Leaf senescence is a highly regulated developmental process that is coordinated by several factors. Many senescence-associated genes (SAGs) have been identified, but their roles during senescence remain unclear. A sweet potato (Ipomoea batatas) SAG, named SPA15, whose function was unknown, was identified previously. To understand the role of SPA15 in leaf senescence further, the orthologue of SPA15 in Arabidopsis thaliana was identified and characterized, and it was named ARABIDOPSIS A-FIFTEEN (AAF). AAF was expressed in early senescent leaves and in tissues with highly proliferative activities. AAF was localized to the chloroplasts by transient expression in Arabidopsis mesophyll protoplasts. Overexpression of AAF (AAF-OX) in Arabidopsis promoted, but the T-DNA insertion mutant (aaf-KO), delayed age-dependent leaf senescence. Furthermore, stress-induced leaf senescence caused by continuous darkness was enhanced in AAF-OX but suppressed in aaf-KO. Transcriptome analysis of expression profiles revealed up-regulated genes related to pathogen defence, senescence, and oxidative stress in 3-week-old AAF-OX plants. Indeed, elevated levels of reactive oxygen species (ROS) and enhanced sensitivity to oxidative and dark stress were apparent in AAF-OX but reduced in aaf-KO. ETHYLENE INSENSITIVE2 (EIN2) was required for the dark- and ROS-induced senescence phenotypes in AAF-OX and the induction of AAF expression by treatment with the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid. The results indicate the functional role of AAF is an involvement in redox homeostasis to regulate leaf senescence mediated by age and stress factors during Arabidopsis development.

These authors contributed equally to this work.Keywords: Arabidopsis, leaf senescence, oxidative stress, ethylene, EIN2, reactive oxygen species ARABIDOPSIS A-FIFTEEN (AAF) encodes a plastid protein and was originally identified as a SENESCENCE-ASSOCIATED GENE. Previously, we found that overexpression of AAF (AAF-OX) in Arabidopsis led to accumulated reactive oxygen species and promoted leaf senescence induced by oxidative stress, which was suppressed by a null mutant, ein2-5, in ethylene response pathway. Whether AAF function is involved in ethylene biosynthesis and/or the response pathway remained unknown. Here we show that neither overexpression (AAF-OX) nor a null mutant (aaf-KO) of AAF generates a higher level of ethylene than the wild type and display a typical triple-response phenotype in etiolated seedlings treated with 1-aminocyclopropane-1-carboxylic acid (ACC). Nevertheless, ein2-5 suppresses the phenotypes of early flowering and age-dependent leaf senescence in AAF-OX plants. We reveal that a functional ethylene response is essential for AAF function in leaf senescence and floral induction, but AAF is unlikely a regulatory component integral to the ethylene pathway.Ethylene is a gaseous plant hormone with versatile functions regulating seed germination, seedling development, fruit ripening, fiber elongation, petal abscission, leaf senescence and response to stress.1 Ethylene at 1 ppm applied to dark-grown (etiolated) seedlings produces a phenotype termed the triple response, with inhibition of elongation in hypocotyls and roots, radical swelling of hypocotyls and an exaggerated curvature of the apical hook. 2Most ethylene-related mutants were identified by the tripleresponse phenotype, and genetic analyses were subsequently used to propose a relatively linear ethylene signaling pathway.