Monocotyledonous C4 NADP(+)-malate dehydrogenase is efficiently synthesized, targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco.
Chloroplastic NADP(+)-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbon-fixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic protein-import machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of L-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.
Institut de Biotechnologie des Plantes, Unité de Recherche Associée CNRS D 1128, Université Paris-Sud, Orsay, France.
F
F
Gallardo
M
M
Miginiac-Maslow
R S
RS
Sangwan
P
P
Decottignies
E
E
Keryer
F
F
Dubois
E
E
Bismuth
S
S
Galvez
B
B
Sangwan-Norreel
P
P
Gadal
D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D018548:Cotyledon / Q000201:enzymology*; D018506:Gene Expression Regulation, Plant; D007150:Immunohistochemistry; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D006109:Poaceae / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D018749:RNA, Plant / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D014026:Nicotiana; D014170:Transformation, Genetic
D002264:Carboxylic Acids; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D013213:Starch; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)