Heteroexpression of Osa-miR319b improved switchgrass biomass yield and feedstock quality by repression of PvPCF5

Abstract

Background: Switchgrass (Panicum virgatum L.), a C 4 perennial grass, has been recognized as one of the most potentially important lignocellulose biofuel crops. MicroRNA319 (miR319) plays a key role in plant development, abiotic resistance, and cell wall biosynthesis by repressing expression of its target TCP genes. We hypothesized miR319-TCP pathway could play important roles in switchgrass feedstock characteristics for biofuel production, and produced switchgrass transgenic plants overexpressing miR319 (by ectopic expressing Osa-MIR319b gene), blocking miR319 (by overexpressing a target mimicry of miR319/MIM319) and repression of miR319 target gene PvPCF5. Plant phenotype, biomass yield, and feedstock quality of transgenic plants were analyzed.Results: Overexpression of miR319 in switchgrass promoted leaf elongation and expansion of transgenic plants, increased plant height, stem diameter, and resulted in a significant increase in plant biomass yield. Transgenic plants overexpressing of miR319 reduced lignin content, showed significantly higher enzymatic hydrolysis efficiency compared to the wild type plant. However, opposite results were observed in the MIM319 plants. Furthermore, suppression of miR319 target gene PvPCF5 activity also reduced lignin content, increased lignin monomer S/G ratio and the proportion of β-O-4 linkages, while significantly improving the sugar production per plant. Quantitative real-time (qRT-PCR) analysis indicated that expression of PvMYB58/63B and PvHCT with predicted TCP binding sites in their promoter regions was negatively regulated by miR319-PvPCF5 module. Conclusions:MiR319-PvPCF5 module plays positive roles in regulating biomass yield and quality of switchgrass. It can be utilized as a candidate molecular tool in regulating biomass yield and feedstock quality. The finding could also be transferred to other grasses for forage quality improvement through genetic manipulation.

MiR396‐GRF module associates with switchgrass biomass yield and feedstock quality

“…It should be noted that the expression of miR396 was not tightly associated with how much its targeted PvGRFs were repressed in the tissues analysed, further suggesting a possible complicated relationship between miR396 and PvGRFs in switchgrass. Similarly, a loose negative effect of miR319 on the expression of its targeted genes in different tissues has previously been reported in switchgrass and rice (Liu et al, 2020;Yang et al, 2013).…”

“…The individual rPvGRFs were subcloned into pZh01, respectively. To repress the function of PvGRFs, the 3’ ends of PvGRFs were linked to a sequence encoding the SRDX domain and cloned into pZh01 (Liu et al, 2020 ; Wu et al, 2016 ). All of these genes were under the control of the CaMV 35S promoter.…”

“…To visualize lignin content in switchgrass stem, switchgrass hand‐cut cross sections of the middle of 1NE3 stem were stained with 1% phloroglucinol/HCl for 1 min. Digital images were captured under a light microscope (Nikon, Shizuoka, Japan C‐DSS230) (Liu et al, 2020 ). The middle parts of 1NE3 stems were fixed in 2.5% glutaraldehyde.…”

“…The enzymatic hydrolysis efficiency (%) was determined by the ratio of the summation of glucose and xylose released by enzymatic hydrolysis to the amount of sugar (glucose, xylose) present in the cells wall composition. The solubility sugar MiR396-GRF mediates biomass and feedstock quality 1533 yield = the per plant biomass (g) 9 CWR content 9 cell wall carbohydrate yield (g/g CWR) 9 enzymatic hydrolysis efficiency (Liu et al, 2020). Three biological replicates (with five technical repeats each) were used in the experiments.…”

Profiling of MicroRNAs Involved in Mepiquat Chloride-Mediated Inhibition of Internode Elongation in Cotton (Gossypium hirsutum L.) Seedlings

“…miR319 has been demonstrated to promote cell proliferation during leaf and petal development ( Nag et al, 2009 ; Liu et al, 2020 ); however, the effect of miR319 on stem elongation varies with plant species. Overexpression of sha-miR319d in tomato reduced plant height ( Shi et al, 2019 ), whereas overexpression of osa-miR319b in switchgrass promoted stem elongation and increased plant height ( Liu et al, 2020 ). Knockdown of miR319 using artificial miRNA target mimics (MIM) completely suppressed stem elongation in Arabidopsis ( Todesco et al, 2010 ).…”