Characterization and transcriptional regulation of chlorophyll b reductase gene NON-YELLOW COLORING 1 associated with leaf senescence in perennial ryegrass (Lolium perenne L.)

Chlorophyll (Chl) degradation leads to leaf senescence and adversely affects biomass production of forage grasses and aesthetic appearance of turfgrasses. The objectives of this study were to characterize the function of a Chl catabolic gene, NON-YELLOW COLORING 1 (LpNYC1), and to understand its transcriptional regulatory pattern involved in leaf senescence in perennial ryegrass (Lolium perenne L.). The LpNYC1 was initially cloned using the RACE-PCR method. Its encoded protein was localized in chloroplasts and its expression pattern was correlated with the progression of leaf senescence. LpNYC1 shared the same biological function with Arabidopsis NYC1, as overexpression of its encoding gene accelerated chlorophyll degradation in Nicotiana benthamiana and rescued the stay-green phenotype of the Arabidopsis nyc1 null mutant. A yeast one-hybrid cDNA library was prepared from senescent leaves of perennial ryegrass. Using the LpNYC1 promoter as bait, five putative transcription factors upstream of LpNYC1 were pooled out using this method, among which three (LpABI5, LpABF3, and LpEIN3) were orthologous to Arabidopsis transcription factors involved in the ABA and ethylene signaling pathways. The expression of LpNYC1 was highly inducible by ABA and ethephon (ethylene releasing reagent) but was suppressed by treatment with AVG (ethylene biosynthesis inhibitor). Furthermore, LpABI5, LpABF3, LpEIN3 directly activated the expression of LpNYC1 by binding to its promoter. The current result laid the groundwork for future in-depth analysis of the molecular regulation of LpNYC1 and Chl metabolism during leaf senescence in perennial grass species.