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Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa
Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain un...
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Published in: | Plant physiology (Bethesda) 2004-10, Vol.136 (2), p.3234-3244 |
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description | Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants. |
doi_str_mv | 10.1104/pp.104.047449 |
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Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.</description><identifier>ISSN: 0032-0889</identifier><identifier>EISSN: 1532-2548</identifier><identifier>DOI: 10.1104/pp.104.047449</identifier><identifier>PMID: 15466227</identifier><identifier>CODEN: PPHYA5</identifier><language>eng</language><publisher>Rockville, MD: American Society of Plant Biologists</publisher><subject>Alfalfa ; Amino Acid Sequence ; amino acid sequences ; Amino acids ; Biochemical Processes and Macromolecular Structures ; Biological and medical sciences ; catechol oxidase ; Catechol Oxidase - chemistry ; Catechol Oxidase - genetics ; Catechol Oxidase - metabolism ; Chloroplasts ; Clover ; Complementary DNA ; DNA, Plant - chemistry ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; forage legumes ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; gene expression regulation ; Gene Expression Regulation, Plant ; Medicago sativa ; Medicago sativa - genetics ; Metabolism ; Molecular Sequence Data ; nucleotide sequences ; Organisms, Genetically Modified ; Oxidases ; plant physiology ; Plant physiology and development ; plant proteins ; Plants ; Plants, Genetically Modified ; Polyphenols ; postharvest physiology ; proteolysis ; RNA ; RNA, Plant - chemistry ; RNA, Plant - metabolism ; Sequence Alignment ; sequence analysis ; tissue distribution ; transgenic plants ; Trifolium - enzymology ; Trifolium - genetics ; Trifolium pratense</subject><ispartof>Plant physiology (Bethesda), 2004-10, Vol.136 (2), p.3234-3244</ispartof><rights>Copyright 2004 American Society of Plant Biologists</rights><rights>2004 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c539t-4bf16accca5c4fc67cae6f6bfd4c31b9e6a52a8b0b1eaf3ff205b342cfe6baf3</citedby><cites>FETCH-LOGICAL-c539t-4bf16accca5c4fc67cae6f6bfd4c31b9e6a52a8b0b1eaf3ff205b342cfe6baf3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/4356672$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/4356672$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,58238,58471</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=16193338$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15466227$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sullivan, M.L</creatorcontrib><creatorcontrib>Hatfield, R.D</creatorcontrib><creatorcontrib>Thoma, S.L</creatorcontrib><creatorcontrib>Samac, D.A</creatorcontrib><title>Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa</title><title>Plant physiology (Bethesda)</title><addtitle>Plant Physiol</addtitle><description>Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.</description><subject>Alfalfa</subject><subject>Amino Acid Sequence</subject><subject>amino acid sequences</subject><subject>Amino acids</subject><subject>Biochemical Processes and Macromolecular Structures</subject><subject>Biological and medical sciences</subject><subject>catechol oxidase</subject><subject>Catechol Oxidase - chemistry</subject><subject>Catechol Oxidase - genetics</subject><subject>Catechol Oxidase - metabolism</subject><subject>Chloroplasts</subject><subject>Clover</subject><subject>Complementary DNA</subject><subject>DNA, Plant - chemistry</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>forage legumes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>gene expression regulation</subject><subject>Gene Expression Regulation, Plant</subject><subject>Medicago sativa</subject><subject>Medicago sativa - genetics</subject><subject>Metabolism</subject><subject>Molecular Sequence Data</subject><subject>nucleotide sequences</subject><subject>Organisms, Genetically Modified</subject><subject>Oxidases</subject><subject>plant physiology</subject><subject>Plant physiology and development</subject><subject>plant proteins</subject><subject>Plants</subject><subject>Plants, Genetically Modified</subject><subject>Polyphenols</subject><subject>postharvest physiology</subject><subject>proteolysis</subject><subject>RNA</subject><subject>RNA, Plant - chemistry</subject><subject>RNA, Plant - metabolism</subject><subject>Sequence Alignment</subject><subject>sequence analysis</subject><subject>tissue distribution</subject><subject>transgenic plants</subject><subject>Trifolium - enzymology</subject><subject>Trifolium - genetics</subject><subject>Trifolium pratense</subject><issn>0032-0889</issn><issn>1532-2548</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><recordid>eNqFkk1v1DAQhi0EosvCkRsCX-CWxV_xJsdqWz6kCg6UczSZHe-6ysbBzlYtf4K_jLeJ6BHJ0mvNPPNqNDOMvZZiJaUwH4dhlWUlzNqY-glbyFKrQpWmesoWQuS_qKr6jL1I6UYIIbU0z9mZLI21Sq0X7M-mC73vdxz6Lcc9RMCRov8Now89D45HyvEu3FLkQ-juhz31oePhzm8hEceLb-fpoZbuhkgpzVXZxd8SH2IYyfc8v8uE-2yMew8cQ-cfisYIfdpR75FD507vJXuWNdGrWZfs-tPl9eZLcfX989fN-VWBpa7HwrROWkBEKNE4tGsEss62bmtQy7YmC6WCqhWtJHDaOSXKVhuFjmybA0v2YbLNHf46Uhqbg09IXQc9hWNqrK0rpSv9X1DWlZSmkhksJhBjSCmSa4boDxDvGyma06aaYWhOMm0q829n42N7oO0jPa8mA-9nABLm0eRRoU-PnJW11rnFJXszcTdpDPFf3ujS2rXK6XdT2kFoYBezxc8fKh-CELXNB1Hpvzklsmc</recordid><startdate>20041001</startdate><enddate>20041001</enddate><creator>Sullivan, M.L</creator><creator>Hatfield, R.D</creator><creator>Thoma, S.L</creator><creator>Samac, D.A</creator><general>American Society of Plant Biologists</general><general>American Society of Plant Physiologists</general><scope>FBQ</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20041001</creationdate><title>Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa</title><author>Sullivan, M.L ; Hatfield, R.D ; Thoma, S.L ; Samac, D.A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c539t-4bf16accca5c4fc67cae6f6bfd4c31b9e6a52a8b0b1eaf3ff205b342cfe6baf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Alfalfa</topic><topic>Amino Acid Sequence</topic><topic>amino acid sequences</topic><topic>Amino acids</topic><topic>Biochemical Processes and Macromolecular Structures</topic><topic>Biological and medical sciences</topic><topic>catechol oxidase</topic><topic>Catechol Oxidase - chemistry</topic><topic>Catechol Oxidase - genetics</topic><topic>Catechol Oxidase - metabolism</topic><topic>Chloroplasts</topic><topic>Clover</topic><topic>Complementary DNA</topic><topic>DNA, Plant - chemistry</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>forage legumes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression</topic><topic>gene expression regulation</topic><topic>Gene Expression Regulation, Plant</topic><topic>Medicago sativa</topic><topic>Medicago sativa - genetics</topic><topic>Metabolism</topic><topic>Molecular Sequence Data</topic><topic>nucleotide sequences</topic><topic>Organisms, Genetically Modified</topic><topic>Oxidases</topic><topic>plant physiology</topic><topic>Plant physiology and development</topic><topic>plant proteins</topic><topic>Plants</topic><topic>Plants, Genetically Modified</topic><topic>Polyphenols</topic><topic>postharvest physiology</topic><topic>proteolysis</topic><topic>RNA</topic><topic>RNA, Plant - chemistry</topic><topic>RNA, Plant - metabolism</topic><topic>Sequence Alignment</topic><topic>sequence analysis</topic><topic>tissue distribution</topic><topic>transgenic plants</topic><topic>Trifolium - enzymology</topic><topic>Trifolium - genetics</topic><topic>Trifolium pratense</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sullivan, M.L</creatorcontrib><creatorcontrib>Hatfield, R.D</creatorcontrib><creatorcontrib>Thoma, S.L</creatorcontrib><creatorcontrib>Samac, D.A</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Plant physiology (Bethesda)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sullivan, M.L</au><au>Hatfield, R.D</au><au>Thoma, S.L</au><au>Samac, D.A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa</atitle><jtitle>Plant physiology (Bethesda)</jtitle><addtitle>Plant Physiol</addtitle><date>2004-10-01</date><risdate>2004</risdate><volume>136</volume><issue>2</issue><spage>3234</spage><epage>3244</epage><pages>3234-3244</pages><issn>0032-0889</issn><eissn>1532-2548</eissn><coden>PPHYA5</coden><abstract>Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.</abstract><cop>Rockville, MD</cop><pub>American Society of Plant Biologists</pub><pmid>15466227</pmid><doi>10.1104/pp.104.047449</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Alfalfa Amino Acid Sequence amino acid sequences Amino acids Biochemical Processes and Macromolecular Structures Biological and medical sciences catechol oxidase Catechol Oxidase - chemistry Catechol Oxidase - genetics Catechol Oxidase - metabolism Chloroplasts Clover Complementary DNA DNA, Plant - chemistry Enzymes Escherichia coli Escherichia coli - genetics forage legumes Fundamental and applied biological sciences. Psychology Gene Expression gene expression regulation Gene Expression Regulation, Plant Medicago sativa Medicago sativa - genetics Metabolism Molecular Sequence Data nucleotide sequences Organisms, Genetically Modified Oxidases plant physiology Plant physiology and development plant proteins Plants Plants, Genetically Modified Polyphenols postharvest physiology proteolysis RNA RNA, Plant - chemistry RNA, Plant - metabolism Sequence Alignment sequence analysis tissue distribution transgenic plants Trifolium - enzymology Trifolium - genetics Trifolium pratense |
title | Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa |
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