Arabidopsis Plastidial Folylpolyglutamate Synthetase Is Required for Seed Reserve Accumulation and Seedling Establishment in Darkness

<div><p>Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (<i>atdfb-3</i>) of the plastidial folylpolyglutamate synthetase gene (<i>AtDFB</i>) was defective in seed reserves and skotomorphogenesis. Lower carbon (C) and higher nitrogen (N) content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that <i>atdfb-3</i> seeds contained less total amino acids and individual Asn and Glu as well as NO₃⁻. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in <i>atdfb-3</i> in darkness under sufficient NO₃⁻ conditions, and further enhanced under NO₃⁻ limited conditions. The strong expression of <i>AtDFB</i> in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO₃⁻ during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in <i>atdfb-3</i> seedlings with NO₃⁻ as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in <i>atdfb-3</i> etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in <i>atdfb-3</i>. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis.</p></div

Metabolite profiles in WT and <i>atdfb-3</i> seeds.

<p>Propanoic acid (3∶0), tetradecanoic acid (14∶0), hexadecanoic acid (16∶0), octadecanoic acid (18∶0), 9,12-octadecadienoic acid (18∶2), linolenic acid (18∶3), eicosanoic acid (20∶0), 11-eicosenoic acid (20∶1), docosanoic acid (22∶0), and tetracosanoic acid (24∶0). n = 5, and each replicate consisted of 20 mg of pooled seeds. Data represent means ± SD. White bars with * indicate a significant difference at <i>P</i><0.05, and ** indicates a highly significant difference at <i>P</i><0.01 (Student’s <i>t</i>-test).</p

Contents of N-rich metabolites in WT and <i>atdfb-3</i> seeds.

<p>(A) Soluble protein. (B) Total amino acids. (C) Individual amino acids. (D) NO₃⁻ content. Data represent means ± SD. n = 3. A, each replicate consisted of 50 mg pooled plant material; B and C, each replicate consisted of 300 mg pooled plant material; D, each replicate consisted of 1 g pooled plant material. Bars with * indicate a significant difference at <i>P</i><0.05, and ** indicates a highly significant difference at <i>P</i><0.01 (Student’s <i>t</i>-test).</p

Histochemical localization of <i>AtDFB</i> promoter activity and <i>AtDFB</i> expression patterns.

<p>(A) Schematic diagram of the <i>AtDFB: GUS</i> construct. LB and RB indicate the left and right borders, respectively, and Kana indicates the kanamycin resistance gene. (B) GUS staining of 2- and 3-day-old etiolated seedlings under 9.4 N or 0.3 N conditions.</p

N Metabolites in 2-day-old WT and <i>atdfb-3</i> germinating seeds under 9.4 N or 0.3 N.

<p>(A) C content. (B) N content. (C) Soluble protein content. Data represent means ± SD. A–B, n = 4, and each replicate consisted of 10 mg DW pooled plant material; C, n = 3, and each replicate consisted of 50 mg pooled plant material. A significant difference at <i>P</i><0.05 is indicated by *, and a highly significant difference at <i>P</i><0.01 is indicated by ** (Student’s <i>t</i>-test).</p

C and N contents in mature WT, <i>atdfb-3</i> and <i>AtDFB</i> complemented (COM) seeds.

<p>(A) C content. (B) N content. Data represent means ± SD. n = 4, and each replicate consisted of 10 mg DW of pooled plant material. Bars with ** indicate a highly significant difference at <i>P</i><0.01 (Student’s <i>t</i>-test).</p

Altered biochemical characteristics of N reduction and assimilation in 6-day-old etiolated WT, <i>atdfb-3</i> and <i>AtDFB</i> complemented (COM) seedlings on 9.4 N or 0.3 N medium.

<p>(A) NO₃⁻ content. (B) NO₂⁻ content. (C) NiR activity. (D) GS activity. (E) GOGAT activity. (F) Altered transcript levels of genes involved in nitrate transport, reduction, and N assimilation. Data are means ± SD (n = 3). In panel (A) and (B), each replicate consisted of 1 g of pooled plant material. In panel (C) and (D), each replicate consisted of 200 mg of pooled plant material. In panel (E), each replicate consisted of 500 mg of pooled plant material. A significant difference at <i>P</i><0.05 is indicated by *, and a highly significant difference at <i>P</i><0.01 is indicated by ** (Student’s <i>t</i>-test).</p

Folate profiles in 6-day-old WT, <i>atdfb-3</i>, and <i>AtDFB</i> complemented (COM) seedlings grown on 9.4 N or 0.3 N medium.

<p>(A) Levels of individual folates and total folates in seedlings. The folate species detected were: 5-formyl-THF (5-F-THF), 5-methyl-THF (5-M-THF), 5,10-methenyl-THF (5,10-CH = THF), 10-formyl-THF (10-CHO-THF), 5,10-methylene THF (5,10-CH₂-THF), tetrahydrofolate (THF), and dihydrofolate (DHF). Note that 10-formyl-THF (10-CHO-THF) and 5,10-CH = THF are grouped together and THF and 5,10-methylene THF (5,10-CH₂-THF) are grouped together because the procedure used for folate analysis results in inter-conversion of these pairs of folate species. (B) Relative LC-MS peak areas of folylpolyglutamates (5-M-THF-Glu_n and 5-F-THF-Glu_n, n = 5, 6, 7, or 8). Data are means ± SD (n = 5). Each replicate consisted of 100 mg of pooled plant material. A significant difference at <i>P</i><0.05 is indicated by *, and a highly significant difference at <i>P</i><0.01 is indicated by ** (Student’s <i>t</i>-test).</p

Maladjustment time window in <i>atdfb-3</i> etiolated seedlings.

<p>(A) Images of 6-day-old etiolated WT and <i>atdfb-3</i> seedlings grown on different amounts of nitrogen (N). Scale bar, 3 mm. (B) Hypocotyl lengths of 6-day-old etiolated WT and <i>atdfb-3</i> seedlings grown on different amounts of nitrogen (N). (C) Effects of carbon (C)-free or phosphate (P)-free media on the hypocotyl lengths of 6-day-old WT and <i>atdfb-3</i> etiolated seedlings. (D) FE-SEM image of hypocotyl cells of 6-day-old etiolated WT and <i>atdfb</i>-3 seedlings grown on 9.4 N and 0.3 N medium. Scale bar, 100 µm. (E) Hypocotyl cell length in 6-day-old etiolated WT and <i>atdfb</i>-3 seedlings on 9.4 N and 0.3 N medium. (F) Hypocotyl length of 6-day-old WT and <i>atdfb-3</i> etiolated seedlings. Both genotypes seedlings were grown on 9.4 N medium for 0 to 6 days and then transferred to 0.3 N medium for the remaining days (0 to 6 days). (G) Hypocotyl length of 6-day-old WT and <i>atdfb-3</i> etiolated seedlings. Seedlings of both genotypes were grown on 0.3 N medium for 0 to 6 days and then transferred onto 9.4 N medium for the remaining days (0 to 6 days). (H) Hypocotyl length of 6-day-old WT and <i>atdfb-3</i> etiolated seedlings with or without endosperm in 9.4 N and 0.3 N media. Data represent the means ± SD (in panel B, C, E, F, G and H, n = 15). White bars with * indicate a significant difference at <i>P</i><0.05, and ** indicates a highly significant difference at <i>P</i><0.01 (Student’s <i>t</i>-test).</p

Seed characteristics in WT and <i>atdfb-3</i>.

<p>(A) Number of seeds per silique. (B) Seed length (black bars) and width (white bars). (C) Seed weight. Data represent means ± SD. A, n = 30; B, n = 3, and each replicate contained 30 seeds. Seeds were viewed using a ZEISS Imager M1 DIC microscope and measured using ImageJ; C, n = 5, and each replicate consisted of a pool from 10 plants. Bars with ** indicate a highly significant difference at <i>P</i><0.01 (Student’s <i>t</i>-test).</p