a. Objectives (a) Identification and characterization of the cotton fiber FRKs; (b) Generating transgenic cotton plants overproducing either substrate inhibited tomato FRK or tomato FRK without substrate inhibition; (c) Generating transgenic cotton plants with RNAi suppression of fiber expressed FRKs; (d) Generating Arabidopsis plants that over express FRK1, FRK2, or both genes, as additional means to assess the contribution of FRK to cellulose synthesis and biomass production. b. Background to the topic: Cellulose synthesis and fiber elongation are dependent on sugar metabolism. Previous results suggested that FRKs (fructokinase enzymes that specifically phosphorylate fructose) are major players in sugar metabolism and cellulose synthesis. We therefore hypothesized that increasing fructose phosphorylation may enhance fiber elongation and cellulose synthesis in cotton plants. Accordinlgy, the objectives of this research were: c. Major conclusions and achievements: Two cotton FRKs expressed in fibers, GhFRK2 and GhFRK3, were cloned and characterized. We found that GhFRK2 enzyme is located in the cytosol and GhFRK3 is located within plastids. Both enzymes enable growth on fructose (but not on glucose) of hexose kinase deficient yeast strain, confirming the fructokinase activity of the cloned genes. RNAi constructs with each gene were prepared and sent to the US collaborator to generate cotton plants with RNAi suppression of these genes. To examine the effect of FRKs using Arabidopsis plants we generated transgenic plants expressing either LeFRK1 or LeFRK2 at high level. No visible phenotype has been observed. Yet, plants expressing both genes simultaneously are being created and will be tested. To test our hypothesis that increasing fructose phosphorylation may enhance fiber cellulose synthesis, we generated twenty independent transgenic cotton plant lines overexpressing Lycopersicon (Le) FRK1. Transgene expression was high in leaves and moderate in developing fiber, but enhanced FRK activity in fibers was inconsistent between experiments. Some lines exhibited a 9-11% enhancement of fiber length or strength, but only one line tested had consistent improvement in fiber strength that correlated with elevated FRK activity in the fibers. However, in one experiment, seed cotton mass was improved in all transgenic lines and correlated with enhanced FRK activity in fibers. When greenhouse plants were subjected to severe drought during flowering and boll development, no genotypic differences in fiber quality were noted. Seed cotton mass was improved for two transgenic lines but did not correlate with fiber FRK activity. We conclude that LeFRK1 over-expression in fibers has only a small effect on fiber quality, and any positive effects depend on optimum conditions. The improvement in productivity for greenhouse plants may have been due to better structural development of the water-conducting tissue (xylem) of the stem, since stem diameters were larger for some lines and the activity of FRK in the outer xylem greater than observed for wild-type plants. We are testing this idea and developing other transgenic cotton plants to understand the roles of FRK in fiber and xylem development. We see the potential to develop a cotton plant with improved stem strength and productivity under drought for windy, semi-arid regions where cotton is grown. d. Implications, scientific and agricultural: FRKs are probably bottle neck enzymes for biomass and wood synthesis and their increased expression has the potential to enhance wood and biomass production, not only in cotton plants but also in other feed and energy renewable plants.
Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology
