Dicarboxylates such as malate, oxaloacetate, succinate, and 2-oxoglutarate are metabolic workhorses. They shuttle reducing power between cellular compartments, feed the tricarboxylic acid cycle, and underpin processes ranging from fatty acid breakdown to amino acid synthesis and photorespiration. The mitochondrial dicarboxylate carrier, or DIC, belongs to the large SLC25 mitochondrial carrier family and sits in the inner mitochondrial membrane, exchanging these acids between the organelle and the cytosol. Arabidopsis carries three DIC genes. AtDIC1 and AtDIC2 share about 70 percent of their amino acid residues and are broadly expressed, while AtDIC3, sharing only 55 to 60 percent identity with its siblings, is an ancient acquisition found exclusively in the mustard family and shows a far more restricted expression pattern. AtDIC2 has already been shown to be essential: it exports cytosolic malate in exchange for mitochondrial citrate, and plants lacking it grow poorly, a defect that neither AtDIC1 nor AtDIC3 can rescue.

To probe the neglected pair, the team obtained T-DNA insertion lines for AtDIC1 and AtDIC3 from the Arabidopsis Biological Resource Center, confirmed the insertion sites by sequencing, and verified near-complete knockout of transcript levels in the strongest alleles, atdic1-1 and atdic3-1, with reductions of 97.9 and 99.9 percent respectively. They then crossed the two null alleles to build a double mutant. Expression profiling showed that AtDIC1 is ubiquitous, peaking in rosettes and flowers, while AtDIC3 transcripts concentrate in post-anthesis flowers and siliques, hinting at a specialized reproductive role. During germination, AtDIC1 and AtDIC2 expression climbed steadily over the first four days after stratification, exactly when seed metabolism reactivates, whereas AtDIC3 stayed flat until day four.

The germination experiments delivered the first surprise. All mutant seeds sprouted more slowly than wild type under normal conditions, and the delay was sharpest in atdic1, particularly on sucrose-free medium, where the mutants also reached a lower final germination percentage. That detail matters: without an external sugar supply, a seed must mobilize its own lipid reserves through gluconeogenesis, a process that early work had already implicated DIC proteins in. The results position AtDIC1 as a key enabler of metabolic flexibility during the earliest days of a seedling’s life, when mitochondria must be reactivated before photosynthesis can take over. Under salt and osmotic stress, germination delays persisted longer, again most prominently in the atdic1 lines, and the double mutant mirrored the atdic1 phenotype rather than exceeding it, suggesting AtDIC1 dominates this developmental window.

Root growth told a different and strikingly isoform-specific story. Under control conditions, primary roots of every mutant grew normally. But when seedlings faced 100 millimolar NaCl or 200 millimolar mannitol, the two genes diverged sharply. The atdic3 mutants showed significantly shortened primary roots under both stresses, a clear stress-sensitive phenotype, while atdic1 roots were actually longer than wild type under salt stress. The authors propose that AtDIC3, despite its low basal expression, is selectively recruited under adverse conditions, possibly acting as a malate-citrate antiporter that sustains mitochondrial metabolic flexibility and redox homeostasis in actively dividing root cells. Intriguingly, a similar paradox has been seen before: the mitochondrial Complex I mutant ndufs4 grows slowly overall yet tolerates salt and osmotic stress better than wild type in its roots.

Then came the finding that turns the textbook expectation of mitochondrial dysfunction on its head. Far from being stunted, the mutant plants accumulated more shoot biomass than wild type. Rosette areas expanded noticeably by 30 days after sowing and peaked at 40 days, when the strongest knockdown lines carried significantly more fresh and dry weight. Gas exchange measurements revealed significantly higher net carbon dioxide assimilation rates in both single mutants, with the largest gain in atdic3, while stomatal conductance remained unchanged. Chlorophyll fluorescence told the mechanistic story: the effective quantum efficiency of photosystem II, Y(II), was significantly elevated in all mutants, accompanied by higher electron transport rates, and atdic1 also showed increased maximum PSII efficiency in the light-adapted state. The mutants were not breathing harder through their stomata; their photochemistry was simply running better.

Metabolomic profiling of 35-day-old rosettes, detecting 102 metabolites of which 86 differed from wild type, revealed genotype-specific fingerprints. The atdic1 mutant accumulated more sugars, whereas atdic3 was characterized by amino acid buildup, with roughly threefold higher serine, alanine, and proline levels than atdic1. Pathway analysis flagged linoleic acid metabolism, alkaloid biosynthesis, phenylalanine metabolism, ascorbate metabolism, and amino acid biosynthesis as the most affected routes. Redox assays sharpened the distinction: disrupting AtDIC3, but not AtDIC1, significantly lowered the NADH/NAD+ ratio while raising NADPH/NADP+, marking AtDIC3 as a genuine regulator of cellular redox balance. Proline accumulation is especially noteworthy given its established role in redox buffering and stress tolerance. Meanwhile, atdic1 showed stronger perturbations in sugar and ascorbate metabolism, with ascorbate rising 2.33-fold, echoing earlier findings in tomato plants with reduced mitochondrial malate dehydrogenase, which likewise grew larger shoots, flowered early, and photosynthesized better.

Carbon partitioning shifted in every mutant line. At the midpoint of the light period, sucrose levels fell markedly below wild type while starch accumulated significantly above it, a signature that downstream carbon utilization is constrained and that photoassimilates are being stored rather than exported. Respirometry added another layer: oxygen consumption rose progressively across the mutant lines, reaching significance in atdic3 and the double mutant, and atdic3 leaves uniquely paired this heightened respiratory flux with significantly higher ATP content and a more oxidized NADH pool. Marker gene analysis showed elevated VDAC1 and COX15 transcripts, the latter known to respond to electron transport chain inhibition, yet the classic mitochondrial stress marker AOX1a was not induced, indicating that these mutants are not suffering the respiratory chain collapse typically associated with sick mitochondria.

The double mutant sealed the interpretation. Its rosette area, biomass, germination kinetics, and root stress responses closely resembled the single mutants, with no additive effects, and AtDIC2 was strongly upregulated in every mutant background, roughly six- to seven-fold in atdic3. This transcriptional compensation, however, proved only partial: it could not rescue germination defects, redox imbalance, or the altered carbon partitioning. All mutants except atdic3-2 flowered three to four days early, and every mutant line produced significantly fewer seeds than wild type, even while atdic3 seeds that did form were individually heavier. The picture that emerges is one of trade-offs: losing AtDIC1 compromises early carbon mobilization, losing AtDIC3 destabilizes redox and amino acid metabolism, and both losses trigger compensatory photosynthetic enhancement that funnels carbon into vegetative growth at the expense of reproduction. For a field that has long treated impaired growth as the hallmark of mitochondrial dysfunction, these oversized, seed-poor Arabidopsis plants are a vivid reminder that how mitochondria trade their metabolites can matter as much as how they make their ATP, and that engineering those trades might one day let breeders tune the balance between vegetative vigor and yield.

Subject of Research: Functional characterization of mitochondrial dicarboxylate carriers AtDIC1 and AtDIC3 in Arabidopsis thaliana metabolism and development

Article Title: Knockout of mitochondrial dicarboxylate carriers AtDIC1 and AtDIC3 affects germination, biomass accumulation, and carbon partitioning in Arabidopsis thaliana

Article References: de Lara Campos Arcuri, M., Barreto, P., Marinho, A. N., Mantoan, L. P. B., Budzinski, I. G. F., Campos, F. G., & Maia, I. G. (2026). Knockout of mitochondrial dicarboxylate carriers AtDIC1 and AtDIC3 affects germination, biomass accumulation, and carbon partitioning in Arabidopsis thaliana. Plant Cell Reports, 45(10), Article 309. https://doi.org/10.1007/s00299-026-04000-7

Image Credits: AI Generated

DOI: 10.1007/s00299-026-04000-7

Keywords: Arabidopsis thaliana, mitochondrial dicarboxylate carrier, AtDIC1, AtDIC3, plant mitochondria, seed germination, carbon partitioning, photosynthesis, redox balance, starch metabolism, T-DNA mutants, abiotic stress