Physiological roles of the beta-substituted alanine synthase gene family in Arabidopsis

Abstract

The beta-substituted alanine (Ala) synthase (Bsas) family in the large superfamily of pyridoxal 5'-phosphate-dependent enzymes comprises cysteine (Cys) synthase (CSase) [O-acetyl-serine (thiol) lyase] and beta-cyano-Ala synthase (CASase) in plants. Nine genomic sequences encode putative Bsas proteins in Arabidopsis thaliana. The physiological roles of these Bsas isoforms in vivo were investigated by the characterization of T-DNA insertion mutants. Analyses of gene expression, activities of CSase and CASase, and levels of Cys and glutathione in the bsas mutants indicated that cytosolic Bsas1;1, plastidic Bsas2;1, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsas1;1 has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;1 is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-of-flight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry. The level of gamma-glutamyl-beta-cyano-Ala decreased in the mutant bsas3;1, indicating the crucial role of Bsas3;1 in beta-cyano-Ala metabolism in vivo.

Figure 1.

Gene expression analysis of Bsas genes in the leaf and root. A, Quantitative real-time PCR analysis. Total RNA was extracted from leaves and roots of 2-week-old wild-type plants. Data are the means of triplicate determinations ±sd (bars). B, Expression analysis using the Genevestigator tool based on DNA microarray analysis (https://www.genevestigator.ethz.ch). C, Expression analysis performed using the number of ESTs in the TAIR database (http://www.arabidopsis.org).

Figure 2.

Bsas transcripts in bsas mutants by RT-PCR analysis. Total RNA was extracted from 2-week-old homozygous bsas mutants and the wild type. RT-PCR was performed with specific primers for Bsas genes and a tubulin gene used as the control.

Figure 3.

CSase activity in bsas mutants. Total extracts of soluble proteins were prepared from leaves and roots of 2-week-old plants and CSase activity was determined by measuring the formation of Cys in the leaf (A) and root (B). Data represent the mean of four experiments with 15 plants in each (±sd). Differences between the wild type and bsas mutants analyzed using Student's t test were statistically significant (*, P < 0.01; **, P < 0.005; ***, P < 0.001).

Figure 4.

CASase activity in bsas mutants. Total extracts of soluble proteins were prepared from leaves and roots of 2-week-old plants and CASase activity was determined by measuring the formation of H₂S from Cys in leaf (A) and root (B). Data represent the mean of three experiments with 15 plants in each (±sd). Differences between the wild type and bsas mutants analyzed using Student's t test were statistically significant (*, P < 0.01; **, P < 0.005; ***, P < 0.001).

Figure 5.

Accumulation of Cys and GSH in bsas mutants. Thiols were extracted from leaves and roots of 2-week-old plants. Cys and GSH in leaf (A and B) and root (C and D) were determined by HPLC analysis. Data represent the mean of five experiments with 15 plants in each (±sd). Differences between the wild type and bsas mutants analyzed using Student's t test were statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.005).

Figure 6.

A, Comparison of the metabolite accumulation between the wild type and bsas mutants. The mean peak areas of the wild type and each bsas mutant are shown on the x and y axes, respectively. Data represent the mean of eight experiments for the wild type and three experiments for bsas mutants with 20 plants. Differences between the wild type and bsas mutants analyzed using Welch's t test were statistically significant (black triangles [P < 0.05] and white triangles [P > 0.05] for GC-TOF/MS; black diamonds [P < 0.05] and white diamonds [P > 0.05] for CE-TOF/MS). The black line represents the diagonal line (y = x). The black circle on the plot of the wild type and bsas3;1 indicates γ-glutamyl-β-cyano-Ala. B, Accumulation of γ-glutamyl-β-cyano-Ala in bsas mutants. γ-Glutamyl-β-cyano-Ala contents were measured in leaves by CE-TOF/MS. Data represent the mean of eight experiments for the wild type and three experiments for bsas mutants with 20 plants in each (±sd). Differences between the wild type and bsas mutants analyzed using Welch's t test were statistically significant (***, P < 0.001).

Figure 7.

Accumulation of γ-glutamyl-β-cyano-Ala in the leaf of the wild type and bsas3;1 mutant treated with β-cyano-Ala. Plants grown for 3 weeks on GM-agar medium were transferred to GM-agar medium or medium containing β-cyano-Ala (100 μm and 1 mm) and grown for 3 d. γ-Glutamyl-β-cyano-Ala was analyzed by fluorescence HPLC. Data represent the mean of three experiments with 15 plants in each (±sd).

Figure 8.

Schematic representation of Cys and β-cyano-Ala metabolism in Arabidopsis. A, Schematic representation of Bsas isoforms involved in Cys metabolism in the cytosol, plastid, and mitochondrion. Solid arrows show enzymatic reactions. γ-EC, γ-Glutamyl-Cys. B, Metabolism of β-cyano-Ala. β-Cyano-Ala is metabolized to γ-glutamyl-β-cyano-Ala by GGT or to Asn and Asp by the gene product of NIT4.