Characterization of the branched-chain amino acid aminotransferase enzyme family in tomato

Abstract

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.

Figure 1.

Synthetic pathways to BCAAs in plants. 1, Thr deaminase. 2, Acetolactate synthase. 3, Acetolactate isomeroreductase. 4, Dihydroxy acid dehydratase. 5, BCAT. 6, 2-Isopropylmalate synthase. 7, Isopropylmalate isomerase. 8, Isopropylmalate dehydrogenase.

Figure 2.

Quantification of SlBCAT RNA in different tissue types. Analysis was performed on three biological and three technical replicates for each sample. Values represent percentage of total mRNA per sample ± sd, calculated from a standard curve for each gene. Note differences in y axes. Expression of SlBCAT5 and SlBCAT6 was below the limit of detection.

Figure 3.

HPLC profile of BCAA content in different tissues of tomato. Br, Breaker stage fruit; FW, fresh weight.

Figure 4.

Subcellular localization of SlBCAT proteins. Each cDNA was fused to E-GFP at the C terminus and expressed in N. benthamiana leaf protoplasts. The left column shows GFP fluorescence, the middle column shows marker fluorescence, and the right column shows merging of GFP and marker. Chlorophyll autofluorescence was used to show the presence of chloroplasts for SlBCAT3, -4, -5, and -6. MitoTracker Orange dye was used to show mitochondria for SlBCAT1 and -2. Bars = 10 μm.

Figure 5.

Characterization of SlBCAT1 antisense lines. A, RT-PCR analyses of SlBCAT1 expression in transgenic plants. B, Content of BCAAs in red tomato fruits (40 dpa). Data represent means ± se from three independent biological replicates with two technical replicates for each. Asterisks show statistically significant changes according to Student's t test (P < 0.05). FW, Fresh weight; WT, wild type.

Figure 6.

Map positions of SlBCATs and BCAA QTLs. cM, Centimorgan.

Figure 7.

Mapping of the gene encoding SlBCAT4 and characterization of respective ILs. A, Schematic presentation of the introgressed region. B, Analysis of BCAA content in fruits by GC-MS. Data represent means ± se from six independent biological replicates. C, DNA-blot analysis of ILs. Ten micrograms of genomic DNA was digested with BfrI restriction enzyme, blotted, and hybridized with radiolabeled gene-specific probe. D, Analysis of the level of expression by RT-PCR. E, qRT-PCR analyses of BCAT1 and BCAT4 transcripts in tomato fruits of S. lycopersicum cv M82 and different ILs. Data represent means ± se from three independent biological replicates with two technical replicates for each point.