Molecular cloning and characterization of a brassinosteriod biosynthesis-related gene PtoCYP90D1 from Populus tomentosa

Brassinosteroids (BRs), one of the major classes of phytohormones are essential for various processes of plant growth, development, and adaptations to biotic and abiotic stresses. In Arabidopsis, AtCYP90D1 acts as a bifunctional cytochrome P450 monooxygenase, catalyzing C-23 hydroxylation in the brassinolide biosynthetic pathway. The present study reports the functional characterizations of PtoCYP90D1, one of the AtCYP90D1 homologous genes from Populus tomentosa. The qRT-PCR analysis showed that PtoCYP90D1 was highly expressed in roots and old leaves. Overexpression of PtoCYP90D1 (PtoCYP90D1-OE) in poplar promoted growth and biomass yield, as well as increased xylem area and cell layers. Transgenic plants exhibited a significant increase in plant height and stem diameter as compared to the wild type. In contrast, the CRISPR/Cas9-generated mutation of PtoCYP90D1 (PtoCYP90D1-KO) resulted in significantly decreased biomass production in transgenic plants. Further studies revealed that cell wall components increased significantly in PtoCYP90D1-OE lines but not in PtoCYP90D1-KO lines, as compared to wild-type plants. Overall, the findings indicate a positive role of PtoCYP90D1 in improving growth rate and elevating biomass production in poplar, which will have positive implications for its versatile industrial or agricultural applications.