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Summary 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. [ABSTRACT FROM AUTHOR]

Background: Salt stress seriously impacts the plantation and productivity of global alfalfa (Medicago sativa). Previously, we genetically defined the M. sativa DROUGHT-INDUCED UNKNOWN PROTEIN 1 (MsDIUP1) as a drought tolerance gene of alfalfa. However, whether it is associated with salt tolerance needs to be explored. Results: In this study, we re-classified MsDIUP1 as a putative novel late embryogenesis abundant protein, and found its promoter activity was strongly induced by salt and ABA treatment. Heterologous overexpression of MsDIUP1 in Arabidopsis led to enhanced salt tolerance and ABA insensitivity, with a higher fresh weight, lateral root number, primary root length, and survival rate compared to the wild-type (WT) plants. Similarly, homologous overexpression of MsDIUP1 also enhanced the salt tolerance of alfalfa over-expression (OE) plants, accompanied by reduced oxidative damage, higher K+/Na+ ratio, increased osmoprotectant accumulation, and improved photosynthetic capacity relative to the WT plants. In reverse, disruption of MsDIUP1 expression caused a salt-hypersensitive phenotype of alfalfa RNA interference (RNAi) plants, with higher oxidative damage, less osmoprotectant accumulation, and lower photosynthetic pigment content than that of the WT. These physiological discoveries were in accordance with differential transcript profiles of the relevant genes associated with stress signaling, antioxidant defense, and osmotic adjustment in the salt-treated alfalfa WT, OE, and RNAi lines. Furthermore, we found that alfalfa OE plants showed no apparent alteration in their forage quality compared with the WT plants. Conclusions: Overall, these results reveal that MsDIUP1 improves plant salt tolerance via regulating stress signaling, antioxidant defense, ion homeostasis, osmotic adjustment, and photosynthesis. [ABSTRACT FROM AUTHOR]

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38 % more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust ( Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2 years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11-16 % less lignin, 33-40 % lower S/G (syringyl-to-guaiacyl) ratios, and 15-42 % higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. These results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late‐acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual‐purpose fodder and biofuel crop.We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild‐type and miR156 overexpressing (miR156OE) switchgrass. Co‐expression analysis, quantitative real‐time polymerase chain reaction (qRT‐PCR), transient dual luciferase assay, and chromatin immunoprecipitation‐qPCR were all used to determine the activation of PvLBO by miR156‐targeted Squamosa Promoter Binding Protein‐like 2 (PvSPL2) in regulating tillering of switchgrass.PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2.We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156‐SPL module in switchgrass. [ABSTRACT FROM AUTHOR]

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs. [ABSTRACT FROM AUTHOR]

Summary: How bacteria adjust gene expression to cope with variable environments remains open to question. Here, we investigated the way global gene expression changes in E. coli correlated with the metabolism of seven carbon substrates chosen to trigger a large panel of metabolic pathways. Coarse‐grained analysis of gene co‐expression identified a novel regulation pattern: we established that the gene expression trend following immediately the reduction of growth rate (GR) was correlated to its initial expression level. Subsequent fine‐grained analysis of co‐expression demonstrated that the Crp regulator, coupled with a change in GR, governed the response of most GR‐dependent genes. By contrast, the Cra, Mlc and Fur regulators governed the expression of genes responding to non‐glycolytic substrates, glycolytic substrates or phosphotransferase system transported sugars following an idiosyncratic way. This work allowed us to expand additional genes in the panel of gene complement regulated by each regulator and to elucidate the regulatory functions of each regulator comprehensively. Interestingly, the bulk of genes controlled by Cra and Mlc were, respectively, co‐regulated by Crp‐ or GR‐related effect and our quantitative analysis showed that each factor took turns to work as the primary one or contributed equally depending on the conditions. [ABSTRACT FROM AUTHOR]

The GRAS gene family is a large plant-specific family of transcription factors that are involved in diverse processes during plant development. Medicago truncatula is an ideal model plant for genetic research in legumes, and specifically for studying nodulation, which is crucial for nitrogen fixation. In this study, 59 MtGRAS genes were identified and classified into eight distinct subgroups based on phylogenetic relationships. Motifs located in the C-termini were conserved across the subgroups, while motifs in the N-termini were subfamily specific. Gene duplication was the main evolutionary force for MtGRAS expansion, especially proliferation of the LISCL subgroup. Seventeen duplicated genes showed strong effects of purifying selection and diverse expression patterns, highlighting their functional importance and diversification after duplication. Thirty MtGRAS genes, including NSP1 and NSP2, were preferentially expressed in nodules, indicating possible roles in the process of nodulation. A transcriptome study, combined with gene expression analysis under different stress conditions, suggested potential functions of MtGRAS genes in various biological pathways and stress responses. Taken together, these comprehensive analyses provide basic information for understanding the potential functions of GRAS genes, and will facilitate further discovery of MtGRAS gene functions. [ABSTRACT FROM AUTHOR]

Transgenic Panicum virgatum L. silencing ( KD) or overexpressing ( OE) specific genes or a small RNA ( GAUT4- KD, mi RNA156- OE, MYB4- OE, COMT- KD and FPGS- KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4- KD, MYB4- OE, COMT- KD and FPGS- KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies. [ABSTRACT FROM AUTHOR]