Expression of Exogenous GFP-CesA6 in Tobacco Enhances Cell Wall Biosynthesis and Biomass Production

SIMPLE SUMMARY: Cellulose is synthesized at the plasma membrane by an enzymatic complex constituted by different cellulose synthase (CesA) proteins. The overexpression of CesA genes has been assessed for increasing cellulose biosynthesis and plant biomass. In this study, we analyzed transgenic tobacco plants (F(3)1 line), stably expressing the Arabidopsis CesA6 fused to GFP, for possible variations in the cellulose biosynthesis. We found that F(3)1 plants were bigger than the wild-type (wt), showing significant increases of stem height, root length, and leaf area. They bloomed about 3 weeks earlier and yielded more flowers and seeds than wt. In the F(3)1 leaves, the expression of the exogenous GFP-CesA6 prompted the overexpression of all CesAs involved in the synthesis of primary cell wall cellulose and of other proteins responsible for plant cell wall building and remodeling. Instead, secondary cell wall CesAs were not affected. In the F(3)1 stem, showing a 3.3-fold increase of the secondary xylem thickness, both primary and secondary CesAs expression was differentially modulated. Significantly, the amounts of cellulose and matrix polysaccharides increased in the transformed seedlings. The results evidence the potentiality to overexpress primary CesAs in tobacco for biomass production increase. ABSTRACT: Improved cellulose biosynthesis and plant biomass represent important economic targets for several biotechnological applications including bioenergy and biofuel production. The attempts to increase the biosynthesis of cellulose by overexpressing CesAs proteins, components of the cellulose synthase complex, has not always produced consistent results. Analyses of morphological and molecular data and of the chemical composition of cell walls showed that tobacco plants (F(3)1 line), stably expressing the Arabidopsis CesA6 fused to GFP, exhibits a “giant” phenotype with no apparent other morphological aberrations. In the F(3)1 line, all evaluated growth parameters, such as stem and root length, leaf size, and lignified secondary xylem, were significantly higher than in wt. Furthermore, F(3)1 line exhibited increased flower and seed number, and an advance of about 20 days in the anthesis. In the leaves of F(3)1 seedlings, the expression of primary CesAs (NtCesA1, NtCesA3, and NtCesA6) was enhanced, as well as of proteins involved in the biosynthesis of non-cellulosic polysaccharides (xyloglucans and galacturonans, NtXyl4, NtGal10), cell wall remodeling (NtExp11 and XTHs), and cell expansion (NtPIP1.1 and NtPIP2.7). While in leaves the expression level of all secondary cell wall CesAs (NtCesA4, NtCesA7, and NtCesA8) did not change significantly, both primary and secondary CesAs were differentially expressed in the stem. The amount of cellulose and matrix polysaccharides significantly increased in the F(3)1 seedlings with no differences in pectin and hemicellulose glycosyl composition. Our results highlight the potentiality to overexpress primary CesAs in tobacco plants to enhance cellulose synthesis and biomass production.