title: Sweet potato SPAP1 is a typical aspartic protease and participates in ethephon-mediated leaf senescence.
abstract: Plant aspartic proteases are generally divided into three categories: typical, nucellin-like, and atypical aspartic proteases based on their gene and protein structures. In this report, a full-length cDNA SPAP1 was cloned from sweet potato leaves, which contained 1515 nucleotides (504 amino acids) and exhibited high amino acid sequence identity (ca. 51-72%) with plant typical aspartic proteases, including tomato LeAspP, potato StAsp, and wheat WAP2. SPAP1 also contained conserved DTG and DSG amino acid residues within its catalytic domain and plant specific insert (PSI) at the C-terminus. The cDNA corresponding to the mature protein (starting from the 66th to 311th amino acid residues) without PSI domain was constructed with pET30a expression vector for fusion protein and antibody production. RT-PCR and protein blot hybridization showed that SPAP1 expression level was the highest in L3 mature leaves, then gradually declined until L5 completely yellow leaves. Ethephon, an ethylene-releasing compound, also enhanced SPAP1 expression at the time much earlier than the onset of leaf senescence. Exogenous application of SPAP1 fusion protein promoted ethephon-induced leaf senescence, which could be abolished by pre-treatment of SPAP1 fusion protein with (a) 95 °C for 5 min, (b) aspartic protease inhibitor pepstatin A, and (c) anti-SPAP1 antibody, respectively. Exogenous SPAP1 fusion protein, whereas, did not significantly affect leaf senescence under dark. These data conclude that sweet potato SPAP1 is a functional typical aspartic protease and participates in ethephon-mediated leaf senescence. The SPAP1-promoted leaf senescence and its activity are likely not associated with the PSI domain. Interaction of ethephon-inducible components for effective SPAP1 promotion on leaf senescence is also suggested.
authors.affiliation: Department of Biological Sciences, National Sun Yat-sen University, 804 Kaohsiung, Taiwan. Electronic address: hjchen@faculty.nsysu.edu.tw.
authors.forename: Hsien-Jung
authors.initials: HJ
authors.lastname: Chen
authors.affiliation: Department of Biological Sciences, National Sun Yat-sen University, 804 Kaohsiung, Taiwan.
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Huang
authors.affiliation: Graduate Institute of Chinese Pharmaceutical Sciences, China Medical University, 404 Taichung, Taiwan.
authors.forename: Guan-Jhong
authors.initials: GJ
authors.lastname: Huang
authors.affiliation: Graduate Institute of Chinese Pharmaceutical Sciences, China Medical University, 404 Taichung, Taiwan.
authors.forename: Shyh-Shyun
authors.initials: SS
authors.lastname: Huang
authors.affiliation: Department of Biotechnology, Fooyin University, 831 Kaohsiung, Taiwan.
authors.forename: Te-Jin
authors.initials: TJ
authors.lastname: Chow
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Nankang, 115 Taipei, Taiwan. Electronic address: boyhlin@gate.sinica.edu.tw.
authors.forename: Yaw-Huei
authors.initials: YH
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003624:Darkness; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006358:Hot Temperature; D027723:Ipomoea batatas / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010802:Phylogeny; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D013997:Time Factors
chemical_list: D009943:Organophosphorus Compounds; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D057055:Aspartic Acid Proteases; C005073:ethephon
keywords: Aspartic protease; Ethephon; Leaf senescence; Pepstatin A; Sweet potato