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Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were non-flowering. These two groups of plants produced 58%-101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment., (© 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Summary: MiRNAs have been reported to be the key regulators involving a wide range of biological processes in diverse plant species, but their functions in switchgrass, an important biofuel and forage crop, are largely unknown. Here, we reported the novel function of miR528, which has expanded to four copies in switchgrass, in controlling biomass trait of tillering number and regrowth rate after mowing. Blocking miR528 activity by expressing short tandem target mimic (STTM) increased tiller number and regrowth rate after mowing. The quadruple pvmir528 mutant lines derived from genome editing also showed such improved traits. Degradome and RNA‐seq analysis, combined with in situ hybridization assay revealed that up‐regulation of two miR528 targets coding for Cu/Zn‐SOD enzymes, might be responsible for the improved traits of tillering and regrowth in pvmir528 mutant. Additionally, natural variations in the miR528‐SOD interaction exist in C3 and C4 monocot species, implying the distinct regulatory strength of the miR528‐SOD module during monocot evolution. Overall, our data illuminated a novel role of miR528 in controlling biomass traits and provided a new target for genetic manipulation‐mediated crop improvement. [ABSTRACT FROM AUTHOR]

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop. Modifying plant lignin pathway to enhance saccharification efficiency is often associated with growth penalty. Here, the authors show that overexpression of Pag-miR408 in poplar leads to enhanced saccharification efficiency and growth in both laboratory and field conditions, and laccase genes are the targets of Pag-miR408. [ABSTRACT FROM AUTHOR]

Background: Lignocellulose is a valuable carbon source for the production of biofuels and biochemicals, thus having the potential to substitute fossil resources. Consolidated bio-saccharification (CBS) is a whole-cell-based catalytic technology previously developed to produce fermentable sugars from lignocellulosic agricultural wastes. The deep-sea yeast strain Rhodotorulapaludigena P4R5 can produce extracellular polyol esters of fatty acids (PEFA) and intracellular single-cell oils (SCO) simultaneously. Therefore, the integration of CBS and P4R5 fermentation processes would achieve high-value-added conversion of lignocellulosic biomass. Results: The strain P4R5 could co-utilize glucose and xylose, the main monosaccharides from lignocellulose, and also use fructose and arabinose for PEFA and SCO production at high levels. By regulating the sugar metabolism pathways for different monosaccharides, the strain could produce PEFA with a single type of polyol head. The potential use of PEFA as functional micelles was also determined. Most importantly, when sugar-rich CBS hydrolysates derived from corn stover or corncob residues were used to replace grain-derived pure sugars for P4R5 fermentation, similar PEFA and SCO productions were obtained, indicating the robust conversion of non-food corn plant wastes to high-value-added glycolipids and lipids. Since the produced PEFA could be easily collected from the culture via short-time standing, we further developed a semi-continuous process for PEFA production from corncob residue-derived CBS hydrolysate, and the PEFA titer and productivity were enhanced up to 41.1 g/L and 8.22 g/L/day, respectively. Conclusions: Here, we integrated the CBS process and the P4R5 fermentation for the robust production of high-value-added PEFA and SCO from non-food corn plant wastes. Therefore, this study suggests a feasible way for lignocellulosic agro-waste utilization and the potential application of P4R5 in industrial PEFA production. [ABSTRACT FROM AUTHOR]

MiR156/ SQUAMOSA PROMOTER BINDING-LIKE s (SPL s) module is the key regulatory hub of juvenile-to-adult phase transition as a critical flowering regulator. In this study, a miR156-targeted PvSPL6 was identified and characterized in switchgrass (Panicum virgatum L.), a dual-purpose fodder and biofuel crop. Overexpression of PvSPL6 in switchgrass promoted flowering and reduced internode length, internode number, and plant height, whereas downregulation of PvSPL6 delayed flowering and increased internode length, internode number, and plant height. Protein subcellular localization analysis revealed that PvSPL6 localizes to both the plasma membrane and nucleus. We produced transgenic switchgrass plants that overexpressed a PvSPL6 - GFP fusion gene, and callus were induced from inflorescences of selected PvSPL6-GFPOE transgenic lines. We found that the PvSPL6-GFP fusion protein accumulated mainly in the nucleus in callus and was present in both the plasma membrane and nucleus in regenerating callus. However, during subsequent development, the signal of the PvSPL6-GFP fusion protein was detected only in the nucleus in the roots and leaves of plantlets. In addition, PvSPL6 protein was rapidly transported from the nucleus to the plasma membrane after exogenous GA3 application, and returned from the plasma membrane to nucleus after treated with the GA3 inhibitor (paclobutrazol). Taken together, our results demonstrate that PvSPL6 is not only an important target that can be used to develop improved cultivars of forage and biofuel crops that show delayed flowering and high biomass yields, but also has the potential to regulate plant regeneration in response to GA3. [ABSTRACT FROM AUTHOR]

Gene mutations linked to lignin biosynthesis are responsible for the brown midrib (bm) phenotypes. The bm mutants have a brown-reddish midrib associated with changes in lignin content and composition. Maize bm1 is caused by a mutation of the cinnamyl alcohol dehydrogenase gene ZmCAD2. Here, we generated two new bm1 mutant alleles (bm1-E1 and bm1-E2) through EMS mutagenesis, which contained a single nucleotide mutation (Zm cad2-1 and Zm cad2-2). The corresponding proteins, ZmCAD2-1 and ZmCAD2-2 were modified with Cys103Ser and Gly185Asp, which resulted in no enzymatic activity in vitro. Sequence alignment showed that CAD proteins have high similarity across plants and that Cys103 and Gly185 are conserved in higher plants. The lack of enzymatic activity when Cys103 was replaced for other amino acids indicates that Cys103 is required for its enzyme activity. Enzymatic activity of proteins encoded by CAD genes in bm1-E plants is 23–98% lower than in the wild type, which leads to lower lignin content and different lignin composition. The bm1-E mutants have higher saccharification efficiency in maize and could therefore provide new and promising breeding resources in the future. [ABSTRACT FROM AUTHOR]

Summary: Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate‐expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs. [ABSTRACT FROM AUTHOR]

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Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS. [ABSTRACT FROM AUTHOR]