Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis

Abstract

Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis.

Figure 1.

Seed germination analysis of mMDH mutants. A, Single mutants of mMDH (mmdh1-2 and mmdh2-1), mMDH double mutant (mmdh1-2-mmdh2-1), and complemented line (mmdh1mmdh2 35S: MMDH1) seeds were grown on agar plates for 19 d under short-day conditions. The seed germination rates were calculated as percentage of radicle emergence (n = 20–50) observed with a dissecting microscope, and the cumulative germination rate is shown for each genotype. WT, Wild type. B, Seed viability assay of wild-type (i), mmdh1-2mmdh2-1 (ii), and heat-killed wild-type (iii) seeds. The embryos excised from seeds were stained with TZ chloride solution and examined with a light microscope. Examples of mmdh1-2-mmdh2-1 embryos arrested between torpedo-shaped (circled in red) and bent-cotyledon (circled in blue) stages are highlighted.

Figure 2.

Length, relative growth rate, and respiratory rate of mMDH mutant roots. A, Primary root length of mMDH mutant seedlings (means ± se; n = 24–72) compared with the wild type (WT) grown on vertical agar plates for 10 d under short-day conditions. Student’s t-test analysis showed significant differences from the wild type: **, P < 0.01. B, Growth rate calculations from root length measurements at days 10 and 16 under short-day conditions (means ± se; n = 16–49). Student’s t test analysis showed significant differences from the wild type: **, P < 0.01. C, Whole-root respiration rates (means ± se; n = 4) from 21-d-old seedlings. Student’s t test analysis showed significant differences from the wild type: *, P < 0.05 and **, P < 0.01. FW, Fresh weight. D, OCRs of root expanded (EXP) and root tip (TIP) regions excised from 12-d-old seedlings, grown as in A. Values represent the OCR adjusted with root volume in mm³ (means ± se; n = 8–21). Student’s t-test analysis shows significant differences from the wild type: *, P < 0.05 and **, P < 0.01; significant differences between mutants and the complemented line also are indicated (orange asterisks): *, P < 0.05 and **, P < 0.01.

Figure 3.

Seed phenotypes and respiration during seed development. A and B, Representative images of the phenotypes of developing siliques (A) and seeds of the wild type (WT), mmdh1-2mmdh2-1, and mmdh1mmdh2 35S: MMDH1 (B) at developmental stages G, GR, and R. C, Seed respiration at different developmental stages. Values represent the mean OCR per seed (means ± se; n = 14–23). Student’s t-test analysis showing significant differences from the wild type is marked with black asterisks, while significant differences between mmdh1-2mmdh2-1 and the complemented line are indicated by orange asterisks, for each maturation stage: *, P < 0.05 and **, P < 0.01. D, Percentage of wild-type and mMDH mutant seed viability at different maturation stages based on TZ staining. Total number of seeds is as follows: for the wild type, G (66), GR (52), and R (81); for mmdh1-2-mmdh2-1, G (65), GR (73), and R (76); and for mmdh1mmdh2 35S: MMDH1, G (63), GR (61), and R (73).

Figure 4.

Seed metabolite changes over development and in mMDH mutants relative to the wild type (WT) at different seed maturation stages. Metabolite amount in the wild type was normalized to the internal standard ribitol and then globally normalized to the average abundance across all stages in each sample before averaging from biological replicates (averages ± se; n = 3–5). Student’s t-test analysis was used to obtain significant differences of individual metabolites between G and GR or R stage: *, P < 0.05 and **, P < 0.01. Light gray boxes denote metabolites that were not measured in this study.

Figure 5.

Seed metabolite changes over development and in mMDH mutants relative to the wild type (WT) at different seed maturation stages. Metabolite amount in mutants was normalized to the internal standard ribitol before normalization to the average abundance across all stages in each sample, and then biological replicates were averaged (averages ± se; n = 3–5). Values represent average ratios of normalized metabolite levels between mutants and the wild type at the corresponding stage: blue (low) to red (high). Gray boxes indicate that metabolite ratios could not be determined, as the compounds were identified in the mutants but not in the wild type at the respective stage. Boldface and boxed numbers denote significant differences of individual metabolites between mutants and the wild type at the corresponding maturation stage according to one-way ANOVA with posthoc analysis at P < 0.05.

Figure 6.

Seed viability after accelerated aging. Seeds were aged at 60% RH and 45°C for 35 to 45 d prior to seed germination assays in long-day lighting regimes. A, Light microscope observation of seed viability by TZ staining. Images of representative seed embryos before aging are as follows: a, wild type; b, mmdh1-2mmdh2-1; c, mmdh1mmdh2 35S: MMDH1; and d, heat-killed wild type. Images of representative seed embryos after 35 d of aging treatment are as follows: e, wild type; f, mmdh1-2mmdh2-1; g, mmdh1mmdh2 35S: MMDH1; and h, heat-killed wild type. B, Seed survival curves for the wild type (WT; blue), mmdh1-2mmdh2-1 (red), and mmdh1mmdh2 35S: MMDH1 (green). The percentage of seed germination was determined at day 7 after seeds were sown on agar plates. The fitted seed survival curve was plotted by Probit analysis based on the cumulative normalized seed germination data of each genotype.