six sub-units each containing six cellulose synthase (CESA) proteins. The CESA proteins utilise UDP-glucose to polymerize growing glucan chains that coalesce to form cellulose microfibrils. Three distinct CESA proteins form the rosette complexes during primary cell wall formation and three different CESA proteins form complexes during secondary cell wall deposition. The exact means by which the CESA proteins interact within a rosette complex remains unknown. Elucidating rosette protein complex assembly and better characterization of CESA protein activity is required in order to increase cellulose biosynthesis in commercially important trees. Because of the difficulties to characterise genes and proteins in tree species, Arabidopsis thaliana has been used to study xylogenesis. Although it is a herbaceous weed, Arabidopsis has been shown to undergo secondary growth under certain conditions. A literature study of cellulose biosynthesis in plants has highlighted several scientific questions: Will over-expression of a heterologous secondary cell wall CESA protein in Arabidopsis lead to increased cellulose biosynthesis? What effect will the over-expression of a heterologous protein have on the growth and development of Arabidopsis? Will it have an effect on cell wall chemistry in stem tissues? What effect will expression of the transgene have on endogenous Arabidopsis gene expression? The aim of this M.Sc study was to functionally characterise the Eucalyptus secondary cell wall associated cellulose synthase gene, EgCesA1, in Arabidopsis. The EgCesA1 coding sequence was constitutively expressed in wild-type Arabidopsis plants. Three transgenic lines expressing EgCesA1 was generated. Hypocotyl and inflorescence vascular cell wall phenotypes were compared between transgenic and wild-type plants. Chemical analysis of inflorescence tissues were performed to detect changes in monosaccharide and lignin content of transgenic plants compared to wild-type plants. Transcript levels of EgCesA1 and endogenous Arabidopsis genes involved in cell wall biogenesis were quantified and compared between wild-type and transgenic lines. No significant changes in cell wall morphology could be detected, despite small alterations in inflorescence cell wall chemical composition in transgenic plants. Expression of EgCesA1 did not appear to have a statistically significant effect on endogenous gene transcript levels. It was concluded that constitutive expression of a single transgenic CesA gene is insufficient to increase cellulose biosynthesis. Copyright