Preferences help
enabled [disable] Abstract
Number of results
2004 | 51 | 3 | 609-624
Article title

Multifunctional role of plant cysteine proteinases

Title variants
Languages of publication
Cysteine proteinases also referred to as thiol proteases play an essential role in plant growth and development but also in senescence and programmed cell death, in accumulation of storage proteins such as in seeds, but also in storage protein mobilization. Thus, they participate in both anabolic and catabolic processes. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. In this review an attempt was undertaken to illustrate these multiple roles of cysteine proteinases and the mechanisms underlying their action.
Physical description
  • Plant Physiology and Biochemistry Department, Plant Breeding and Acclimatization Institute, Radzików, Warszawa, Poland
  • Biochemistry Department, Warsaw Agricultural University, Warszawa, Poland
  • Amor Y, Babiychuk E, Inze D, Levine A. (1998) The involvement of poly(ADP-ribose) polymerase in the oxidative stress responses in plant. FEBS Lett.; 440: 1-7.
  • Barrett AJ. (1986) The classes of proteolytic enzymes. In Plant Proteolytic Enzymes. Dalling MJ, ed, vol. 1: pp 1-16. CRC Press, Boca Raton, Fl.
  • Barrett AJ. (1994) Classification of peptidases. Methods Enzymol.; 244: 1-15.
  • Becker C, Shutov AD, Nong VH, Denyuk VI, Jung R, Horstmann C, Fischer J, Nielsen NC, Müntz K. (1995) Purification, cDNA cloning and characterisation of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds. Eur J Biochem.; 228: 456-62.
  • Beers EP, Woffenden BJ, Zhao C. (2000) Plant proteolytic enzymes: possible roles during programmed cell death. Plant Mol Biol.; 44: 399-415.
  • Beers EP, Jones AM, Dickerman AW. (2004) The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis. Phytochemistry.; 65: 43-58.
  • Bhandari V, Palfree RGE, Bateman A. (1992) Isolation and sequence of granulin precursor cDNA from human bone narrow reveals tandem cysteine-rich granulin domain. Proc Natl Acad Sci USA.; 89: 1715-9.
  • Botella MA, Xu Y, Prabhe TN, Zhao Y, Narasimahan ML, Wilson KA, Nielsen SS, Bressau RA, Hasegawa PM. (1996) Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate. Plant Physiol.; 112: 1201-10.
  • Bottari A, Capocchi A, Galleschi A, Jopova A, Saviozzi E. (1996) Asparaginyl endopeptidase during maturation and germination of durum wheat. Physiol Plant.; 97: 475-80.
  • Cercós M, Harris N, Carbonell J. (1993) Immunolocalization of a thiol-protease induced during the senescence of unpollinated pea ovaries. Physiol Plant.; 88: 275-80.
  • Chen GH, Huang LT, Yap MN, Lee RH, Huang YJ, Cheng MC, Chen SCG. (2002) Molecular characterization of a senescence associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet potato. Plant Cell Physiol.; 43: 984-91.
  • Chrispeels MJ, Herman EM. (2000) Endoplasmatic reticulum-derived compartments function in storage and as mediators of vacuolar remodelling via a new type of organelle, precursor protease vesicles. Plant Physiol.; 123: 1227-33.
  • Coupe AS, Sinclair BK, Watson BK, Heyes JA, Eason JR. (2003) Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets. J Exp Bot.; 54: 1045-56.
  • Cowgill SE, Wright C, Atkinson HJ. (2002) Transgenic potatoes with enhanced levels of nematode resistance do not have altered susceptibility to nontarget aphids. Mol Ecol.; 11: 821-7.
  • Davy A, Sorensen MB, Svendsen Ib, Cameron-Mills V, Simpson DJ. (2000) Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis. Plant Physiol.; 122: 137-46.
  • de Barros EG, Larkins BA. (1994) Cloning of a cDNA encoding a putative cysteine protease gliadin during wheat germination. Phytochemistry.; 43Z: 39-44.
  • del Pozo O, Lam E. (1998) Caspases and programmed cell deathin the hypersensitive response of plants to pathogens. Curr Biol.; 8: 1129-32.
  • D'Silva J, Poirier GG. (1998) Activation of cysteine proteases in cowpea plants during the hypersensitive response of programmed cell death. Exp Cell Res.; 245: 389-99.
  • Dubin A. (2003) Defense against own arms: staphylococcal cysteine proteases and their inhibitors. Acta Biochim Polon.; 50: 715-24.
  • Dubin G, Krajewski M, Popowicz G, Stec-Niemczyk J, Bochtler M, Potempa J, Dubin A, Holak TA. (2003) A novel class of cysteine protease inhibitors: solution structure of staphostatin A from Staphylococcus aureus. Biochemistry.; 42: 13449-56.
  • Eason JR, Ryan DJ, Pinkey TT, Donoghue EMO. (2002) Programmed cell death during flower senescence: isolation and characterization of cysteine proteinases from Sandersonia aurantiaca. Funct Plant Biol.; 29: 1055-64.
  • Fernandes KVS, Sabelli PA, Barratt DHP, Richardson M, Xavier Filho J, Shevery PR. (1993) The resistance of cowpea seeds to bruchid beetles is not related to level of cysteine proteinase inhibitors. Plant Mol Biol.; 23: 215-9.
  • Fischer J, Becker C, Hillmer S, Horstmann C, Neubohn B, Schlereth A, Senyuk V, Shutov A, Müntz K. (2000) The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intercellular localization and functions in globulin proteolysis. Plant Mol Biol.; 43: 83-101.
  • Frigerio L, Pastres A, Prada A, Vitale A. (2001) Influence of KDEL on the fate of trimeric or assembly-defective phaseolin: selective use of an alternative route to vacuoles. Plant Cell.; 13: 1109-26.
  • Funk V, Kositsup B, Zhao Ch, Beers EP. (2002) The Arabidopsis xylem peptidase XCP1 is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiol.; 128: 84-94.
  • Gietl C, Schmid M, Simpson D. (2000) Ricinosomes and aleurain-containing vacuoles (ACV): proteinase storing organelles. In: Annual Plant Reviews: Vacuolar Compartment. Robinson DG, Rogers JC, eds, vol 5, pp 90-111. Sheffield Academy Press, Sheffield.
  • Goetting-Minesky MP, Mullin BC. (1994) Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase. Proc Natl Acad Sci.; 91: 9891-5.
  • Grudkowska M, Wiśniewski K, Zagdańska B. (2002) Aktywność endoproteinaz cysteinowych wskaźnikiem odporności siewek pszenicy ozimej na mróz i suszę. Biul IHAR.; 223/224: 45-55.
  • Gruis DF, Selinger DA, Curran JM, Jung R. (2002) Redundant proteolytic mechanisms process seed storage proteins in the absence of seed-type members of the vacuolar processing enzyme family of cysteine proteases. Plant Cell.; 14: 2863-82.
  • Gruis DF, Schulze J, Jung R. (2004) Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases. Plant Cell.; 16: 270-90.
  • Guerrero FD, Jones JT, Mullet JE. (1990) Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol.; 15: 11-26.
  • Grzonka Z, Jankowska E, Kasprzykowski F, Kasprzykowska R, Łankiewicz L, Wiczk W, Wieczerzak E, Ciarkowski J, Drabik P, Janowski R, Kozak E, Jaskólski M, Grubb A. (2001) Structural studies of cysteine proteases and theirs inhibitors. Acta Biochim Polon.; 48: 1-20.
  • Hara-Nishimura I, Inoue K, Nishimura M. (1991) A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into mature forms. FEBS Lett.; 294: 89-94.
  • Hara-Nishimura I, Shimada T, Hiraiwa N, Nishimura M. (1995) Vacuolar processing enzyme responsible for maturation of seed protein. J Plant Physiol.; 145: 6412-7.
  • Hara-Nishimura I, Kinoshita T, Hiraiwa N, Nishimura M. (1998a) Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles. J Plant Physiol.; 152: 668-74.
  • Hara-Nishimura I, Shimada T, Hatano K, Takeuchi Y, Nishimura M. (1998b) Transport of storage proteins to protein-storage vacuoles is mediated by large precursor-accumulating vesicles. Plant Cell.; 10: 825-36.
  • Hayashi Y, Yamada K, Matsushima R, Nishizawa N, Nishimura M, Hara-Nishimura I. (2001) A proteinase-storing body that prepares for cell death or stress in the epidermal cells of Arabidopsis. Plant Cell Physiol.; 42: 894-9.
  • Herman EM, Larkins BA. (1999) Protein storage bodies. Plant Cell.; 11: 601-13.
  • Ho SL, Tong WF, Yu SM. (2000) Multiple mode of regulation of cysteine proteinase gene expression in rice. Plant Physiol.; 122: 57-66.
  • Huang YJ, To KY, Yap MN, Chiang WJ, Suen DF, Chen SCG. (2001) Cloning and characterization of leaf senescence up-regulated genes in sweet potato. Physiol Plant.; 113: 384-91.
  • Jung R, Scott MP, Nam Y-W, Beaman TW, Bassuner R, Saalbach I, Müntz K, Nielsen NC. (1998) The role of proteolysis in the processing and assembly of 11S seed globulins. Plant Cell.; 10: 343-57.
  • Kinoshita T, Nishimura M, Hara-Nishimura I. (1995a) Homologues of vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana. Plant Mol Biol.; 29: 81-9.
  • Kinoshita T, Nishimura M, Hara-Nishimura I. (1995b) The sequence and the expression of the γ-VPE gene, one member of a family of three genes for vacuolar processing enzymes in Arabidopsis thaliana. Plant Cell Physiol.; 36: 1555-62.
  • Kinoshita T, Yamada K, Hiraiwa N, Kondo M, Nishimura M, Hara-Nishimura I. (1999) Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stress conditions. Plant J.; 19: 43-53.
  • Koehler S, Ho TD. (1988) Purification and characterization of gibberelic acid-induced cysteine endoproteases in barley aleurone layers. Plant Physiol.; 87: 251-8.
  • Koehler SM, Ho TD. (1990a) A major gibberellic acid-induced barley aleurone cysteine proteinase which digests hordein. Plant Physiol.; 94: 251-8.
  • Koehler SM, Ho TD. (1990b) Hormonal regulation, processing and secretion of cysteine proteinases in barley aleurone layers. Plant Cell.; 2: 769-83.
  • Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, Shinozaki K. (1993) Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene.; 129: 175-82.
  • Korthout HAAJ, Berecki G, Bruin W, van Duijn B, Wang M. (2000) The presence and subcellular localization of caspase 3-like proteinases in plant cells. FEBS Lett.; 475: 139-44.
  • Kuo A, Cappelluti S, Cervantes-Cervantes M, Rodriguez M, Bush DS. (1996) Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells. Plant Cell.; 8: 259-69.
  • Kuroyanagi M, Nishimura M, Hara-Nishimura I. (2002) Activation of Arabidopsis vacuolar processing enzyme by self-catalytic removal of an auto-inhibitory domain of the C-terminal propeptide. Plant Cell Physiol.; 43: 143-51.
  • Kuriyama H, Fukuda H. (2002) Developmental programmed cell death in plants. Curr Opin Plant Biol.; 5: 568-73.
  • Kuwabara T, Hashimoto Y. (1990) Purification of a dithiothreitol-sensitive tetrameric protease from spinach PS II membranes. Plant Cell Physiol.; 31: 581-9.
  • Lam E, del Pozo O. (2000) Caspase-like protease involvement in the control of plant cell death. Plant Mol Biol.; 44: 417-28.
  • Lid SE, Gruis D, Jung R, Lorentzen JA, Ananiev E, Chamberlin M, Niu X, Meeley R, Nichols S, Olsen OA. (2002) The defective kernel1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. Proc Natl Acad Sci USA.; 99: 5460-5.
  • Lindhal M, Yang DH, Andersson B. (1995) Regulatory proteolysis of the major light-harvesting chlorophyll a/b protein of photosystem II by a light-induced membrane-associated enzymatic system. Eur J Biochem.; 231: 503-9.
  • Linnestad C, Doan DNP, Brown RC, Lemmon BE, Meyer DJ, Jung R, Olsen OA. (1998) Nucellain, a barley homolog of the dicot vacuolar-processing protease, is localized in the nucellar cell walls. Plant Physiol.; 118: 1169-80.
  • Margis R, Reis EM, Villeret V. (1998) Structural and phylogenetic relationships among plant and animal cystatins. Arch Biochem Biophys.; 359: 24-30.
  • Margis R, Margis-Pinheiro M. (2003) Phytocalpains: orthologous calcium-dependent cysteine proteinases. Trends Plant Sci.; 8: 58-62.
  • Menard R, Storer AC. (1998) Papain. In Handbook of Proteolytic Enzymes. Barrett AJ, Rawlings ND, Woessner JF, eds, pp 555-7. Academic Press, New York.
  • Minami A, Fukuda H. (1995) Transient and specific expression of a cysteine endoproteinase associated with autolysis during differentiation of Zinia mesophyll cells into tracheary elements. Plant Cell Physiol.; 36: 1599-606.
  • Müntz K, Blattner FR, Shutov AD. (2002) Legumains - a family of asparagine-specific cysteine endopeptidases involved in propolypeptide processing and protein breakdown in plants. J Plant Physiol.; 160: 1281-93.
  • Müntz K, Shutov AD. (2002) Legumains and their functions in plants. Trends Plant Sci.; 7: 340-4.
  • Nielsen NC, Jung R, Nam YW, Beaman TW, Oliveira LO, Bassuner R. (1995) Synthesis and assembly of 11S globulins. Plant Physiol.; 145: 641-7.
  • Okamoto T, Minamikawa T. (1995) Purification of a processing enzyme (VmPE-1) that is involved in post-translational processing of a plant cysteine proteinase (SH-EP). Eur J Biochem.; 231: 300-5.
  • Okamoto T, Minamikawa T. (1998) A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: characterization, regulation of gene expression, and posttranslational processing. J Plant Physiol.; 152: 675-82.
  • Okamoto T, Minamikawa T. (1999) Molecular cloning and characterization of Vinga mungo processing enzyme (VmPE-1), and asparaginyl endopeptidase possibly involved in post-translational processing of vacuolar cysteine endopeptidase (SH-EP). Plant Mol Biol.; 39: 63-73.
  • Okamoto T, Shimada T, Hara-Nishimura I, Nishimura M, Minamikawa T. (2003) C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle in efficient vacuolar transport of sulfhydryl-endopeptidase. Plant Physiol.; 132: 1892-900.
  • Pechan T, Ye L, Chang Y, Mitra A, Lin L, Davis FM, Williams WB, Luthe DS. (2000) A unique 33-kD cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other Lepidoptera. Plant Cell.; 12: 1031-40.
  • Pernas M, Sanchez-Mong R, Salcedo G. (2000) Biotic and abiotic stress can induce cystatin expression in chestnut. FEBS Lett.; 467: 206-10.
  • Poulle M, Jones BL. (1988) A proteinase from germinating barley. Purification and some physical properties. J Plant Physiol.; 88: 1454-60.
  • Rahbe Y, Deraison C, Bonade-Bottino M, Girard C, Nardon Ch, Jouanin L. (2003) Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape. Plant Sci.; 164: 441-50.
  • Rabiza-Świder J, Rybka Z, Skutnik E, Łukaszewska A. (2003) Proteolysis and expression of the cysteine protease gene in senescing cut leaves of Hosta Undulata Erromena and Zantedeschia aethiopica Spr. treated with BA or GA3. Acta Physiol Plant.; 25: 319-24.
  • Rawlings ND, Barrett AJ. (1993) Evolutionary families of peptidases. Biochem J.; 290: 205-18.
  • Royo E, Zouhar J, Carter C, Kovalewa V, Raikhel NV. (2003) A unique mechanism for protein processing and degradation in Arabidopsis thaliana. Proc Natl Acad Sci USA.; 100: 7389-94.
  • Rzychon M, Sabat A, Kosowska K, Potempa J, Dubin A. (2003) Staphostatins: an expanding new group of inhibitors with a unique specificity for the regulation of staphopains, Staphylococcus ssp. Cysteine proteinases. Mol Microbiol.; 49: 1051-66.
  • Safadi F, Mykles DI, Reddy ASN. (1997) Partial purification and characterization of a Ca2+-dependent proteinase from Arabidopsis roots. Arch Biochem Biophys.; 348: 143-51.
  • Schaffer MA, Fischer RL. (1988) Analysis of mRNAs that accumulate in response to low temperature identifies a thiol protease gene in tomato. Plant Physiol.; 87: 431-6.
  • Schaffer MA, Fischer RL. (1990) Transcriptional activation by heat and cold of a thiol protease gene in tomato. Plant Physiol.; 93: 1486-91.
  • Schlereth A, Becker C, Horstmann C, Tiedemann J, Müntz K. (2000) Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.). J Exp Bot.; 51: 1423-33.
  • Schlereth A, Standhardt D, Mock HP, Müntz K. (2001) Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination. Planta.; 212: 718-27.
  • Schmid M, Simpson D, Gietl Ch. (1999) Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes. Proc Nat Acad Sci USA.; 96: 14159-64.
  • Scott MP, Jung R, Müntz K, Nielsen NC. (1992) A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major storage protein of soybean. Proc Natl Acad Sci USA.; 89: 658-62.
  • Shimada T, Fuji K, Tamura K, Kondo M, Nishimura M, Hara-Nishimura I. (2003) Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana. Proc Natl Acad Sci USA.; 100: 16095-100.
  • Simpson DJ. (2001) Proteolytic degradation of cereal prolamins - the problem with proline. Plant Sci.; 161: 825-38.
  • Subbaiah CC, Kollipara KP, Sachs MM. (2000) A Ca2+-dependent cysteine protease is associated with anoxia-induced root tip death in maize. J Exp Bot.; 51: 721-30.
  • Sun YL, Zhu HZ, Zhou J, Dai YR, Zhai ZH. (1999) Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplast. Cell Mol Life Sci.; 55: 310-6.
  • Tanaka T, Minamikawa T, Yamauchi D, Ogushi Y. (1993) Expression of an endopeptidase (EP-C1) in Phaseolus vulgaris plants. Plant Physiol.; 101: 421-8.
  • Tiedemann J, Schlereth A, Müntz K. (2001) Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds. Planta.; 212: 728-38.
  • Turk V, Bode W. (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett.; 285: 213-9.
  • Toyooka K, Okamoto T, Minamikawa T. (2000) Mass transport of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplasmatic reticulum-derived vesicle is involved in protein mobilization in germinating seeds. J Cell Biol.; 148: 453-63.
  • Uren AG, O'Rourke K, Arrand L, Pisabarro MT, Seshagiri B, Koonin EV, Dixit VM. (2000) Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell.; 6: 961-7.
  • Van der Vyver C, Schneidereit J, Driscoll S, Turner J, Kunert K, Foyer C. (2003) Oryzacystatin I expression in transformed tobacco produces a conditional growth phenotype and enhances chilling tolerance. Plant Biotech J.; 1: 101-12.
  • Vierstra RD. (2003) The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends Plant Sci.; 8: 135-42.
  • Vincent JL, Brewin NJ. (2000) Immunolocalization of cysteine protease in vacuoles, vesicles, and symbiosomes of pea nodule cells. Plant Physiol.; 123: 521-30.
  • Waldron C, Wegrich LM, Merlo PAO, Walsh TA. (1993) Characterization of genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor. Plant Mol Biol.; 23: 801-12.
  • Wang C, Barry JK, Min Z, Tordsen G, Rao AG, Olsen O-A. (2003) The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase. J Biol Chem.; 278: 34467-74.
  • Wiederanders B. (2003) Structure-function relationships in class CA1 cysteine peptidase propeptides. Acta Biochim Polon.; 50: 691-713.
  • Wiśniewski K, Zagdańska B. (2001) Genotype-dependent proteolytic response of spring wheat to water deficiency. J Exp Bot.; 52: 1455-63.
  • Woltering EJ, van der Bent A, Hoeberichts FA. (2002) Do plant caspases exist? Plant Physiol.; 130: 1764-9.
  • Yamada K, Matsushima R, Nishimura M, Hara-Nishimura I. (2001) A slow maturation of a cysteine protease with a granulin domain in the vacuoles of senescencing Arabidopsis leaves. Plant Physiol.; 127: 1626-34.
  • Yamaguchi-Shinozaki K, Koizumi M, Urao S, Shinozaki K. (1992) Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol.; 33: 217-24.
  • Zagdańska B, Wiśniewski K. (1996) Endoproteinase activities in wheat leaves upon water deficit. Acta Biochim Polon.; 43: 515-9.
  • Zhang N, Jones BL. (1995) Characterization of germinated barley endoproteolytic enzymes by two dimensional gel electrophoresis. J Cereal Sci.; 21: 145-53.
  • Zhao Y, Botella MA, Subramanian L, Niu X, Nielsen SS, Bressan RA, Hasegawa PM. (1996) Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol.; 111: 1299-306.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.