Explore TCA Cycle Functions in Plant Metabolism

Tricarboxylic acid cycle (TCA cycle), also known as Krebs Cycle or Citric Acid Cycle, is a basic metabolic pathway in all aerobic organisms, including plants. Although TCA cycle is usually related to the energy production of animals, its role in plants is more diversified. Plants not only rely on TCA cycle for energy generation, but also rely on it for biosynthesis, stress response and signal transduction. This paper will discuss the importance of TCA cycle in plants, and explain in detail its functions in energy metabolism, carbon skeleton provision, stress adaptation and integration with other metabolic pathways.

Energy production

TCA cycle is the core of cell energy metabolism, which generates ATP by oxidizing and decomposing carbon sources (such as glucose, fatty acids and amino acids) to support plant growth and various metabolic activities.

• As a critical subunit of the mitochondrial pyruvate carrier (MPC) complex, MPC1 mediates the transport of pyruvate from the cytosol into mitochondria, serving as the central component for mitochondrial pyruvate uptake in plants. Loss of MPC1 disrupts the stable expression of MPC3 and MPC4 in plant mitochondria, impairing pyruvate import efficiency and compromising mitochondrial respiratory capacity. Notably, mpc1 mutants exhibit significantly reduced oxygen consumption during pyruvate-dependent respiration, underscoring MPC1's essential role in this process.MPC1 is indispensable for the structural assembly of the MPC complex. While transcript levels of MPC3 and MPC4 remain detectable in mpc1 mutants, their corresponding proteins are absent in mitochondria, suggesting rapid degradation due to instability. Importantly, transgenic expression of MPC1 in mpc1 plants restores mitochondrial accumulation of both MPC3 and MPC4, confirming its requirement for complex stabilization.Experimental evidence using isolated mitochondria revealed that mpc1 mutants exhibit defective pyruvate uptake, leading to restricted accumulation of pyruvate-derived metabolites in the TCA cycle. Furthermore, pharmacological inhibition of MPC activity with UK-5099—a specific MPC blocker—reproduced the respiratory deficiency observed in mpc1 mutants, conclusively validating MPC1's central role in pyruvate-dependent mitochondrial respiration (Le XH et al., 2021).

MPC1 is required for the uptake and usage of pyruvate by Arabidopsis mitochondria (Le XH et al., 2021).

Carbon skeleton supply

TCA cycle not only provides energy, but also provides carbon skeleton for plants to synthesize other important compounds.

• Plants need to adjust their metabolic network according to the availability and form of nitrogen in soil. Nitrogen is an important nutrient element for plant growth and development. This study aims to deeply understand the absorption efficiency of plants under different nitrogen sources and the relationship between nitrogen assimilation kinetics and TCA cycle. Under the two nitrogen sources, the [13C] amino acid in roots increased logarithmically, but the content of [13C] amino acid in ammonium nitrogen nutrition (RA) was higher, reaching the maximum value of 15.4 μmol/g dry weight after 6 hours, while it was 5.1 μmol/g dry weight in nitrate nutrition (RN). Under ammonium nitrogen nutrition, the level of [13C] amino acids accumulated in roots is relatively high, especially [13C]Gln and [13C]Asn increased significantly in the first two hours, and then remained stable. Under the nutrition of ammonium nitrogen (NH4+), wheat roots assimilate ammonia by enhancing GS and GOGAT pathways. In this process, amino acids such as Gln and Asn are synthesized and accumulated in order to deal with the toxicity of ammonium nitrogen more effectively. TCA cycle provides the necessary carbon skeleton in ammonium nitrogen assimilation, and promotes the detoxification of ammonium nitrogen and the synthesis of amino acids by cooperating with GS/GOGAT pathway. Gln is the first labeled amino acid in the assimilation process. With the passage of time, the increase of Glu and the decrease of Gln indicate the synergistic effect of GS and GOGAT. Other amino acids such as Asp, Gly and Ala are also involved by transamination. Under the stress of ammonium nitrogen, GABA(γ- aminobutyric acid), as an amino acid, may help to alleviate ammonium toxicity. When wheat roots receive 15NO3− (nitrate), the assimilation efficiency of nitrate is lower than that of ammonium nitrogen. TCA cycle not only provides the necessary carbon skeleton for ammonia assimilation, but also can flexibly respond to different nitrogen sources and provide suitable metabolic intermediates to support the synthesis and detoxification of amino acids (Vega-Mas I et al., 2019).

TCA cycle flux model inferred from [13C]Pyr marker of wheat roots (Vega-Mas I et al., 2019).

Plant development and growth

The tricarboxylic acid (TCA) cycle plays a pivotal role in plant development, influencing root morphogenesis, cotyledon expansion, floral organ formation, and fruit maturation. Experimental evidence demonstrates that modulating TCA cycle activity impacts root respiration efficiency, biomass partitioning, and photosynthetic capacity—key determinants of overall plant vigor.

• A genome-wide investigation in maize identified 91 TCA-associated genes, systematically categorized into enzyme subclasses such as SDH, αKGDHC,IDH, ACO, MDH, and SCoAL. Phylogenetic comparisons of TCA-related proteins across maize, Arabidopsis thaliana, and tomato revealed conserved subgroup clustering, with maize-specific gene duplications suggesting lineage-specific evolutionary adaptations.Spatiotemporal expression profiling uncovered ubiquitous TCA gene activity in maize roots, leaves, floral tissues, and ears, with pronounced transcriptional enrichment in anthers and ears—a pattern potentially linked to elevated energy demands during male reproductive development.Five maize TCA genes—ZmIDH (Zm00001d008244), ZmSCoAL (Zm00001d017258), ZmαKGDH (Zm00001d025258), ZmACO (Zm00001d027558), and Zm00001D044—were heterologously expressed in Arabidopsis. Transgenic lines validated by antibiotic selection, PCR amplification, and subcellular localization exhibited distinct root phenotypes:ZmIDH and ZmACO overexpression caused marked taproot shortening .ZmSCoAL, ZmαKGDH, and ZmMDH transgenics displayed moderate root inhibition.ZmACO-overexpressing plants maintained wild-type root elongation.Under saline conditions, ZmαKGDH- and ZmACO-expressing lines exhibited hypersensitivity, evidenced by reduced germination rates. Notably, ZmIDH overexpression induced reproductive abnormalities, including shortened siliques and increased seed abortion, indicative of fertilization defects (Liu et al., 2019).

Coping with biotic and abiotic stresses

TCA cycle plays an important role in plants coping with different types of stresses (such as salt stress, drought, phosphorus deficiency, etc.). The activities of some TCA enzymes are regulated by these stresses, thus affecting the stress resistance of plants.

• The coordinated up-regulation of the TCA cycle of Phaeodactylum tricornutum is the most significant reaction under nitrogen starvation and other growth inhibition stresses. By providing 13C-labeled acetate to diatom cells, the researchers further verified the activation of TCA cycle under nitrogen starvation. The results showed that under the condition of nitrogen deficiency, the accumulation of 13C label in citrate increased significantly, reflecting the activation of TCA cycle. It was found that during nitrogen starvation, the expression of 19 of the 24 genes encoding TCA cycle was significantly up-regulated, which was verified by qRT-PCR analysis. In order to explore how bZIP14 regulates TCA cycle, the researchers constructed a P. tricornutum cell line that overexpressed bZIP14. In bZIP14 overexpressed cell lines, the levels of amino acids were higher, and the levels of TCA cycle-related organic acids (such as pyruvate, succinate and citrate) increased slightly. In addition, the levels of γ -butyric acid (GABA) and glutamic acid also showed significant differences, suggesting that bZIP14 has an important regulatory role in nitrogen metabolism and TCA cycle. It was found that the expression of bZIP14 transcript and TCA cycle gene showed a circadian rhythm, which was usually up-regulated at dusk (at the end of light stage) and down at night. This result has been consistently observed in different species, including T. pseudonana and N. oceanica, and further shows that bZIP14 may be universal in regulating TCA cycle in cross-species circadian rhythm regulation (Matthijs M et al., 2017).

Photosynthesis and photorespiration

TCA cycle is closely related to photosynthesis and photorespiration. By interacting with other metabolic pathways, TCA cycle can help plants optimize energy use under different environmental conditions.

• In this study, the expression of Sl SDH2-2 gene in transgenic tomato was controlled by using 35S promoter, and its effects on photosynthesis rate, stomatal regulation and TCA cycle were discussed. The levels of malic acid and fumarate in transgenic plants decreased significantly, while the levels of carbohydrates such as starch, sucrose, glucose and fructose increased significantly. Under the control of 35S promoter, the transgenic tomato plants with antisense expression of Sl SDH2-2 gene encoding iron-sulfur subunit of succinate dehydrogenase showed enhanced photosynthetic rate. These transgenic plants showed high carbon dioxide assimilation ability (increased by about 25%), and mature transgenic plants showed significantly increased biomass. In transgenic tomatoes, the rate of TCA cycle decreased, and the level of metabolites related to TCA cycle changed. Compared with wild-type plants, transgenic plants show higher transpiration rate and stomatal conductance, and the stomatal aperture is enlarged. Transgenic plants showed enhanced assimilation rate and stomatal conductance under higher light intensity (PFD). Even under different light intensities, the photosynthetic performance of transgenic plants is still higher than that of wild plants. When the Sl SDH2-2 gene was specifically expressed in guard cells by antisense RNA, no change of stomatal aperture and enhancement of photosynthesis were observed. Carbon isotope analysis (δ13C) revealed that the stomatal function of transgenic plants was enhanced, which was related to the lower (more negative) δ13C value, indicating that the photosynthetic efficiency was improved. The role of TCA cycle intermediates in photosynthetic metabolism was emphasized (Araújo WL et al., 2011).

Hormone signal transduction

TCA cycle is also involved in plant hormone signal transduction, especially in plant growth regulation and response to external stimuli. For example, the metabolic pathway related to plant hormones may be regulated by the enzyme activity of TCA cycle, which affects the physiological response of plants.

• PDC plays a key role in connecting glycolytic pathway and TCA cycle. The effects of TCA cycle damage on intracellular vesicle transport were studied by analyzing TCA cycle inhibitors (such as 3-BP) and PDC mutants (such as mab1-1 mutant). MtPDC is involved in many processes in plant development. For example, E1 component mutants (iar4 and mab1) show root growth defects, E2 component mutants will affect the size of plant organs, and E3 component mutants will inhibit root growth, especially in arsenate environment. In plants, PIN-FORMED protein (PINs) is a carrier protein involved in auxin efflux. It was found that mab1 mutation affected the abundance of PINs by reducing the recovery in vacuoles and enhancing the degradation. ARA6 is a plant-specific RAB5 GTP enzyme, which is involved in endosome transport. It is found that the damage of TCA cycle will lead to the accumulation of ARA6 in cells and regulate the degradation of PIN protein by affecting the endocytosis pathway. MAB4 is a NPH3-like protein, which can inhibit the internalization of PIN from plasma membrane. It was found that in 3-BP treated roots and mab1-1 mutants, the level of MAB4 decreased seriously, which led to the internalization of PIN2. TCA cycle damage can also lead to the accumulation of reactive oxygen species (such as H2O2) in the root tip, thus affecting the stability of MAB4. H2O2 treatment decreased the level of MAB4, increased the internalization of PIN2, and led to the aggregation of positive compartments of ARA6, which further proved that TCA cycle injury promoted the degradation of PIN protein in vacuoles by destroying the endocytosis pathway mediated by MAB4 and ARA6. TCA cycle injury inhibits the internalization of PIN protein and promotes its degradation by increasing ROS level, thus affecting auxin transport and root development (Song X et al., 2024).

References

1. Le XH, Lee CP, Millar AH. "The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism." Plant Cell. 2021 ;33(8):2776-2793. https://pubmed.ncbi.nlm.nih.gov/34137858/
2. Vega-Mas I, Cukier C, Coleto I, González-Murua C, Limami AM, González-Moro MB, Marino D. "Isotopic labelling reveals the efficient adaptation of wheat root TCA cycle flux modes to match carbon demand under ammonium nutrition." Sci Rep. 2019 ;9(1):8925. doi: 10.1038/s41598-019-45393-8
3. Liu Y, Qu J, Zhang L, Xu X, Wei G, Zhao Z, Ren M, Cao M. "Identification and characterization of the TCA cycle genes in maize." BMC Plant Biol. 2019 ;19(1):592. doi: 10.1186/s12870-019-2213-0
4. Matthijs M, Fabris M, Obata T, Foubert I, Franco-Zorrilla JM, Solano R, Fernie AR, Vyverman W, "Goossens A. The transcription factor bZIP14 regulates the TCA cycle in the diatom Phaeodactylum tricornutum." EMBO J. 2017 ;36(11):1559-1576. doi: 10.15252/embj.201696392
5. Araújo WL, Nunes-Nesi A, Osorio S, Usadel B, Fuentes D, Nagy R, Balbo I, Lehmann M, "Studart-Witkowski C, Tohge T, Martinoia E, Jordana X, Damatta FM, Fernie AR. Antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase enhances photosynthesis and growth in tomato via an organic acid-mediated effect on stomatal aperture." Plant Cell. 2011 ;23(2):600-27. doi: 10.1105/tpc.110.081224
6. Song X, Ohbayashi I, Sun S, Wang Q, Yang Y, Lu M, Liu Y, Sawa S, Furutani M. "TCA cycle impairment leads to PIN2 internalization and degradation via reduced MAB4 level and ARA6 components in Arabidopsis roots." Front Plant Sci. 2024 ;15:1462235. doi: 10.3389/fpls.2024.1462235