EXPRESSION OF FERREDOXIN-DEPENDENT GLUTAMATE SYNTHASE IN DARK-GROWN PINE-SEEDLINGS

被引:30
作者
GARCIAGUTIERREZ, A [1 ]
CANTON, FR [1 ]
GALLARDO, F [1 ]
SANCHEZJIMENEZ, F [1 ]
CANOVAS, FM [1 ]
机构
[1] UNIV MALAGA,FAC CIENCIAS,BIOQUIM & BIOL MOLEC LAB,E-29071 MALAGA,SPAIN
关键词
PINUS; GYMNOSPERM; CONIFER; AMINO ACID BIOSYNTHESIS; GLUTAMATE SYNTHASE; CDNA CLONING; LIGHT REGULATION;
D O I
10.1007/BF00019183
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a lambda gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.
引用
收藏
页码:115 / 128
页数:14
相关论文
共 49 条
  • [11] LIGHT-INDEPENDENT SYNTHESIS OF LHC IIB POLYPEPTIDES AND ASSEMBLY OF THE MAJOR PIGMENTED COMPLEXES DURING THE INITIAL-STAGES OF PINUS-PALUSTRIS SEEDLING DEVELOPMENT
    CANOVAS, F
    MCLARNEY, B
    SILVERTHORNE, J
    [J]. PHOTOSYNTHESIS RESEARCH, 1993, 38 (01) : 89 - 97
  • [12] ACCUMULATION OF GLUTAMINE-SYNTHETASE DURING EARLY DEVELOPMENT OF MARITIME PINE (PINUS-PINASTER) SEEDLINGS
    CANOVAS, FM
    CANTON, FR
    GALLARDO, F
    GARCIAGUTIERREZ, A
    DEVICENTE, A
    [J]. PLANTA, 1991, 185 (03) : 372 - 378
  • [13] CANTON, 1993, THESIS U MALAGA
  • [14] MOLECULAR CHARACTERIZATION OF A CDNA CLONE ENCODING GLUTAMINE-SYNTHETASE FROM A GYMNOSPERM, PINUS-SYLVESTRIS
    CANTON, FR
    GARCIAGUTIERREZ, A
    GALLARDO, F
    DEVICENTE, A
    CANOVAS, FM
    [J]. PLANT MOLECULAR BIOLOGY, 1993, 22 (05) : 819 - 828
  • [15] CANTON FR, 1992, METABOLISMO NITROGEN, P180
  • [16] CHLOROPHYLL BIOSYNTHESIS - RECENT ADVANCES AND AREAS OF CURRENT INTEREST
    CASTELFRANCO, PA
    BEALE, SI
    [J]. ANNUAL REVIEW OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY, 1983, 34 : 241 - 278
  • [17] A COMPREHENSIVE SET OF SEQUENCE-ANALYSIS PROGRAMS FOR THE VAX
    DEVEREUX, J
    HAEBERLI, P
    SMITHIES, O
    [J]. NUCLEIC ACIDS RESEARCH, 1984, 12 (01) : 387 - 395
  • [18] ELMLINGER MW, 1991, PLANTA, V183, P374, DOI 10.1007/BF00197736
  • [19] GALLARDO F, 1993, PLANT PHYSIOL BIOCH, V31, P189
  • [20] IMMUNOCHEMICAL ANALYSIS OF CHLOROPLAST POLYPEPTIDES FROM MARITIME PINE
    GARCIAGUTIERREZ, A
    CANTON, FR
    GALLARDO, F
    CANOVAS, FM
    [J]. PHYTOCHEMISTRY, 1993, 34 (02) : 337 - 341