Characterization of the branched-chain amino acid aminotransferase enzyme family in tomato.
Branched-chain amino acids (BCAAs) are synthesized in plants from branched-chain keto acids, but their metabolism is not completely understood. The interface of BCAA metabolism lies with branched-chain aminotransferases (BCAT) that catalyze both the last anabolic step and the first catabolic step. In this study, six BCAT genes from the cultivated tomato (Solanum lycopersicum) were identified and characterized. SlBCAT1, -2, -3, and -4 are expressed in multiple plant tissues, while SlBCAT5 and -6 were undetectable. SlBCAT1 and -2 are located in the mitochondria, SlBCAT3 and -4 are located in chloroplasts, while SlBCAT5 and -6 are located in the cytosol and vacuole, respectively. SlBCAT1, -2, -3, and -4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, but SlBCAT1 and -2 were less effective than SlBCAT3 and -4 in growth restoration. All enzymes were active in the forward (BCAA synthesis) and reverse (branched-chain keto acid synthesis) reactions. SlBCAT3 and -4 exhibited a preference for the forward reaction, while SlBCAT1 and -2 were more active in the reverse reaction. While overexpression of SlBCAT1 or -3 in tomato fruit did not significantly alter amino acid levels, an expression quantitative trait locus on chromosome 3, associated with substantially higher expression of Solanum pennellii BCAT4, did significantly increase BCAA levels. Conversely, antisense-mediated reduction of SlBCAT1 resulted in higher levels of BCAAs. Together, these results support a model in which the mitochondrial SlBCAT1 and -2 function in BCAA catabolism while the chloroplastic SlBCAT3 and -4 function in BCAA synthesis.
Maloney
Gregory S
GS
Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611, USA.
Kochevenko
Andrej
A
Tieman
Denise M
DM
Tohge
Takayuki
T
Krieger
Uri
U
Zamir
Dani
D
Taylor
Mark G
MG
Fernie
Alisdair R
AR
Klee
Harry J
HJ
D000597:Amino Acids, Branched-Chain / Q000096:biosynthesis / Q000737:chemistry; D053898:Biosynthetic Pathways; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000254:growth & development; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D007178:Inbreeding; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics*; D005810:Multigene Family; D009928:Organ Specificity / Q000235:genetics; D020161:Physical Chromosome Mapping; D030821:Plants, Genetically Modified; D021381:Protein Transport; D040641:Quantitative Trait Loci / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
D000597:Amino Acids, Branched-Chain; D018076:DNA, Complementary; D012333:RNA, Messenger; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase