CRISPR/Cas9-mediated knockout of PvTCP19/22 enhances tiller number and biomass yield in switchgrass
Ying Sun, Ruijuan Yang, Min Liu, Yuchen Liu, Xiaoyi Yuan, Longhao Chen, Siyi Zhao, Xiaochun Qin, Chuanen Zhou, Chunxiang Fu, Xiaoshan Wang, Wenwen Liu
Abstract
Controlling tiller number is a promising strategy to improve biomass yield for switchgrass, which is an important lignocellulosic energy crop used for biofuel production. Therefore, the functions of PvTCP19 and its paralog PvTCP22, two members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family, were investigated for increasing tiller number and biomass yield. These data revealed that PvTCP19 localizes in the nucleus and is highly expressed in leaf organ. Using a Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR Associated 9 (CRISPR/Cas9) system to knock out PvTCP19 , both PvTCP19 and PvTCP22 were edited efficiently in most of the positive transgenic lines (50/53) and the tetra-allelic homozygous editing efficiency was 80 % (40/50). The knockout mutants demonstrated significant improvements: tiller number increased, dry biomass yield rose by 92 %, and soluble sugar content increased by 120 % compared with those of control plants. Additionally, the mutants exhibited superior nitrogen and phosphorus use efficiencies. Transcriptome analysis revealed that PvTCP19/22 likely repress the expression of DWARF AND LOW-TILLERING ( PvDLT ) and activate the transcription of DORMANCY-ASSOCIATED ( PvDRM ) genes, which are key regulators of tiller outgrowth and bud dormancy. These findings establish PvTCP19/22 as potential pivotal inhibitors of tillering downstream of the strigolactone (SL) signaling pathway. Overall, this study identified PvTCP19/22 as valuable targets for CRISPR/Cas9-based genetic enhancement of switchgrass, paving the way for increased biomass yield and sugar production. ● Forty PvTCP19/22 knockout mutants were generated by using CRISPR/Cas9 system. ● PvTCP19/22 knockout mutants improved tiller number, biomass and sugar yields. ● Mutation of PvTCP19/22 affected nitrogen and phosphorus utilization.