Summary: Salinity‐induced accumulation of certain microRNAs accompanied by gaseous phytohormone ethylene production has been recognized as a mechanism of plant salt tolerance. MicroRNA319 (miR319) has been characterized as an important player in abiotic stress resistance in some C3 plants, such as Arabidopsis thaliana and rice. However, its role in the dedicated biomass plant switchgrass (Panicum virgatum L.), a C4 plant, has not been reported. Here, we show crosstalk between miR319 and ethylene (ET) for increasing salt tolerance. By overexpressing Osa‐MIR319b and a target mimicry form of miR319 (MIM319), we showed that miR319 positively regulated ET synthesis and salt tolerance in switchgrass. By experimental treatments, we demonstrated that ET‐mediated salt tolerance in switchgrass was dose‐dependent, and miR319 regulated the switchgrass salt response by fine‐tuning ET synthesis. Further experiments showed that the repression of a miR319 target, PvPCF5, in switchgrass also led to enhanced ethylene accumulation and salt tolerance in transgenic plants. Genome‐wide transcriptome analysis demonstrated that overexpression of miR319 (OE‐miR319) down‐regulated the expression of key genes in the methionine (Met) cycle but promoted the expression of genes in ethylene synthesis. The results enrich our understanding of the synergistic effects of the miR319‐PvPCF5 module and ethylene synthesis in the salt tolerance of switchgrass, a C4 bioenergy plant.

Perennial ryegrass (Lolium perenne) is one of the most widely used forage and turf grasses in the world due to its desirable agronomic qualities. However, as a cool-season perennial grass species, high temperature is a major factor limiting its performance in warmer and transition regions. In this study, a de novo transcriptome was generated using a cDNA library constructed from perennial ryegrass leaves subjected to short-term heat stress treatment. Then the expression profiling and identification of perennial ryegrass heat response genes by digital gene expression (DGE) analyses was performed. The goal of this work was to produce expression profiles of high temperature stress responsive genes in perennial ryegrass leaves and further identify the potentially important candidate genes with altered levels of transcript, such as those genes involved in transcriptional regulation, antioxidant responses, plant hormones & signal transduction, and cellular metabolism. The de novo assembly of perennial ryegrass transcriptome in this study obtained more total and annotated unigenes compared to previously published ones. Many DEGs identified were genes that are known to respond to heat stress in plants, including HSFs, HSPs, and antioxidant related genes. In the meanwhile, we also identified 4 gene candidates mainly involved in C4 carbon fixation, and 1 TOR gene. Their exact roles in plant heat stress response need to dissect further. This study would be important by providing the gene resources for improving heat stress tolerance in both perennial ryegrass and other cool-season perennial grass plants.

The microRNA393 (miR393) family is one of the conserved miRNA families in the plant kingdom. MiR393 was reported to regulate rice tillering and abiotic stress resistance positively through an auxin signaling pathway. However, little is known about the function of miR393 in switchgrass (Panicum virgatum L.), an important bioenergy C₄ grass plant. We tested the expression level of miR393 and its four putative target genes (PvAFB1, PvAFB2, PvAFB3 and PvTIR1) in switchgrass, and found that these genes all responded to cold stress and exogenous 1-naphthaleneacetic acid (NAA) treatment. To investigate the function of miR393 in switchgrass, we enhanced miR393 expression by introducing an Osa-miR393a gene into switchgrass. The results showed that cold tolerance of the transgenic T₀ and T₁ generation plants was highly improved. Cold tolerance-related genes PvCOR47, PvICE1 and PvRAV1 were negatively regulated by exogenous NAA, and the expression of these genes was significantly higher in transgenic plants than in wild-type plants. The transgenic T₁ seedlings were more tolerant to exogenous NAA treatment, accumulating less H₂O₂ after cold treatments. It was also observed that the miR393/target module regulates cold tolerance responses in Arabidopsis. In addition, transgenic plants overexpressing miR393 had significantly more tillers and higher biomass yield per plant in greenhouse and field tests. Forage quality analyses revealed that the soluble sugar contents of transgenic plants were increased markedly. Overall, the results suggested that overexpression of miR393 improved cold tolerance and tillering of switchgrass through regulation of auxin signaling transduction.

Summary: Improving plant biomass yield and/or feedstock quality for highly efficient lignocellulose conversion has been the main research focus in genetic modification of switchgrass (Panicum virgatum L.), a dedicated model plant for biofuel production. Here, we proved that overexpression of miR396 (OE‐miR396) leads to reduced plant height and lignin content mainly by reducing G‐lignin monomer content. We identified nineteen PvGRFs in switchgrass and proved thirteen of them were cleaved by miR396. MiR396‐targeted PvGRF1, PvGRF9 and PvGRF3 showed significantly higher expression in stem. By separately overexpressing rPvGRF1, 3 and 9, in which synonymous mutations abolished the miR396 target sites, and suppression of PvGRF1/3/9 activity via PvGRF1/3/9‐SRDX overexpression in switchgrass, we confirmed PvGRF1 and PvGRF9 played positive roles in improving plant height and G‐lignin content. Overexpression of PvGRF9 was sufficient to complement the defective phenotype of OE‐miR396 plants. MiR396‐PvGRF9 modulates these traits partly by interfering GA and auxin biosynthesis and signalling transduction and cell wall lignin, glucose and xylan biosynthesis pathways. Moreover, by enzymatic hydrolysis analyses, we found that overexpression of rPvGRF9 significantly enhanced per plant sugar yield. Our results suggest that PvGRF9 can be utilized as a candidate molecular tool in modifying plant biomass yield and feedstock quality.