Arabidopsis methionine gamma-lyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase

Abstract

The canonical pathway for isoleucine biosynthesis in plants begins with the conversion of threonine to 2-ketobutyrate by threonine deaminase (OMR1). However, demonstration of methionine gamma-lyase (MGL) activity in Arabidopsis (Arabidopsis thaliana) suggested that production of 2-ketobutyrate from methionine can also lead to isoleucine biosynthesis. Rescue of the isoleucine deficit in a threonine deaminase mutant by MGL overexpression, as well as decreased transcription of endogenous Arabidopsis MGL in a feedback-insensitive threonine deaminase mutant background, shows that these two enzymes have overlapping functions in amino acid biosynthesis. In mgl mutant flowers and seeds, methionine levels are significantly increased and incorporation of [(13)C]Met into isoleucine is decreased, but isoleucine levels are unaffected. Accumulation of free isoleucine and other branched-chain amino acids is greatly elevated in response to drought stress in Arabidopsis. Gene expression analyses, amino acid phenotypes, and labeled precursor feeding experiments demonstrate that MGL activity is up-regulated by osmotic stress but likely plays a less prominent role in isoleucine biosynthesis than threonine deaminase. The observation that MGL makes a significant contribution to methionine degradation, particularly in reproductive tissue, suggests practical applications for silencing the expression of MGL in crop plants and thereby increasing the abundance of methionine, a limiting essential amino acid.

Figure 1.

Pathways for Ile and Gly biosynthesis in plants. 2-Ketobutyrate, a precursor of Ile biosynthesis, can be produced from either Thr or Met. Thr deaminase competes with Thr aldolase for a common substrate. Gly can be synthesized from Thr, Ser, 3-phosphoglycerate (3-PGA), and glyoxylate. The names of Arabidopsis enzymes relevant to this project are indicated in parentheses.

Figure 2.

Relative mRNA accumulation of genes related to Met synthesis (ATMS1, ATMS2, ATMS3, and CGS) and degradation (SAM-1, SAM-2, SAM-3, and SAM-4) in flowers (A) and siliques (B and C) of wild-type Col-0 and mgl-2 plants. Flowers and siliques of 30-d-old plants were used to extract total RNA and used for real-time quantitative PCR to obtain relative mRNA expression levels. Expression levels of all genes were normalized with respect to the internal control UBQ10. The y axes are in arbitrary units. Note different scales for each y axis. Data are mean ± sd of n = 3 samples. *, P < 0.05, Student's t test.

Figure 3.

Relative amounts of MGL (A) and OMR (B) transcripts in fresh and drought-stressed flowers of wild-type Col-0, mgl-2, and omr1-5. Relative expression levels on the y axis are in arbitrary units. Values are mean ± sd of n = 3 samples. *, P < 0.05 relative to fresh tissue for each genotype, Student's t test.

Figure 4.

Salt sensitivity and drought-induced amino acid accumulation. A, Salt sensitivity of amino acid mutants. Seeds were planted on MS medium with 100 mm NaCl. Roots of 10-d-old seedlings were measured. Mean ± sd of n = 7. B, Amino acid accumulation in drought-stressed wild-type and mgl-2 flowers. Amino acids were quantified from fresh and dehydrated flowers of wild-type Col-0 and mgl-2. Fold-change due to dehydration was calculated. Mean ± sd of n = 4 to 6. *, P < 0.05, relative to wild-type Col-0, Student's t test.

Figure 5.

¹³C incorporation from Met into Ile. Flower stalks of 30-d-old plants of Col-0, mgl-2, and icl-2 plants were labeled with [¹³C₅ ¹⁵N]Met for 24 h. Siliques and flowers were harvested and dehydrated for 18 h before extracting amino acids. [¹³C₄]Ile recovered from siliques and flowers was measured by HPLC-mass spectrometry. Mean ± sd of n = 3. *, P < 0.05 relative to wild-type Col-0, Student's t test.

Figure 6.

Amino acid profiles of a Thr deaminase knockdown mutant. Amino acid abundance in leaves (A) and flowers (B) of the wild type and omr1-9. Mean ± sd of n = 4 to 6. *, P < 0.05, Student's t test. C, Rescue of omr1-9 seed amino acid phenotypes by MGL overexpression. Lines omr1-9 MGL-A and omr1-9 MGL-B are independent p35S:MGL transformations. Mean ± sd of n =3. Only amino acids with significant changes in abundance, as well as Met, are shown. *, P < 0.05 relative to wild-type Col-0, Student's t test.

Figure 7.

Met incorporation into Ile in Thr deaminase mutants. Flower stalks of Thr deaminase feedback-insensitive (omr1-5) and knockdown (omr1-9) mutants were labeled with [¹³C₅ ¹⁵N]Met. Accumulation of [¹³C₄]Ile was measured in siliques and flowers. Mean ± sd of n = 3. *, P < 0.05, relative to wild-type Col-0, Student's t test.