Genome-wide analysis of branched-chain amino acid levels in Arabidopsis seeds

Abstract

Branched-chain amino acids (BCAAs) are three of the nine essential amino acids in human and animal diets and are important for numerous processes in development and growth. However, seed BCAA levels in major crops are insufficient to meet dietary requirements, making genetic improvement for increased and balanced seed BCAAs an important nutritional target. Addressing this issue requires a better understanding of the genetics underlying seed BCAA content and composition. Here, a genome-wide association study and haplotype analysis for seed BCAA traits in Arabidopsis thaliana revealed a strong association with a chromosomal interval containing two branched-chain amino acid transferases, BCAT1 and BCAT2. Linkage analysis, reverse genetic approaches, and molecular complementation analysis demonstrated that allelic variation at BCAT2 is responsible for the natural variation of seed BCAAs in this interval. Complementation analysis of a bcat2 null mutant with two significantly different alleles from accessions Bayreuth-0 and Shahdara is consistent with BCAT2 contributing to natural variation in BCAA levels, glutamate recycling, and free amino acid homeostasis in seeds in an allele-dependent manner. The seed-specific phenotype of bcat2 null alleles, its strong transcription induction during late seed development, and its subcellular localization to the mitochondria are consistent with a unique, catabolic role for BCAT2 in BCAA metabolism in seeds.

Figure 1.

Compartmentalization of BCAA Biosynthesis and Degradation. Key enzymes and metabolites of BCAA biosynthesis and degradation in the chloroplast and mitochondrion, respectively, are abbreviated and given in boxes. BCAT enzymes are highlighted by black boxes. AHAS, acetohydroxyacid synthase; 2A2HB, 2-aceto-2-hydroxybutanoate; 2AL, 2-acetolactate; BCKDH, branched-chain keto acid dehydrogenase; DHAD, dihydroxyacid dehydratase; 2,3DH3MB, 2,3-dihydroxy-3-methylbutanoate; 2,3DH3MP, 2,3-dihydroxy-3-methylpentanoate; Enol, enoyl-CoA hydratase; 2-IPM, 2-isopropylmalate; 3-IPM, 3-isopropylmalate; IPMDH, isopropylmalate dehydrogenase; IPMI, isopropylmalate isomerase; IPMS, isopropylmalate synthase; KARI, ketol acid reductoisomerase; 3MOB, 3-methyl-2-oxobutanoate; 3MOP, 3-methyl-2-oxopentanoate; 4MOP, 4-methyl-2-oxopentanoate; 2OB, 2-oxobutanoate; Pyr, pyruvate; TCA, tricarboxylic acid; TD, Thr deaminase. Known functions for specific BCATs are indicated, and steps without a specific associated BCAT are indicated by “BCAT?”. BCAT2 localization and catabolic role were deduced in this article.

Figure 2.

GWAS for the Five BCAA Traits with Significant SNP–Trait Associations. Scatterplots of association results from a unified mixed-model analysis for five BCAA traits across the five Arabidopsis chromosomes are shown. Negative log₁₀-transformed P values from the GWAS analysis are plotted against the genomic physical position. P values for SNPs that are statistically significant for a trait at 5% FDR are colored in red. Traits are as follows: Ile (A), Ile/total (B), Ile/BCAAs (C), Leu/total (D), and Val/BCAAs (E). All five traits shared significant SNP–trait associations on chromosome 1. Five additional chromosomal regions with significant SNP–trait associations are marked by different red shapes, with the same shapes indicating a corresponding chromosomal region influencing multiple traits.

Figure 3.

GWAS for Ile/BCAAs with Significant SNP–Trait Associations. (A) Scatterplot of association results from a unified mixed-model analysis of Ile/BCAAs on chromosome 1. Negative log₁₀-transformed P values from the GWAS analysis are plotted against the genomic physical position. P values for SNPs that are statistically significant at 5% FDR are colored in red. (B) Graphical representation of genes and haploblocks within the genomic region spanning SNPs that showed significant associations across at least four BCAA traits (chromosome 1,3,274,080 to 3,297,645). Gene models are represented by black (exons) and white (untranslated regions) connected boxes. Haploblocks are represented with gray, red, and white squares; white indicates the haploblock containing SNP5373 that includes a large part of BCAT2; red represents the most significant haploblock associations across all five BCAA traits. (C) Average levels of Ile, Ile/total, Leu/total, Val/BCAAs, and Ile/BCAAs from three biological replicates of the population. Black and white bars represent the trait average from accessions with the CA-GA (white) and TT-AG (black) haplotypes for haploblocks 8 and 9, respectively. In the case of Ile, Ile/total, Leu/total, and Ile/BCAAs, the CA-GA haplotype results in higher levels; lower levels are seen in the case of Val/BCAA. *P < 1.0E-04, **P < 1.5E-16 by Student’s t test. Error bars represent se (n > 80).

Figure 4.

Seed and Leaf FAA Ratios in BCAT Mutants Compared with the Wild Type (Col-0 or WS-4). FAA levels were measured from dry seeds (A) and leaves harvested from 4-week-old plants (B). Values represent fold changes in FAA for bcat2-2 (dark gray bars), bcat2-1 (black bars), bcat1-1 (dotted bars), bcat1-2 (striped bars), and erd4 (white bars; seeds only) relative to their respective wild type (WT). Averages and se were calculated from five measurements. Statistical analyses are presented in Supplemental Data Set 4 online.

Figure 5.

Complementation of bcat2-1 with the Bay and Sha Alleles. Absolute levels of free BCAAs (A) and ratios of Ile/total and Leu/total (B) and Ile/BCAAs and Val/BCAAs (C) are shown for dry seeds of Col-0 (white bars), bcat2-1 (black bars), bcat2-1/BCAT2-(Sha) (dark gray bars), and bcat2-1/BCAT2-(Bay) (light gray bars). The BCAA levels of bcat2-1/BCAT2-(Bay) were significantly lower than those of Col-0, while bcat2-1/BCAT2-(Sha) levels were indistinguishable from those of Col-0 except for Ile/BCAAs. Means and se were calculated from 9 independent transformation events for bcat2-1/BCAT2-(Bay), 10 for bcat2-1/BCAT2-(Sha), and 11 for Col-0 plants. *P <0.05, **P < 3E-03 calculated from Student’s t test compared with Col-0.

Figure 6.

BCAT1 and BCAT2 Transcript Levels during Seed Maturation and Desiccation. Transcript levels for BCAT2 (A) and BCAT1 (B) were measured from Bay (white bars) and Sha (black bars) accessions over five time points of seed maturation and desiccation using quantitative PCR with ACTIN2 mRNA as an internal control. Error bars represent se (independent biological replicates; n = 3).

Figure 7.

Colocalization of BCAT2 with a Mitochondrial Organelle Marker. YFP was fused in frame to the C terminus of BCAT2, and the construct was transformed into Arabidopsis plants expressing the CFP mitochondrial marker mt-ck CS16262 (Nelson et al., 2007). (A) 35S:BCAT2-YFP is shown in magenta. (B) Mitochondrial marker mt-ck CS16262 is shown in cyan. (C) Chlorophyll autoflorescence is shown in red. (D) Merge of (A), (B), and (C).