Abstract
Chlorophyll (Chl) degradation occurs naturally during leaf maturation and senescence, and can be induced by stresses, both processes involving the regulation of plant hormones. The objective of this study was to determine the functional roles and hormonal regulation of a gene encoding pheophytin pheophorbide hydrolyase (PPH) that catabolizes Chl degradation during leaf senescence in perennial grass species. A PPH gene, LpPPH, was cloned from perennial ryegrass (Lolium perenne L.). LpPPH was localized in the chloroplast. Overexpressing LpPPH accelerated Chl degradation in wild tobacco, and rescued the stay-green phenotype of the Arabidopsis pph null mutant. The expression level of LpPPH was positively related to the extent of leaf senescence. Exogenous application of abscisic acid (ABA) and ethephon (an ethylene-releasing agent) accelerated the decline in Chl content in leaves of perennial ryegrass, whereas cytokinin (CK) and aminoethoxyvinylglycine (AVG; an ethylene biosynthesis inhibitor) treatments suppressed leaf senescence, corresponding to the up- or down-regulation of LpPPH expression. The promoters of five orthologous PPH genes were predicted to share conserved cis-elements potentially recognized by transcription factors in the ABA and CK pathways. Taken together, the results suggested that LpPPH-mediated Chl breakdown could be regulated positively by ABA and ethylene, and negatively by CK, and LpPPH could be a direct downstream target gene of transcription factors in the ABA and CK signaling pathways.
Highlights
Leaf senescence is a highly regulated natural process during the life of a leaf, which involves degradation of various macromolecules, such as chlorophyll (Chl) and proteins (Breeze et al, 2011; Fukao et al, 2012)
Based on the phylogenetic relationship of the pheophorbide hydrolyase (PPH) orthologs, the synonymous and nonsynonymous nucleotide substitution rates were further compared between the closely related pairs, demonstrating that all PPH genes were under purifying selection (Ka/Ks
Transient expression of LpPPH fused to a green fluorescent protein (GFP) tag demonstrated that LpPPH was localized in the chloroplast, and the lack of a transmembrane domain in LpPPH further suggested that this protein may be mainly localized in the chloroplast stroma, which is consistent with the finding for AtPPH (Schelbert et al, 2009)
Summary
Leaf senescence is a highly regulated natural process during the life of a leaf, which involves degradation of various macromolecules, such as chlorophyll (Chl) and proteins (Breeze et al, 2011; Fukao et al, 2012). PPH, named NYC3 in rice (Oryza sativa) or CRN1 in Arabidopsis, is one of the key CCEs, which catalyzes conversion of pheophytin a to. The PPH transcript level is positively correlated with the extent of leaf senescence during the natural leaf maturation process (Schelbert et al, 2009) or is induced by stresses, such as dark or shade (Wei et al, 2011; Liu and Guo, 2013). The regulatory mechanisms of PPH expression associated with natural or stress-induced leaf senescence are not well understood
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