The mitochondrial folylpolyglutamate synthetase gene is required for nitrogen utilization during early seedling development in arabidopsis

Abstract

Investigations into the biochemical processes and regulatory mechanisms of nitrogen (N) utilization can aid in understanding how N is used efficiently in plants. This report describes a deficiency in N utilization in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant of the mitochondrial folylpolyglutamate synthetase gene DFC, which catalyzes the conjugation of glutamate residues to the tetrahydrofolate during folate synthesis. The mutant seedlings displayed several metabolic changes that are typical of plant responses to low-N stress, including increased levels of starch and anthocyanin synthesis as well as decreased levels of soluble protein and free amino acid, as compared with those in wild-type seedlings when external N was sufficient. More striking changes were observed when dfc seedlings were grown under N-limited conditions, including shorter primary roots, fewer lateral roots, higher levels of glycine and carbon-N ratios, and lower N content than those in wild-type seedlings. Gene expression studies in mutant seedlings revealed altered transcript levels of several genes involved in folate biosynthesis and N metabolism. The biochemical and metabolic changes also suggested that N assimilation is drastically perturbed due to a loss of DFC function. The observation that elevated CO(2) partly rescued the dfc phenotypes suggests that the alterations in N metabolism in dfc may be mainly due to a defect in photorespiration. These results indicate that DFC is required for N utilization in Arabidopsis and provide new insight into a potential interaction between folate and N metabolism.

Figure 1.

Effects of NO₃⁻ on the growth of wild-type (white bars), dfc (black bars), and DFC complementation (gray bars) seedlings. A, Effects of different NO₃⁻ levels (0, 0.3, 3, and 9.4 n) on the primary root length with 30 mm Suc in the medium and NO₃⁻ as the sole N source. B, Wild-type (WT), dfc, and DFC complementation (pDFC::DFC) seedlings grown on 0.3 or 9.4 n medium for 3, 6, and 9 d. C, Cotyledon width of wild-type, dfc, and DFC complementation seedlings grown on 0.3 or 9.4 n medium. D, Cotyledon and leaves of wild-type and dfc seedlings grown on 0.3 or 9.4 n medium for 3, 6, and 9 d. E, Lateral root initiation events of wild-type, dfc, and DFC complementation seedlings grown on 0.3 or 9.4 n medium for 5, 7, and 9 d. F, Fresh weight and dry weight per plant of wild-type, dfc, and DFC complementation seedlings grown on 0.3 or 9.4 n medium for 3, 6, and 9 d. Data are means ± sd (n = 4), and each replicate consisted of a pool of 10 to 15 plants. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test). [See online article for color version of this figure.]

Figure 2.

The maladjustment time window in the dfc mutant (black bars) compared with that in wild-type (white bars) seedlings, and the growth rates of those seedlings on 0.3 or 9.4 n medium. A, Primary root length of wild-type and dfc seedlings grown on the 0.3 n medium for 2 d after transfer from one-half-strength (1/2) MS medium on different days (0–5). B, Primary root length of wild-type and dfc seedlings on day 7 from the start of incubation. Both types of seedlings were grown on one-half-strength MS medium for 0 to 7 d and then transferred onto the 0.3 n medium for the remaining time (7–0 d). C, Primary root growth rates of wild-type and dfc seedlings from days 3 to 6 and from days 6 to 9 on 0.3 or 9.4 n medium. Data are means ± sd (n = 4), and each replicate consisted of a pool of 10 to15 plants. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test).

Figure 3.

Altered C and N metabolite levels in 12-d-old wild-type (white bars) and dfc (black bars) seedlings on 0.3 or 9.4 n medium. The contents of starch (A), anthocyanin (B), Glc (C), Fru (D), soluble protein (E), NH₄⁺ (F), chlorophyll (G), and N (H) as well as C-N ratio (I) in seedlings grown on 0.3 or 9.4 n medium are shown. Data are means ± sd (n = 4), and each replicate consisted of a pool of 30 to 50 plants. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test). FW, Fresh weight.

Figure 4.

Altered transcript levels of the genes involved in N metabolism in wild-type (white bars) and dfc (black bars) seedlings on 0.3 or 9.4 n medium. A, Fold change in expression of the genes involved in NO₃⁻ reduction and N assimilation in 9-d-old seedlings. The transcript level of each gene is normalized to that in the wild type on the 9.4 n medium. B, Fold change in expression of NRT1.1, NRT2.1, and ANR1 on days 4, 6, and 9. The transcript level of each gene is normalized to that of the wild type grown on the 9.4 n medium on day 4.

Figure 5.

Altered biochemical characteristics of N assimilation in 12-d-old wild-type (white bars) and dfc (black bars) seedlings on 0.3 or 9.4 n medium. A, Relative protein content involved in NO₃⁻ reduction and N assimilation (NR, GOGAT, GS1, and GS2) in seedlings detected with the corresponding antibodies. Coomassie blue staining of a large subunit of the Rubisco subunit was used as the loading control. WT, Wild type. B to E, NR (including NRmax and NRact; B), NiR (C), GOGAT (D), and GS (E) activities in the seedlings. Data are means ± sd (n = 4), and each replicate consisted of a pool of 30 to 50 plants. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test).

Figure 6.

Altered amino acid profiles in 12-d-old wild-type (white bars), dfc (black bars), and DFC complementation (gray bars) seedlings on 0.3 or 9.4 n medium. Total free amino acid (A) and some individual amino acid (B and C) contents in seedlings are shown. Data are represented as means ± sd (n = 4), and each replicate consisted of 200 mg of pooled plant material. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test). FW, Fresh weight.

Figure 7.

Altered transcript levels of the genes involved in folate metabolism and the synthesis of one-carbon units in wild-type (white bars) and dfc (black bars) seedlings on 0.3 or 9.4 n medium. A and B, The transcript levels of DFA, DFB, DFC, and DFD in seedlings on day 2 (A) and day 9 (B) are shown as percentages of that of the UBC gene. Black bars with single asterisks indicate significant differences at P < 0.05 (Student’s t test). C and D, Transcript levels of other genes involved in folate metabolism (C) and genes involved in the synthesis of one-carbon units (D) in 9-d-old seedlings are shown, normalized to those of the wild type on the 9.4 n medium. FDF, FORMYTETRAHYDROFOLATE DEFORMYLASE; DHC, METHYLENETETRAHYDROFOLATE DEHYDROGENASE; 5-FCL, 5-FORMYLTETRAHYDROFOLATE CYCLOLIGASE.

Figure 8.

Altered folate profiles in 12-d-old wild-type (WT; white bars), dfc (black bars), and DFC complementation (gray bars) seedlings on 0.3 or 9.4 n medium. A, Various folate types and total folate levels of seedlings. The folate species detected were as follows: THF, 5,10-methenyltetrahydrofolate (5,10-CH=THF), 5-M-THF, 5-F-THF, and dihydrofolate (DHF). Note that THF and 5,10-CH₂-THF are grouped, and 10-formyl-THF (10-CHO-THF) and 5,10-CH=THF are grouped because the procedure used for folate analysis results in interconversions in these two pairs of folate species. B, Folate glutamylation profiles are shown as percentages of each class in the sum of the amount of folylpolyglutamates (5-M-THF-Glu_n and 5-F-THF-Glu_n; n = 1–4). C, LC-MS relative peak areas of folylpolyglutamates (5-M-THF-Glu_n and 5-F-THF-Glu_n; n = 5, 7, or 8) normalized to the levels of the wild type. Data are means ± sd (n = 4), and each replicate consisted of 100 mg of pooled plant material. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test). FW, Fresh weight.

Figure 9.

Effects of elevated CO₂ on wild-type (WT; white bars), dfc (black bars), and DFC complementation (gray bars) seedlings on 0.3 or 9.4 n medium. A, The primary root length of seedlings under N-limited conditions with ambient air or elevated CO₂. B, Wild-type, dfc, and DFC complementation seedlings grown on the 0.3 n medium for 9 d with elevated CO₂. C, Total free amino acid contents of 12-d-old seedlings on 0.3 or 9.4 n medium with elevated CO₂. D, Contents of some individual amino acids in 12-d-old seedlings on the 9.4 n medium with elevated CO₂. E, Contents of some individual amino acids in 12-d-old seedlings on the 0.3 n medium with elevated CO₂. F, The percentage of each folate in the sum of the amount of folylpolyglutamates (5-M-THF-Glu_n and 5-F-THF-Glu_n; n = 1–4) in 12-d-old seedlings on 0.3 or 9.4 n medium with elevated CO₂. Data are means ± sd (n = 4), and each replicate consisted of a pool of 30 to 50 plants. Black bars with single asterisks indicate significant differences at P < 0.05, and those with double asterisks indicate highly significant differences at P < 0.01 (Student’s t test). FW, Fresh weight. [See online article for color version of this figure.]