Please use this identifier to cite or link to this item:
Analysis of gene expression and protein accumulation of an E3 ligase mcCPN1 in ice plant
|引用:||Abas L, Benjamins R, Malenica N, Paciorek T, Wiśniewska J, Moulinier-Anzola JC, Sieberer T, Friml J and Luschnig C (2006) Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol 8: 249-56 Adams P, Nelson D, Yamada S, Chmara W, Jensen RG, Bohnert HJ and Griffiths H (1998) Growth and development of Mesembryanthemum crystallinum (Aizoaceae). New Phytol 138: 171-90 Apse MP, Aharon GS, Snedden WA and Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285: 1256-8 Aravind L and Koonin EV (2000) The U box is a modified RING finger - a common domain in ubiquitination. Curr Biol 10: R132-4 Ardley HC and Robinson PA (2005) E3 ubiquitin ligases. Essays Biochem 41: 15-30 Babst M, Wendland B, Estepa EJ and Emr SD (1998) The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J 17: 2982-93 Bachmair A, Novatchkova M, Potuschak T and Eisenhaber F (2001) Ubiquitylation in plants: a post-genomic look at a post-translational modification. Trends Plant Sci 6: 463-70 Baena-González E, Rolland F, Thevelein JM and Sheen J (2007) A central integrator of transcription networks in plant stress and energy signaling. Nature 448: 938-42 Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G and Reed SI (2001) UBA domains of DNA damage-inducible proteins interact with ubiquitin. Nat Struct Biol 8: 417-22 Bhandal IS and Malik CP (1988) Potassium estimation, uptake, and its role in the physiology and metabolism of flowering plants. Int Rev Cytology 110: 205-254 Blumwald E, Aharom G and Apse MP (2000) Sodium transporter in plant cells. Biochem Biophys Acta 1465: 140-151 Bohner HJ and Cushman J (2000) The ice plant comth: lessons in abiotic stress tolerance. J Plant Growth Regul 19: 334-436 Bohnert HJ, Nelson DE and Jenson RG (1995) Adaptation to environmental stresses. Plant Cell 7: 1099-1111 Chen Y-C (2006) Identification of full-length sequence and analysis of protein domains of mcCPN1 gene in halophyte Mesembryanthemum crystallinum. Bachelor thesis, Department of Life Sciences, National Chung-Hsing University Chen ZJ (2005) Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol 7: 758-65 Chu C, Dai Z, Ku MS and Edwards GE (1990) Induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum by abscisic acid. Plant Physiol 93: 1253-1260 Creutz CE, Tomsig JL, Snyder SL, Gautier MC, Skouri F, Beisson J and Cohen J (1998) The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans. J Biol Chem 273: 1393-402 Cushman JC and Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Plant Biol 3: 117-24 Cushman JC, Michalowski CB and Bohnert HJ (1990) Developmental control of Crassulacean acid metabolism inducibility by salt stress in the common ice plant. Plant Physiol 94: 1137-1142 Damer CK, Bayeva M, Hahn ES, Rivera J and Socec CI (2005) Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium. BMC Cell Biol 6: 46 Damer CK, Bayeva M, Kim PS, Ho LK, Eberhardt ES, Socec CI, Lee JS, Bruce EA, Goldman-Yassen AE and Naliboff LC (2007) Copine A is required for cytokinesis, contractile vacuole function, and development in Dictyostelium. Eukaryot Cell 6: 430-42 Davies JM (1997) Vacuolar energization: pumps, shunts and stress. J Exp Bot 48: 633-641 de Jonge HR, Hogema B and Tilly BC (2000) Protein N-myristoylation: critical role in apoptosis and salt tolerance. Sci STKE 2000: 1-4 Deshaies RJ (1999) SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol 15: 435-67 Dietz KJ, Tavakoli N, Kluge C, Mimura T, Sharma SS, Harris GC, Chardonnens AN and Golldack D (2001) Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J Exp Bot 52: 1969-80 Dong CH, Agarwal M, Zhang Y, Xie Q and Zhu JK (2006) The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci USA 103: 8281-6 Dupré S, Urban-Grimal D and Haguenauer-Tsapis R (2004) Ubiquitin and endocytic internalization in yeast and animal cells. Biochim Biophys Acta 1695: 89-111 Farrás R, Ferrando A, Jásik J, Kleinow T, Okrész L, Tiburcio A, Salchert K, del Pozo C, Schell J and Koncz C (2001) SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase. EMBO J 20: 2742-56 Flowers TJ, Troke RF and Yeo AR (1977) The mechanism of salt tolerance in halophytes. Annu Rev Plant Physiol 28: 89-121 Flowers TJ, Hajibagheri MA and Clipson NCW (1986) Halophytes. Q Rev Biol 61: 313-337 Flowers TJ and Dalmond D (1992) Protein synthesis in halophytes: The influence of potassium, sodium and magnesium in vitro. Plant and Soil 146: 153-161 Freemont PS, Hanson IM and Trowsdale J (1991) A novel cysteine-rich sequence motif. Cell 64: 483-484 Gao YP, Young L, Bonham-Smith P and Gusta LV (1999) Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions. Plant Mol Biol 40: 635-44 Gaxiola RA, Palmgren MG and Schumacher K (2007) Plant proton pumps. FEBS Lett 581: 2204-14 Glickman MH and Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82: 373-428 Greenway H and Munns R (1980) Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant Physio 31: 149-190 Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557-80 Hardie DG and Carling D (1997) The AMP-activated protein kinase--fuel gauge of the mammalian cell? Eur J Biochem 246: 259-73 Hicke L (2001) Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2: 195-201 Hoege C, Pfander B, Moldovan GL, Pyrowolakis G and Jentsch S (2002) RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135-41 Hofmann RM and Pickart CM (2001) In vitro assembly and recognition of Lys-63 polyubiquitin chains. J Biol Chem 276: 27936-43 Hong JK, Choi HW, Hwang IS and Hwang BK (2007) Role of a novel pathogen-induced pepper C3-H-C4 type RING-finger protein gene, CaRFP1, in disease susceptibility and osmotic stress tolerance. Plant Mol Biol 63: 571-88 Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu JK and Harmon AC (2003) The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol 132: 666-80 Jambunathan N and McNellis TW (2003) Regulation of Arabidopsis COPINE 1 gene expression in response to pathogens and abiotic stimuli. Plant Physiol 132: 1370-81 Jambunathan N, Siani JM and McNellis TW (2001) A humidity-sensitive Arabidopsis copine mutant exhibits precocious cell death and increased disease resistance. Plant Cell 13: 2225-40 Jou Y (2007) Characterization of bladder cell-specific mcSKD1 and its interacting protein in halophyte Mesembrayanthemum crystallinum L. Ph D thesis, Department of Life Sciences, National Chung-Hsing University Jou Y, Chiang CP, Jauh GY and Yen HE (2006) Functional characterization of ice plant SKD1, an AAA-type ATPase associated with the endoplasmic reticulum-Golgi network, and its role in adaptation to salt stress. Plant Physiol 141: 135-46 Jou Y, Chou P-H, He M, Hung Y and Yen HE (2004) Tissue-specific expression and functional complementation of a yeast potassium-uptake mutant by a salt-induced ice plant gene mcSKD1. Plant Mol Biol 54: 881-893 Koepp DM, Harper JW and Elledge SJ (1999) How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97: 431-4 Koiwai H, Tagiri A, Katoh S, Katoh E, Ichikawa H, Minami E and Nishizawa Y (2007) RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice. Plant J 51: 92-104 Leyman B, Geelen D, Quintero FJ and Blatt MR (1999) A tobacco syntaxin with a role in hormonal control of guard cell ion channels. Science 283: 537-540 Liu J, Jambunathan N and McNellis TW (2005) Transgenic expression of the von Willebrand A domain of the BONZAI 1/COPINE 1 protein triggers a lesion-mimic phenotype in Arabidopsis. Planta 221: 85-94 Lopez-Molina L, Mongrand S, Kinoshita N and Chua NH (2003) AFP is a novel negative regulator of ABA signaling that promotes ABI5 protein degradation. Genes Dev 17: 410-8 Lovering R, Hanson IM, Borden KL, Martin S, O'Reilly NJ, Evan GI, Rahman D, Pappin DJ, Trowsdale J and Freemont PS (1993) Identification and preliminary characterization of a protein motif related to the zinc-finger. Proc Natl Acad Sci USA 90: 2112-2116 Mass EV (1993) Salinity and citriculture. Tree Physiol 12: 195-216 Mazel A, Leshem Y, Tiwari BS and Levine A (2004) Induction of salt and osmotic stress tolerance by overexpression of an intracellular vesicle trafficking protein AtRab7 (AtRabG3e). Plant Physiol 134: 118-128 Matsushita N, Kitao H, Ishiai M, Nagashima N, Hirano S, Okawa K, Ohta T, Yu DS, McHugh PJ, Hickson ID, Venkitaraman AR, Kurumizaka H and Takata M (2005) A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair. Mol Cell 19: 841-7 Mazzucotelli E, Belloni S, Marone D, De Leonardis A, Guerra D, Di Fonzo N, Cattivelli L and Mastrangelo A (2006) The E3 ubiquitin ligase gene family in plants: regulation by degradation. Curr Genomics 7: 509-22 Miyada CG, Stoltzfus L and Wilcox G (1984) Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression. Proc Natl Acad Sci USA 81: 4120-4. Morris JR and Solomon E (2004) BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair. Hum Mol Genet 13: 807-17 Nalefski EA and Falke JJ (1996) The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 5: 2375-90 Nakayama T, Yaoi T and Kuwajima G (1999) Localization and subcellular distribution of N-copine in mouse brain. J Neurochem 72: 373-9 Nakayama T, Yaoi T, Yasui M and Kuwajima G (1998) N-copine: a novel two C2-domain-containing protein with neuronal activity-regulated expression. FEBS Lett 428: 80-4 Nishizuka Y (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: 661-5 Ogden S, Haggerty D, Stoner CM, Kolodrubetz D and Schleif R (1980) The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. Proc Natl Acad Sci USA 77: 3346-50 Page AM and Hieter P (1999) The anaphase-promoting complex: new subunits and regulators. Annu Rev Biochem 68: 583-609 Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-33 Polge C and Thomas M (2007) SNF1/AMPK/SnRK1 kinases, global regulators at the heart of energy control? Trends Plant Sci 12: 20-8 Qiu L, Joazeiro C, Fang N, Wang HY, Elly C, Altman Y, Fang D, Hunter T and Liu YC (2000) Recognition and ubiquitination of Notch by Itch, a HECT-type E3 ubiquitin ligase. J Biol Chem 275: 35734-7 Rizo J and Sudhof TC (1998) C2-domains, structure and function of a universal Ca2+-binding domain. J Biol Chem 273: 15879-82 Savino M, d''Apolito M, Centra M, van Beerendonk HM, Cleton-Jansen AM, Whitmore SA, Crawford J, Callen DF, Zelante L and Savoia A (1999) Characterization of copine VII, a new member of the copine family, and its exclusion as a candidate in sporadic breast cancers with loss of heterozygosity at 16q24.3. Genomics 61: 219-26 Scheffner M, Huibregtse JM, Vierstra RD and Howley PM (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75: 495-505 Scott A, Chung HY, Gonciarz-Swiatek M, Hill GC, Whitby FG, Gaspar J, Holton JM, Viswanathan R, Ghaffarian S, Hill CP and Sundquist WI (2005) Structural and mechanistic studies of VPS4 proteins. EMBO J 24: 3658-69 Smalle J and Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55: 555-90 Stone SL, Hauksdóttir H, Troy A, Herschleb J, Kraft E and Callis J (2005) Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol 137: 13-30 Stone SL, Williams LA, Farmer LM, Vierstra RD and Callis J (2006) KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell 18: 3415-28 Storey R and Jones RG (1979) Responses of Atriplex spongiosa and Suaeda monoica to Salinity. Plant Physiol 63: 156-162 Studier FW and Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189: 113-30 Sugden C, Crawford RM, Halford NG and Hardie DG (1999) Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5''-AMP. Plant J 19: 433-9 Sun L and Chen ZJ (2004) The novel functions of ubiquitination in signaling. Curr Opin Cell Biol 16: 119-26 Tuteja N (2007) Mechanisms of high salinity tolerance in plants. Methods Enzymol 428: 419-38 Tomsig JL and Creutz CE (2002) Copines: a ubiquitous family of Ca(2+)-dependent phospholipid-binding proteins. Cell Mol Life Sci 59: 1467-77 Tomsig JL, Snyder SL and Creutz CE (2003) Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif. J Biol Chem 278: 10048-54 Treichal S (1986) The influence of NaCl on Δ-pyrrolone-5-carboxylate in proline-accumulating cell suspension cultures of Mesembryanthemum nodiflorum and other halophytes. Physiol Plant 67: 173-181 Tuckwell D (1999) Evolution of von Willebrand factor A (VWA) domains. Biochem Soc Trans 27: 835-40 Wang Y-C (2006) Isolation and characterization of mcSNF-1 gene in halophyte Mesembryanthemum crystallinum L. Bachelor Thesis, Department of Life Sciences, National Chung-Hsing University Whittaker CA and Hynes RO (2002) Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol Biol Cell 13: 3369-87 Wilkinson CR, Seeger M, Hartmann-Petersen R, Stone M, Wallace M, Semple C and Gordon C (2001) Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Nat Cell Biol 3: 939-43 Windheim M, Peggie M and Cohen P (2008) Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology. Biochem J 409: 723-9 Winter V and Hauser MT (2006) Exploring the ESCRTing machinery in eukaryotes. Trends Plant Sci 11: 115-23 Yang F-Y (2006) Analysis of gene expression and protein accumulation of a signal transduction-related mcSNF1 in halophyte Mesembryanthemum crystallinum under salt stress. Master thesis, Department of Life Sciences, National Chung-Hsing University Yang S, Yang H, Grisafi P, Sanchatjate S, Fink GR, Sun Q and Hua J (2006) The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis. Plant J 45: 166-79 Yen HE, Wu S-M, Hong Y-H and Yen S-K (2000) Isolation of 3 salt-induced low-abundance cDNAs from light-grown callus of Mesembryanthemum crystallinum by suppression subtractive hybridization. Physiol Plant 110: 402-409 Yen HE, Grimes HD and Edwards GE (1995) The effects of high salinity, water-deficit, and abscisic acid on phosphoenolpyruvate carboxylase activity and proline accumulation in Mesembryanthemum crystallinum cell cultures. J Plant Physiol 145: 557-56410: 402-4 Yeo A (1999) Predicting the interaction between the effects of salinity and climate change on crop plants. Sci Hortic (Amsterdam) 78: 159-174 Yi C and Deng XW (2005) COP1 - from plant photomorphogenesis to mammalian tumorigenesis. Trends Cell Biol 15: 618-25 Yin XJ, Volk S, Ljung K, Mehlmer N, Dolezal K, Ditengou F, Hanano S, Davis SJ, Schmelzer E, Sandberg G, Teige M, Palme K, Pickart C and Bachmair A (2007) Ubiquitin lysine 63 chain forming ligases regulate apical dominance in Arabidopsis. Plant Cell 19: 1898-911 Zhang HX and Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotech 19:765-8 Zhang X, Garreton V and Chua NH (2005) The AIP2 E3 ligase acts as a novel negative regulator of ABA signaling by promoting ABI3 degradation. Genes Dev 19: 1532-43 Zhang Y, Yang C, Li Y, Zheng N, Chen H, Zhao Q, Gao T, Guo H and Xie Q (2007) SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell 19: 1912-29 Zhao F, Wang Z, Zhang Q, Zhao Y and Zhang H (2006) Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H + antiporter gene from Suaeda salsa. J Plant Res 119: 95-104 Zhu J, Gong Z, Zhang C, Song C-P, Damsz B, Inan G, Koiwa H, Zhu JK, Hasegawa PM and Bressan RA (2002) OSM1/SYP61: a syntaxin protein in Arabidopsis controls abscisic acid-mediated and non-abscisic acid mediated responses to abiotic stress. Plant Cell 14: 3009-3|
|摘要:||McCOPINE1 (mcCPN1)是在耐鹽植物冰花中鑑定到的一個copine蛋白，與鹽逆境相關蛋白mcSKD1 (suppressor of K+ transport defect 1)及mcSNF1 (sucrose non-fermenting 1)具有蛋白質交互作用。已知鹽誘導蛋白mcSKD1參與鹽逆境下intracellular protein trafficking之過程，而蛋白激酶mcSNF1則與鹽逆境訊息傳遞路徑有關。由於mcCPN1蛋白上具有兩個保留性區位，分別為N端copine A domain及C端RING-finger domain，將其歸類為RING-type copine。已知大部分含有RING-finger domain 的蛋白皆具有E3 ubiquitin ligase的活性，在ubiquitination過程中的功能為辨識並標定受質蛋白。本論文之目的在於分析鹽逆境下mcCPN1的基因表現及蛋白累積的情形，並進一步偵測其E3 ligase的活性，以期對mcCPN1蛋白的作用有更深入的了解。
為了解mcCPN1與鹽逆境的相關性，分別偵測鹽處理的細胞及植株中mcCPN1基因及蛋白累積的情況。此部分的分析是以RT-PCR及北方墨點法偵測mcCPN1基因表現量，並以西方墨點法偵測mcCPN1蛋白的累積量。在細胞層次上，鹽處理後的冰花培養細胞中mcCPN1基因皆呈現穩定的表現。然而在蛋白的累積量上，鹽處理七天後，mcCPN1在細胞中的累積量會隨著鹽濃度增加而增加，反之，鹽處理三天卻會減緩mcCPN1的累積量，進一步以0 mM及200 mM鹽處理冰花培養細胞一周後，分析不同時間點收集的細胞中mcCPN1蛋白累積量，發現mcCPN1蛋白的累積在細胞層次與細胞的增生具有較大的相關性。在冰花植株中，mcCPN1基因在根部與葉部中亦呈現穩定表現，然而200 mM鹽處理後，mcCPN1蛋白累積量均上升，顯示mcCPN1蛋白在冰花植株中可能參與鹽逆境的反應。經由上述結果推測冰花mcCPN1蛋白參與了細胞增生及鹽逆境的反應。
由於mcCPN1蛋白累積量在鹽誘導下及細胞增生過程中產生變化，進一步針對其蛋白活性做測試，首先利用in vitro ubiquitination assay證實mcCPN1確實具有E3 ligase的活性，並嚐試以mcSKD1及mcSNF1蛋白當作mcCPN1的受質蛋白，結果顯示mcSKD1可被mcCPN1蛋白辨識並標定ubiquitin，証實mcSKD1為mcCPN1的受質蛋白之一。此外，in vitro下mcSNF1會對減緩mcSKD1被mcCPN1標定ubiquitin的程度。由以上結果可知mcCPN1為一E3 ubiquitin ligase且mcSKD1為其受質蛋白，且mcSNF1可能在mcCPN1主導的mcSKD1 ubiquitination過程中扮演調控的角色。
綜合本論文之結果，E3 ligase mcCPN1可能參與冰花的細胞增生和鹽逆境下的反應，且其蛋白累積受到轉譯或後轉譯層次的調控。在鹽逆境下，mcCPN1可能會藉由控制鹽逆境相關蛋白mcSKD1之protein ubiquitination進而參與冰花鹽逆境下之反應機制。|
McCOPINE1 (mcCPN1), a RING-type copine, was identified from halophyte ice plant (Mesembryanthemum crystallinum L.) and known to interact with salt stress-induced proteins mcSKD1 (suppressor of K+ transport defect 1) and mcSNF1 (sucrose non-fermenting 1). McSKD1 is a salt-induced gene and involves in intracellular protein trafficking, whereas mcSNF1 is predicted to be a protein kinase and possibly involves in salt-stress signaling. Our previous study showed that mcCPN1 contains two conserved domains, N-terminal copine A domain and C-terminal RING (really interesting new gene)-finger domain. RING-finger proteins usually possess E3 ligase catalytic activity executing the last step of ubiquitination in recognizing and ubiquitinating substrate proteins. Thus, the main focus of this thesis is to examine the expression and accumulation of mcCPN1 under salt stress, along with the analysis of its E3 ligase activity in order to access its function in salt-tolerant mechanism of ice plant. To understand the role of mcCPN1 in salt-tolerant mechanism, gene expression and protein accumulation of mcCPN1 were examined at both cellular and intact plant levels. RT-PCR and Northern blotting were used to analyze mcCPN1 expression and anti-mcCPN1 antibody was used to detect mcCPN1 accumulation. At cellular level, gene expression of mcCPN1 was constitutive after addition of salt, whereas a significant change was found in the accumulation of mcCPN1. Long-term salt stress induced mcCPN1 accumulation; however, short-term salt stress suppressed it. Temperal accumulation of mcCPN1 indicated that cell proliferation has a profound influence on mcCPN1 accumulation. At intact plant level, constitutive expression of mcCPN1 was found in salt-stressed roots and leaves, whereas protein accumulation of mcCPN1 was salt-induced suggesting that mcCPN1 might participate in the salt stress response of ice plant. Due to the reason that the changes in the accumulation mcCPN1 were found at cellular and intact plant level, the catalytic activity of mcCPN1 was further examined. The activity of mcCPN1 was demonstrated by the in vitro ubiquitination assay. When using mcCPN1 interacting protein mcSKD1 or mcSNF1 as the in vitro substrate, mcSKD1 can be ubiquitinated but mcSNF1 can not. Furthermore, mcSNF1 reduced the amount of ubiquitinated mcSKD1 in the in vitro system. These results suggested that mcSKD1 is one of the substrate proteins for E3 ligase mcCPN1 and mcSNF1 might play a regulatory role in mcCPN1-mediated mcSKD1 ubiquitination. Taken together, these results indicated that the E3 ligase mcCPN1 participates in the cell proliferation and salt stress response in ice plant and the accumulation of mcCPN1 was regulated at translational and/or post-translational level. The involvement of mcCPN1 in salt-tolerant mechanism of ice plant is possibly by way of recognizing and ubiquitinated salt stress-related protein mcSKD1.
|Appears in Collections:||生命科學系所|
Show full item record
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.