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Article: Brassica juncea HMG-CoA synthase: Localization of mRNA and protein

TitleBrassica juncea HMG-CoA synthase: Localization of mRNA and protein
Authors
Keywords3-Hydroxy-3-methylglutaryl-coenzyme A synthase
Brassica
Green fluorescent protein
Isoprenoid
Mevalonate
Subcellular fractionation
Issue Date2005
PublisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00425
Citation
Planta, 2005, v. 221 n. 6, p. 844-856 How to Cite?
Abstract3-Hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) synthase (HMGS; EC 2.3.3.10) synthesizes HMG-CoA, a substrate for mevalonate biosynthesis in the isoprenoid pathway. It catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA and HS-CoA. In Brassica juncea (Indian mustard), HMGS is encoded by four isogenes (BjHMGS1-BjHMGS4). We have already enzymatically characterized recombinant BjHMGS1 expressed in Escherichia coli, and have identified its residues that are significant in catalysis. To further study HMGS mRNA expression that is developmentally regulated in flowers and seedlings, we have examined its mRNA distribution by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). We observed predominant localization of HMGS mRNA in the stigmas and ovules of flower buds and in the piths of seedling hypocotyls. RT-PCR analysis revealed that BjHMGS1 and BjHMGS2 but not BjHMGS3 and BjHMGS4were expressed in floral buds. To investigate the subcellular localization of BjHMGS1, we fused BjHMGS1 translationally in-frame either to the N- or C-terminus of green fluorescent protein (GFP). BjHMGS1-GFP and GFP-BjHMGS1 fusions were used in particle gun bombardment of onion epidermal cells and tobacco BY-2 cells. The GFP-BjHMGS1 construct was also used in agroinfiltration of tobacco leaves. Both GFP-fusion proteins were observed transiently expressed in the cytosol on confocal microscopy of onion epidermal cells, tobacco BY-2 cells, and agroinfiltrated tobacco leaves. Further, subcellular fractionation of total proteins from transgenic plants expressing GFP-BjHMGS1 derived from Agrobacterium-mediated transformation confirmed that BjHMGS1 is a cytosolic enzyme. We suggest that the presence of BjHMGS isoforms is likely related to the specialization of each in different cellular and metabolic processes rather than to a different intracellular compartmentation of the enzyme. © Springer-Verlag 2005.
Persistent Identifierhttp://hdl.handle.net/10722/68587
ISSN
2015 Impact Factor: 3.239
2015 SCImago Journal Rankings: 1.470
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorNagegowda, DAen_HK
dc.contributor.authorRamalingam, Sen_HK
dc.contributor.authorHemmerlin, Aen_HK
dc.contributor.authorBach, TJen_HK
dc.contributor.authorChye, MLen_HK
dc.date.accessioned2010-09-06T06:05:55Z-
dc.date.available2010-09-06T06:05:55Z-
dc.date.issued2005en_HK
dc.identifier.citationPlanta, 2005, v. 221 n. 6, p. 844-856en_HK
dc.identifier.issn0032-0935en_HK
dc.identifier.urihttp://hdl.handle.net/10722/68587-
dc.description.abstract3-Hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) synthase (HMGS; EC 2.3.3.10) synthesizes HMG-CoA, a substrate for mevalonate biosynthesis in the isoprenoid pathway. It catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA and HS-CoA. In Brassica juncea (Indian mustard), HMGS is encoded by four isogenes (BjHMGS1-BjHMGS4). We have already enzymatically characterized recombinant BjHMGS1 expressed in Escherichia coli, and have identified its residues that are significant in catalysis. To further study HMGS mRNA expression that is developmentally regulated in flowers and seedlings, we have examined its mRNA distribution by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). We observed predominant localization of HMGS mRNA in the stigmas and ovules of flower buds and in the piths of seedling hypocotyls. RT-PCR analysis revealed that BjHMGS1 and BjHMGS2 but not BjHMGS3 and BjHMGS4were expressed in floral buds. To investigate the subcellular localization of BjHMGS1, we fused BjHMGS1 translationally in-frame either to the N- or C-terminus of green fluorescent protein (GFP). BjHMGS1-GFP and GFP-BjHMGS1 fusions were used in particle gun bombardment of onion epidermal cells and tobacco BY-2 cells. The GFP-BjHMGS1 construct was also used in agroinfiltration of tobacco leaves. Both GFP-fusion proteins were observed transiently expressed in the cytosol on confocal microscopy of onion epidermal cells, tobacco BY-2 cells, and agroinfiltrated tobacco leaves. Further, subcellular fractionation of total proteins from transgenic plants expressing GFP-BjHMGS1 derived from Agrobacterium-mediated transformation confirmed that BjHMGS1 is a cytosolic enzyme. We suggest that the presence of BjHMGS isoforms is likely related to the specialization of each in different cellular and metabolic processes rather than to a different intracellular compartmentation of the enzyme. © Springer-Verlag 2005.en_HK
dc.languageengen_HK
dc.publisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00425en_HK
dc.relation.ispartofPlantaen_HK
dc.subject3-Hydroxy-3-methylglutaryl-coenzyme A synthaseen_HK
dc.subjectBrassicaen_HK
dc.subjectGreen fluorescent proteinen_HK
dc.subjectIsoprenoiden_HK
dc.subjectMevalonateen_HK
dc.subjectSubcellular fractionationen_HK
dc.titleBrassica juncea HMG-CoA synthase: Localization of mRNA and proteinen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0032-0935&volume=221&spage=844&epage=856&date=2005&atitle=Brassica+juncea+HMG-CoA+synthase:+localization+of+mRNA+and+proteinen_HK
dc.identifier.emailChye, ML: mlchye@hkucc.hku.hken_HK
dc.identifier.authorityChye, ML=rp00687en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1007/s00425-005-1497-5en_HK
dc.identifier.pmid15770484-
dc.identifier.scopuseid_2-s2.0-23944456631en_HK
dc.identifier.hkuros108448en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-23944456631&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume221en_HK
dc.identifier.issue6en_HK
dc.identifier.spage844en_HK
dc.identifier.epage856en_HK
dc.identifier.isiWOS:000231313200011-
dc.publisher.placeGermanyen_HK
dc.identifier.scopusauthoridNagegowda, DA=8709830800en_HK
dc.identifier.scopusauthoridRamalingam, S=8709830400en_HK
dc.identifier.scopusauthoridHemmerlin, A=6602620485en_HK
dc.identifier.scopusauthoridBach, TJ=24786011400en_HK
dc.identifier.scopusauthoridChye, ML=7003905460en_HK

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