Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass

Abstract

Biofuels developed from biomass crops have the potential to supply a significant portion of our transportation fuel needs. To achieve this potential, however, it will be necessary to develop improved plant germplasm specifically tailored to serve as energy crops. Liquid transportation fuel can be created from the sugars locked inside plant cell walls. Unfortunately, these sugars are inherently resistant to hydrolytic release because they are contained in polysaccharides embedded in lignin. Overcoming this obstacle is a major objective toward developing sustainable bioenergy crop plants. The maize Corngrass1 (Cg1) gene encodes a microRNA that promotes juvenile cell wall identities and morphology. To test the hypothesis that juvenile biomass has superior qualities as a potential biofuel feedstock, the Cg1 gene was transferred into several other plants, including the bioenergy crop Panicum virgatum (switchgrass). Such plants were found to have up to 250% more starch, resulting in higher glucose release from saccharification assays with or without biomass pretreatment. In addition, a complete inhibition of flowering was observed in both greenhouse and field grown plants. These results point to the potential utility of this approach, both for the domestication of new biofuel crops, and for the limitation of transgene flow into native plant species.

Fig. 1.

Overexpression of Cg1 in different plant species. (A) Cg1 overexpression in maize (Left) compared with wild-type sibling (Right). (B) Cg1 overexpression in Arabidopsis (Left) compared with wild-type Columbia (Right). (C) Cg1 overexpression in Brachypodium (Left) compared with wild-type Bd21-3 (Right). Inset shows inflorescence morphology. (D) Cg1 overexpression in switchgrass (Left) compared with wild-type Alamo (Right). All plants are the same age and grown under the same conditions.

Fig. 2.

Histological analysis of Cg1 overexpressors. (A) Plastic sections of juvenile, adult, and Cg1 leaves of maize. (B) Plastic sections of juvenile, adult, and UBI::Cg1 leaves of Brachypodium. (C) Plastic sections of juvenile, adult, and UBI::Cg1 leaves of switchgrass. (D) Epidermal peel of juvenile switchgrass leaf. (E) Epidermal peel of adult switchgrass leaf. (F) Epidermal peel of UBI::Cg1 switchgrass leaf. All plastic sections and epidermal peels were stained with Toluidine Blue. Arrows point to lignified hypodermal sclerenchyma cells.

Fig. 3.

Morphological and molecular analysis of UBI::Cg1 switchgrass plants. (A) Phenotypes of three classes of Cg1 switchgrass transformants. (B) Close up of a severe transformant showing ectopic shoots at each node. (C) Comparison of weakly expressing transformant (Left) and wild type (Right) in the field. Wild-type plant is flowering. (D) Same plants in C, 3 wk postharvest. The transformant displays better recovery. (E) Comparison of first year postharvest dry weights of above-ground biomass of wild type and three classes of transformants. (F) Comparison of tiller numbers of wild type and three classes of transformants. For data in E and F error bars show 1 SE. Sample sizes: WT, n = 4; weak, n = 4; moderate, n = 6; and severe, n = 7. Asterisks indicate significant differences with wild type (Materials and Methods). (G) qPCR analysis of wild type and three classes of transformants using primers corresponding to four different miR156-targeted SPL genes. (H) qPCR analysis using primers corresponding to three different AP1 MADS box genes. (I) qPCR analysis using primers corresponding to a switchgrass glossy15 homolog.

Fig. 4.

Biofuel properties and digestibility of UBI::Cg1 switchgrass. (A) Saccharification assay using dilute base pretreatment of field grown leaves of wild type and three classes of transformants. Asterisks indicate significant differences with wild type. (B) Potassium iodide staining of upper nodes of field grown stems of Cg1 (Left) and wild type (Right). (C) Starch measurements of field grown stems of wild type and two independent transformants of each class. The severe class contained leaf bases. (D) Saccharification assay of stems and leaves of wild type and two independent weak transformants without biomass pretreatment after 24 h of digestion. Method I was done with Accelerase enzyme mixture, and method II was done with the same mixture plus α-amylase and amyloglucosidase. (E) Saccharification assay over 72 h of stems of two field grown wild-type plants and two weakly expressing transformants using method I without pretreatment. (F) Saccharification assay over 72 h of stems of two field grown wild-type plants and two weakly expressing transformants using method II without pretreatment. All transformants in D–F were assayed in duplicate.