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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: 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]

Alkaligrass (Puccinellia tenuiflora) is a monocotyledonous halophyte pasture, which has strong tolerance to saline-alkali, drought, and chilling stresses. We have reported a high-quality chromosome-level genome and stress-responsive proteomic results in P. tenuiflora. However, the gene/protein function investigations are still lacking, due to the absent of genetic transformation system in P. tenuiflora. In this study, we established a higher efficient Agrobacterium-mediated transformation for P. tenuiflora using calluses induced from seeds. Agrobacterium strain EHA105 harbors pANIC 6B vectors that contain GUS reporter gene and Hyg gene for screening. Ten mg·L−1 hygromycin was used for selecting transgenic calluses. The optimized condition of vacuum for 10 min, ultrasonication for 10 min, and then vacuum for 10 min was used for improvement of conversion efficiency. Besides, 300 mg·L−1 timentin was the optimum antibiotics in transformation. PCR amplification exhibited that GUS gene has been successfully integrated into the chromosome of P. tenuiflora. Histochemical GUS staining and qRT-PCR analysis indicated that GUS gene has stably expressed with ß-glucuronidase activity in transgene seedlings. All these demonstrated that we have successfully established an Agrobacterium-mediated transformation system of P. tenuiflora, which provides a good platform for further gene function analysis and lays a solid foundation for molecular breeding. [ABSTRACT FROM AUTHOR]

Changes in atmospheric CO2 concentration have played a central role in algal and plant adaptation and evolution. The commercially important red algal genus, Pyropia (Bangiales) appears to have responded to inorganic carbon (Ci) availability by evolving alternating heteromorphic generations that occupy distinct habitats. The leafy gametophyte inhabits the intertidal zone that undergoes frequent emersion, whereas the sporophyte conchocelis bores into mollusk shells. Here, we analyze a high-quality genome assembly of Pyropia yezoensis to elucidate the interplay between Ci availability and life cycle evolution. We find horizontal gene transfers from bacteria and expansion of gene families (e.g. carbonic anhydrase, anti-oxidative related genes), many of which show gametophyte-specific expression or significant up-regulation in gametophyte in response to dehydration. In conchocelis, the release of HCO3- from shell promoted by carbonic anhydrase provides a source of Ci. This hypothesis is supported by the incorporation of 13C isotope by conchocelis when co-cultured with 13C-labeled CaCO3. The nori producing seaweed Pyropia yezoensis has heteromorphic generations that occupy distinct habitats. Here, via genome assembly, transcriptome analysis, and 13 C isotope labeling, the authors show the interplay between inorganic carbon availability and life cycle evolution in the intertidal environment. [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]

Key message: Two 2-phenylethanol biosynthetic pathways were constructed into Arabidopsis ; 2-phenylethanol biosynthesis led to reduced rate of lignin biosynthesis and increased cellulose-to-glucose conversion in the transgenic plants. Abstract: Lignin is the second most abundant biopolymer on the planet with importance for various agro-industrial activities. The presence of lignin in cell walls, however, impedes biofuel production from lignocellulosic biomass. The phenylpropanoid pathway is responsible for the biosynthesis of lignin and other phenolic metabolites such as 2-phenylethanol. As one of the most used fragrance chemicals, 2-phenylethanol is synthesized in plants from l-phenylalanine which is the first specific intermediate towards lignin biosynthesis. Thus, it is interesting to prove the concept that the phenylpropanoid pathway can be modulated for reduction of lignin as well as production of natural value-added compounds. Here we conferred two 2-phenylethanol biosynthetic pathways constructed from plants and Saccharomyces cerevisiae into Arabidopsis. As anticipated, 2-phenylethanol was accumulated in transgenic plants. Moreover, the transformants showed 12-14 % reduction in lignin content and 9-13 % increase in cellulose content. Consequently, the glucose yield from cell wall hydrolysis was increased from 37.4 % in wild type to 49.9-52.1 % in transgenic plants with hot water pretreatment. The transgenic plants had normal development and even enhanced growth relative to the wild type. Our results indicate that the shunt of l-phenylalanine flux to the artificially constructed 2-phenylethanol biosynthetic pathway most likely reduced the rate of lignin biosynthesis in Arabidopsis. [ABSTRACT FROM AUTHOR]

Key message: MLNAC5 functions as a stress-responsive NAC transcription factor gene and enhances drought and cold stress tolerance in transgenic Arabidopsis via the ABA-dependent signaling pathway. Abstract: NAC transcription factors (TFs) play crucial roles in plant responses to abiotic stress. Miscanthus lutarioriparius is one of Miscanthus species native to East Asia. It has attracted much attention as a bioenergy crop because of its superior biomass productivity as well as wide adaptability to different environments. However, the functions of stress-related NAC TFs remain to be elucidated in M. lutarioriparius. In this study, a detailed functional characterization of MlNAC5 was carried out. MlNAC5 was a member of ATAF subfamily and it showed the highest sequence identity to ATAF1. Subcellular localization of MlNAC5-YFP fusion protein in tobacco leaves indicated that MlNAC5 is a nuclear protein. Transactivation assay in yeast cells demonstrated that MlNAC5 functions as a transcription activator and its activation domain is located in the C-terminus. Overexpression of MlNAC5 in Arabidopsis had impacts on plant development including dwarfism, leaf senescence, leaf morphology, and late flowering under normal growth conditions. Furthermore, MlNAC5 overexpression lines in Arabidopsis exhibited hypersensitivity to abscisic acid (ABA) and NaCl. Moreover, overexpression of MlNAC5 in Arabidopsis significantly enhanced drought and cold tolerance by transcriptionally regulating some stress-responsive marker genes. Collectively, our results indicated that MlNAC5 functions as an important regulator during the process of plant development and responses to salinity, drought and cold stresses. [ABSTRACT FROM AUTHOR]

Two full-length cDNAs encoding NAM, ATAF, and CUC (NAC)-family transcription factors (TFs) were isolated from two different cultivars of Panicum virgatum L. (switchgrass) and named PvNAC1 and PvNAC2. Phylogenetic analysis of PvNAC1 and PvNAC2 grouped them with NAC proteins involved in senescence in annual plant species. Transcript profiling revealed that both PvNAC1 and PvNAC2 are induced during leaf senescence. Expression of a PvNAC1-green fluorescent protein (GFP) fusion in plant cells revealed a nuclear location of the protein, consistent with a role in transcriptional regulation. Expression of PvNAC1 in an Arabidopsis nap stay-green mutant suppressed its senescence defect. Expression of PvNAC1 in wild-type Arabidopsis triggered early leaf senescence and remobilization. Transcriptome analysis implicated leaf protein degradation and nitrogen recycling enzymes in NAC-dependent seed protein increase in Arabidopsis. Overexpression of pvNAC2 in switchgrass resulted in increased aboveground biomass associated with increased transcript levels of key nitrogen metabolism genes in leaves and nitrate and ammonium transporter genes in roots. The results indicate that NAC TFs play conserved roles in leaf senescence in the plant kingdom not only in annual monocots and dicots but also in perennial plants such as switchgrass. PvNAC1 and PvNAC2 hold promise for improving nutrient use efficiency in switchgrass through genetic manipulation. [ABSTRACT FROM AUTHOR]

Key message: The 13 MlNAC genes could respond to various abiotic stresses, suggesting their crucial roles in stress response. Overexpression of MlNAC2 in Arabidopsis led to improved drought tolerance. Abstract: NAC (NAM, ATAF1/2 and CUC2) proteins are plant-specific transcription factors that play crucial roles in plant development, growth and stress responses. In this study, 13 stress-responsive NAC genes were identified from Miscanthus lutarioriparius. Full-length cDNA sequences were obtained for 11 MlNAC genes, which were phylogenetically classified into six subfamilies. Sequence alignment revealed the highly conserved NAC domain in the N-terminus of these MlNACs, while the C-terminus was highly divergent. We performed quantitative real-time RT-PCR to examine the expression profiles of MlNAC genes in different tissues including root, rhizome, mature stem, young stem, leaf and sheath. The 13 MlNAC genes displayed distinct tissue-specific patterns in six tissues examined. To gain further insight into their roles in response to abiotic stresses, expressions of MlNAC genes were analyzed under different stresses and hormone treatments including salt, drought, cold, wounding, abscisic acid, Methyl jasmonate and salicylic acid. The 13 MlNAC genes could respond to at least five stress treatments, and over 100-fold variations in transcript levels of MlNAC1, MlNAC2, MlNAC4, and MlNAC12 were observed in salt, drought and MeJA treatments, which indicated that MlNACs play crucial roles in stress response. Crosstalk among various abiotic stress and hormone responses was also discussed based on the expression of MlNAC genes. Overexpression of MlNAC2 in Arabidopsis (Col-0) led to improved drought tolerance. The water loss rate was significantly lower, and the recovery rate after a 60-min dehydration stress treatment was significantly higher in the MlNAC2 overexpression lines than the control. [ABSTRACT FROM AUTHOR]