Malate Circulation: Linking Chloroplast Metabolism to Mitochondrial ROS

The malate valve has long been proposed to release excess reducing equivalents from the chloroplast, but mutants lacking the proposed key enzyme NADP-dependent malate dehydrogenase show little impairment of chloroplast function, suggesting the involvement of an alternative pathway.New research suggests a central role for a malate valve, employing chloroplast NAD-dependent malate dehydrogenase, which also has additional non-enzymatic functions in chloroplast biogenesis.The new malate circulation model proposes that malate exported from the chloroplast is oxidized in the mitochondrion triggering ROS production, which can modulate growth and ultimately induce cell death.It is proposed that the chloroplast to mitochondrion (CTM) pathway in conjunction with direct import of NAD+ leads to elevated levels of NADH in the mitochondrion. In photosynthetic cells, chloroplasts and mitochondria are the sites of the core redox reactions underpinning energy metabolism. Such reactions generate reactive oxygen species (ROS) when oxygen is partially reduced. ROS signaling leads to responses by cells which enable them to adjust to changes in redox status. Recent studies in Arabidopsis thaliana reveal that chloroplast NADH can be used to generate malate which is exported to the mitochondrion where its oxidation regenerates NADH. Oxidation of this NADH produces mitochondrial ROS (mROS) which can activate signaling systems to modulate energy metabolism, and in certain cases can lead to programmed cell death (PCD). We propose the term 'malate circulation' to describe such redistribution of reducing equivalents to mediate energy homeostasis in the cell. In photosynthetic cells, chloroplasts and mitochondria are the sites of the core redox reactions underpinning energy metabolism. Such reactions generate reactive oxygen species (ROS) when oxygen is partially reduced. ROS signaling leads to responses by cells which enable them to adjust to changes in redox status. Recent studies in Arabidopsis thaliana reveal that chloroplast NADH can be used to generate malate which is exported to the mitochondrion where its oxidation regenerates NADH. Oxidation of this NADH produces mitochondrial ROS (mROS) which can activate signaling systems to modulate energy metabolism, and in certain cases can lead to programmed cell death (PCD). We propose the term 'malate circulation' to describe such redistribution of reducing equivalents to mediate energy homeostasis in the cell. The chloroplast is considered to be the main site of reactive oxygen species (ROS; see Glossary) production, because photosynthetic electron transfer reactions ultimately generate all the reducing equivalents required for assimilation and biosynthesis in the whole cell. Achieving the balance between the production and consumption of reducing power is vital since excess reducing equivalents result in ROS production, leading to oxidative signaling. Under stressful environmental conditions such as excess light or high temperature, elevated ROS signaling can lead to changes in cell function to adjust to the effects of excess reducing equivalents [1.Van Aken O. Van Breusegem F. Licensed to kill: mitochondria, chloroplasts, and cell death.Trends Plant Sci. 2015; 20: 754-766Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2.Czarnocka W. Karpinski S. Friend or foe? Reactive oxygen species production, scavenging and signaling in plant response to environmental stresses.Free Radic. Biol. Med. 2018; 122: 4-20Crossref PubMed Scopus (253) Google Scholar, 3.Waszczak C. et al.Reactive oxygen species in plant signaling.Annu. Rev. Plant Biol. 2018; 69: 209-236Crossref PubMed Scopus (459) Google Scholar]. One way in which the chloroplast is thought to dissipate reducing equivalents is by means of the malate valve [4.Scheibe R. Malate valves to balance cellular energy supply.Physiol. Plant. 2004; 120: 21-26Crossref PubMed Scopus (351) Google Scholar,5.Selinski J. Scheibe R. Malate valves: old shuttles with new perspectives.Plant Biol. 2019; 21: 21-30Crossref PubMed Scopus (84) Google Scholar]. It is proposed that excess NADPH is consumed by plastidial NADP-dependent malate dehydrogenase (plNADP-MDH) to produce malate which is exported from the chloroplast for subsequent oxidation. However, mutants lacking plNADP-MDH do not appear to be compromised under conditions of photosynthetic stress, suggesting that other mechanisms can operate [6.Hebbelmann I. et al.Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.J. Exp. Bot. 2012; 63: 1445-1459Crossref PubMed Scopus (95) Google Scholar, 7.Heyno E. et al.Putative role of the malate valve enzyme NADP-malate dehydrogenase in H2O2 signalling in Arabidopsis.Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 2014; 36920130228Crossref PubMed Scopus (42) Google Scholar, 8.Selinski J. Scheibe R. Lack of malate valve capacities lead to improved N-assimilation and growth in transgenic A. thaliana plants.Plant Signal. Behav. 2014; 9e29057Crossref PubMed Scopus (18) Google Scholar]. New discoveries reveal that excess chloroplast NADH is consumed by plastidial NAD-dependent malate dehydrogenase (plNAD-MDH), and the malate is exported to the mitochondrion. There, malate oxidation generates NADH, which is then oxidized by Complex I (NADH: ubiquinone oxidoreductase), generating ROS which can trigger cell death in extreme circumstances [9.Wu J. et al.Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species.Cell Res. 2015; 25: 621-633Crossref PubMed Scopus (58) Google Scholar,10.Zhao Y. et al.Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.Cell Res. 2018; 28: 448-461Crossref PubMed Scopus (73) Google Scholar]. In this opinion article we propose that chloroplast redox stress is linked by a CTM malate transport system that brings about mROS signaling to modulate energy metabolism in the cell under variable environmental conditions. We further propose the term 'malate circulation' to describe this form of redistribution of reducing equivalents in the cell. Chloroplast ROS production occurs when electron carriers in the photosynthetic electron transport chain (pETC) become over-reduced. This might happen when the supply of reducing equivalents is greater than their consumption by the cell. Mitochondria provide a means to alleviate over-reduction in the cell by transferring electrons from reductants to molecular oxygen [11.Millar A.H. et al.Organization and regulation of mitochondrial respiration in plants.Annu. Rev. Plant Biol. 2011; 62: 79-104Crossref PubMed Scopus (418) Google Scholar, 12.Noguchi K. Yoshida K. Interaction between photosynthesis and respiration in illuminated leaves.Mitochondrion. 2008; 8: 87-99Crossref PubMed Scopus (237) Google Scholar, 13.Sweetlove L.J. et al.Mitochondrial uncoupling protein is required for efficient photosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19587-19592Crossref PubMed Scopus (179) Google Scholar, 14.Vanlerberghe G.C. et al.Alternative oxidase: a respiratory electron transport chain pathway essential for maintaining photosynthetic performance during drought stress.Physiol. Plant. 2016; 157: 322-337Crossref PubMed Scopus (77) Google Scholar]. However, partial reduction of oxygen in the mitochondrion can lead to mROS production and we know relatively little about how this is controlled. The generation of mROS is particularly important in responses of plants to various types of abiotic stresses, such as heat and drought, which are expected to increase in severity due to climate change [14.Vanlerberghe G.C. et al.Alternative oxidase: a respiratory electron transport chain pathway essential for maintaining photosynthetic performance during drought stress.Physiol. Plant. 2016; 157: 322-337Crossref PubMed Scopus (77) Google Scholar, 15.Cvetkovska M. Vanlerberghe G.C. Alternative oxidase impacts the plant response to biotic stress by influencing the mitochondrial generation of reactive oxygen species.Plant Cell Environ. 2013; 36: 721-732Crossref PubMed Scopus (88) Google Scholar, 16.Liu J. et al.Overexpression of ALTERNATIVE OXIDASE1a alleviates mitochondria-dependent programmed cell death induced by aluminium phytotoxicity in Arabidopsis.J. Exp. Bot. 2014; 65: 4465-4478Crossref PubMed Scopus (55) Google Scholar, 17.Vanlerberghe G.C. Alternative oxidase: a mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants.Int. J. Mol. Sci. 2013; 14: 6805-6847Crossref PubMed Scopus (433) Google Scholar, 18.Wang J. Vanlerberghe G.C. A lack of mitochondrial alternative oxidase compromises capacity to recover from severe drought stress.Physiol. Plant. 2013; 149: 461-473Crossref PubMed Scopus (54) Google Scholar, 19.Yu C.C. et al.Mitochondrial ORFH79 is essential for drought and salt tolerance in rice.Plant Cell Physiol. 2015; 56: 2248-2258PubMed Google Scholar, 20.Zhu T. et al.Mitochondrial alternative oxidase-dependent autophagy involved in ethylene-mediated drought tolerance in Solanum lycopersicum.Plant Biotechnol. J. 2018; 16: 2063-2076Crossref PubMed Scopus (57) Google Scholar]. The mitochondrial electron transport chain (mETC) is responsible for the generation of mROS. The rate of mROS production is increased when the respiratory rate is slowed, for example when ADP is limiting, or by inhibition of the respiratory chain, leading to a highly reduced state of mETC components [21.Huang S. et al.The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants.Plant Physiol. 2016; 171: 1551-1559Crossref PubMed Scopus (233) Google Scholar,22.Moller I.M. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species.Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 561-591Crossref PubMed Scopus (1280) Google Scholar]. It is believed that mROS production occurs at the inner mitochondrial membrane and is associated particularly with Complex I, but also with Complex II (succinate dehydrogenase), and Complex III (cytochrome bc1 Complex) [21.Huang S. et al.The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants.Plant Physiol. 2016; 171: 1551-1559Crossref PubMed Scopus (233) Google Scholar]. In photosynthetic tissues, it is suggested that the contribution of mitochondria to total cellular ROS production is relatively low in the light [3.Waszczak C. et al.Reactive oxygen species in plant signaling.Annu. Rev. Plant Biol. 2018; 69: 209-236Crossref PubMed Scopus (459) Google Scholar,21.Huang S. et al.The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants.Plant Physiol. 2016; 171: 1551-1559Crossref PubMed Scopus (233) Google Scholar, 22.Moller I.M. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species.Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 561-591Crossref PubMed Scopus (1280) Google Scholar, 23.Karuppanapandian T. et al.Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms.Aust. J. Crop. Sci. 2011; 5: 709-725Google Scholar, 24.Noctor G. et al.Mitochondrial redox biology and homeostasis in plants.Trends Plant Sci. 2007; 12: 125-134Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar]. Plant mitochondria have alternative oxidases (AOXs), which provide a means of transferring electrons directly from ubiquinone to oxygen to minimize mROS production [21.Huang S. et al.The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants.Plant Physiol. 2016; 171: 1551-1559Crossref PubMed Scopus (233) Google Scholar,22.Moller I.M. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species.Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 561-591Crossref PubMed Scopus (1280) Google Scholar]. Nevertheless, mROS seems to be very important in signaling systems that integrate energy metabolism and stress responses in plants [1.Van Aken O. Van Breusegem F. Licensed to kill: mitochondria, chloroplasts, and cell death.Trends Plant Sci. 2015; 20: 754-766Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,3.Waszczak C. et al.Reactive oxygen species in plant signaling.Annu. Rev. Plant Biol. 2018; 69: 209-236Crossref PubMed Scopus (459) Google Scholar,21.Huang S. et al.The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants.Plant Physiol. 2016; 171: 1551-1559Crossref PubMed Scopus (233) Google Scholar]. Such mROS has been implicated in the induction of programmed cell death (PCD) [1.Van Aken O. Van Breusegem F. Licensed to kill: mitochondria, chloroplasts, and cell death.Trends Plant Sci. 2015; 20: 754-766Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,25.Lam E. et al.Programmed cell death, mitochondria and the plant hypersensitive response.Nature. 2001; 411: 848-853Crossref PubMed Scopus (682) Google Scholar, 26.Mou Z. et al.Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology.Plant Cell. 2000; 12: 405-418Crossref PubMed Scopus (182) Google Scholar, 27.Vanlerberghe G.C. et al.Induction of mitochondrial alternative oxidase in response to a cell signal pathway down-regulating the cytochrome pathway prevents programmed cell death.Plant Physiol. 2002; 129: 1829-1842Crossref PubMed Scopus (168) Google Scholar, 28.Yao N. Greenberg J.T. Arabidopsis ACCELERATED CELL DEATH2 modulates programmed cell death.Plant Cell. 2006; 18: 397-411Crossref PubMed Scopus (187) Google Scholar, 29.Zhu Q. et al.The Arabidopsis thaliana RNA editing factor SLO2, which affects the mitochondrial electron transport chain, participates in multiple stress and hormone responses.Mol. Plant. 2014; 7: 290-310Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. It is suggested that mROS produced during stress and in PCD could occur by inhibition of the mETC [21.Huang S. et al.The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants.Plant Physiol. 2016; 171: 1551-1559Crossref PubMed Scopus (233) Google Scholar,22.Moller I.M. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species.Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 561-591Crossref PubMed Scopus (1280) Google Scholar]. However, it has been shown that AOX overexpression can protect against PCD [14.Vanlerberghe G.C. et al.Alternative oxidase: a respiratory electron transport chain pathway essential for maintaining photosynthetic performance during drought stress.Physiol. Plant. 2016; 157: 322-337Crossref PubMed Scopus (77) Google Scholar,16.Liu J. et al.Overexpression of ALTERNATIVE OXIDASE1a alleviates mitochondria-dependent programmed cell death induced by aluminium phytotoxicity in Arabidopsis.J. Exp. Bot. 2014; 65: 4465-4478Crossref PubMed Scopus (55) Google Scholar,17.Vanlerberghe G.C. Alternative oxidase: a mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants.Int. J. Mol. Sci. 2013; 14: 6805-6847Crossref PubMed Scopus (433) Google Scholar,27.Vanlerberghe G.C. et al.Induction of mitochondrial alternative oxidase in response to a cell signal pathway down-regulating the cytochrome pathway prevents programmed cell death.Plant Physiol. 2002; 129: 1829-1842Crossref PubMed Scopus (168) Google Scholar,30.Li Z. Xing D. Mechanistic study of mitochondria-dependent programmed cell death induced by aluminium phytotoxicity using fluorescence techniques.J. Exp. Bot. 2011; 62: 331-343Crossref PubMed Scopus (99) Google Scholar]. The nature of the reducing equivalents that drive mROS production in response to stress has been unclear, but new evidence described below suggests that malate plays a key role. Evidence for an important role for malate in mROS production comes unexpectedly from analysis of a mutant defective in chloroplast function. The Arabidopsis thaliana mutant mosaic death1 (mod1) is characterized by semi-dwarfism, chlorotic curly leaves, premature senescence, and distorted siliques, when it is grown under prolonged illumination or high temperature [26.Mou Z. et al.Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology.Plant Cell. 2000; 12: 405-418Crossref PubMed Scopus (182) Google Scholar]. The abnormal growth phenotype is closely associated with mosaic cell death. The MOD1 gene encodes the chloroplast enzyme enoyl-acyl carrier protein (ACP) reductase (or ENR) (EC 1.3.1.9). This enzyme is a component of the fatty acid synthase (FAS) complex and catalyzes an essential NADH-dependent reduction step in fatty acid biosynthesis [31.Slabas A.R. et al.Induction, purification and characterization of NADH-specific enoyl acyl carrier protein reductase from developing seeds of oil seed rape (Brassica napus).Biochim. Biophys. Acta. 1986; 877: 271-280Crossref Scopus (60) Google Scholar]. In Arabidopsis, ENR is encoded by a single-copy gene, which in the mod1 mutant contains a single amino acid substitution. The mod1 mutation leads to a dramatic decrease in ENR enzymatic activity in extracts from leaves or siliques, and the mod1 mutant exhibits a decrease in total lipid content [26.Mou Z. et al.Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology.Plant Cell. 2000; 12: 405-418Crossref PubMed Scopus (182) Google Scholar]. To understand this phenotype, suppressor of mod1 (som) mutants were selected from a library of T-DNA insertion mutants created in the mod1 mutant [9.Wu J. et al.Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species.Cell Res. 2015; 25: 621-633Crossref PubMed Scopus (58) Google Scholar]. Analysis of the som3 mutant established that the SOM3 gene encodes a subunit of mitochondrial Complex I. Another mutant, som42, exhibited overexpression of the SOM42 gene which encodes a mitochondria-localized pentatricopeptide repeat (PPR) protein that negatively regulates the amount of transcript for NAD7 encoding a subunit of Complex I. Further mutants with impaired Complex I activity were found to suppress the mod1 phenotype and mROS production [9.Wu J. et al.Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species.Cell Res. 2015; 25: 621-633Crossref PubMed Scopus (58) Google Scholar]. Overexpression of AOX1a in mod1 suppressed mROS levels and cell death [32.Luo L. et al.Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis.Sci. China Life Sci. 2019; 62: 991-1002Crossref PubMed Scopus (16) Google Scholar]. Therefore, the mod1 phenotype depends on production of mROS by mitochondrial Complex I and potentially Complex III. These observations revealed that there appears to be a line of communication directly from the chloroplast to the mitochondrion. To understand the communication between chloroplast and mitochondrion in mod1, further som mutants were screened and selected for those with wild-type Complex I activity. This approach led to the identification of four further genes in which mutations could suppress mROS accumulation and the mod1 phenotype [10.Zhao Y. et al.Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.Cell Res. 2018; 28: 448-461Crossref PubMed Scopus (73) Google Scholar,32.Luo L. et al.Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis.Sci. China Life Sci. 2019; 62: 991-1002Crossref PubMed Scopus (16) Google Scholar]. Three of these genes encode key components of malate shuttle between chloroplast and mitochondrion. These components are plNAD-MDH, chloroplast dicarboxylate transporter 1 (DiT1) and mitochondrial NAD-dependent malate dehydrogenase 1 (mNAD-MDH1) [10.Zhao Y. et al.Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.Cell Res. 2018; 28: 448-461Crossref PubMed Scopus (73) Google Scholar]. These findings implied that in mod1, accumulating chloroplast NADH is oxidized by plNAD-MDH to produce malate which is exported in exchange for oxaloacetate (OAA) via the DiT1 transporter and imported into the mitochondrion. Such malate would then be oxidized by mNAD-MDH1, producing NADH to serve as substrate for Complex I, with resultant mROS production [10.Zhao Y. et al.Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.Cell Res. 2018; 28: 448-461Crossref PubMed Scopus (73) Google Scholar]. In this proposed 'malate circulation', reducing equivalents are transferred in the form of malate from chloroplasts to the mitochondria with concomitant return of OAA to the chloroplasts (Figure 1). In a new development, a loss-of-function mutation in mitochondrial NAD+ transporter 2 (NDT2) was identified as a new suppressor of mod1 [32.Luo L. et al.Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis.Sci. China Life Sci. 2019; 62: 991-1002Crossref PubMed Scopus (16) Google Scholar]. In mod1, the NADH pool in mitochondria is increased compared with that of wild type, and loss of NDT2 activity suppresses the mROS and PCD in mod1. These results suggest that in mod1, the amount of malate transported into mitochondria is such that additional NAD+ is needed to meet the demand for malate oxidation [32.Luo L. et al.Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis.Sci. China Life Sci. 2019; 62: 991-1002Crossref PubMed Scopus (16) Google Scholar]. A malate shuttle from the chloroplast in this way is usually referred to as a malate valve [4.Scheibe R. Malate valves to balance cellular energy supply.Physiol. Plant. 2004; 120: 21-26Crossref PubMed Scopus (351) Google Scholar,5.Selinski J. Scheibe R. Malate valves: old shuttles with new perspectives.Plant Biol. 2019; 21: 21-30Crossref PubMed Scopus (84) Google Scholar]. It is widely believed that the chloroplast malate valve normally employs plNADP-MDH to dissipate excess NADPH during photosynthesis. The plNADP-MDH enzyme is redox-modulated by the ferredoxin-thioredoxin (Fd-Trx) system and is thought to be active only in the excess light and therefore constitutes part of a 'light malate valve' [33.Fickenscher K. Scheibe R. Limited proteolysis of inactive tetrameric chloroplast NADP-malate dehydrogenase produces active dimers.Arch. Biochem. Biophys. 1988; 260: 771-779Crossref PubMed Scopus (24) Google Scholar, 34.MiginiacMaslow M. et al.Light-dependent activation of NADP-malate dehydrogenase: a complex process.Aust. J. Plant Physiol. 1997; 24: 529-542Google Scholar, 35.Miginiac-Maslow M. et al.Light-activation of NADP-malate dehydrogenase: a highly controlled process for an optimized function.Physiol. Plant. 2000; 110: 322-329Crossref Scopus (34) Google Scholar]. However, recent studies in which plNADP-MDH has been 'knocked out' in Arabidopsis suggest that other mechanisms are involved in the response to chloroplast redox stress [6.Hebbelmann I. et al.Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.J. Exp. Bot. 2012; 63: 1445-1459Crossref PubMed Scopus (95) Google Scholar]. In contrast to plNADP-MDH, plNAD-MDH is present in all green and non-green plastids and its activity is not apparently redox-modulated, so is active under both light and dark conditions [4.Scheibe R. Malate valves to balance cellular energy supply.Physiol. Plant. 2004; 120: 21-26Crossref PubMed Scopus (351) Google Scholar,5.Selinski J. Scheibe R. Malate valves: old shuttles with new perspectives.Plant Biol. 2019; 21: 21-30Crossref PubMed Scopus (84) Google Scholar,36.Berkemeyer M. et al.A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L.J. Biol. Chem. 1998; 273: 27927-27933Crossref PubMed Scopus (60) Google Scholar]. The enzyme is assumed to play a role in the export of reducing equivalents from chloroplasts during darkness and from non-green plastids, so it is sometimes considered to be a component of a 'dark malate valve' [4.Scheibe R. Malate valves to balance cellular energy supply.Physiol. Plant. 2004; 120: 21-26Crossref PubMed Scopus (351) Google Scholar,5.Selinski J. Scheibe R. Malate valves: old shuttles with new perspectives.Plant Biol. 2019; 21: 21-30Crossref PubMed Scopus (84) Google Scholar]. The function of plNAD-MDH has eluded investigation because knock-out (null) mutations are embryo-lethal [37.Beeler S. et al.Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.Plant Physiol. 2014; 164: 1175-1190Crossref PubMed Scopus (61) Google Scholar, 38.Schreier T.B. et al.Plastidial NAD-dependent malate dehydrogenase: a moonlighting protein involved in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex.Plant Cell. 2018; 30: 1745-1769Crossref PubMed Scopus (34) Google Scholar, 39.Selinski J. et al.The plastid-localized NAD-dependent malate dehydrogenase is crucial for energy homeostasis in developing Arabidopsis thaliana seeds.Mol. Plant. 2014; 7: 170-186Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar] (see below). Current analysis of the mod1 mutant suggests that plNAD-MDH could have an important function in a malate valve operating in the light in response to elevated NADH [10.Zhao Y. et al.Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.Cell Res. 2018; 28: 448-461Crossref PubMed Scopus (73) Google Scholar]. These results are new and exciting. The null mutants of plNAD-MDH in Arabidopsis are embryo-lethal, implying that this enzyme has an essential metabolic function during early embryogenesis [37.Beeler S. et al.Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.Plant Physiol. 2014; 164: 1175-1190Crossref PubMed Scopus (61) Google Scholar,39.Selinski J. et al.The plastid-localized NAD-dependent malate dehydrogenase is crucial for energy homeostasis in developing Arabidopsis thaliana seeds.Mol. Plant. 2014; 7: 170-186Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. Furthermore, a transgenic line with lowered plNAD-MDH resulting from gene silencing exhibited poor growth, pale leaves, disorganized chloroplasts and low respiration, indicating an important function at multiple stages of plant development [37.Beeler S. et al.Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.Plant Physiol. 2014; 164: 1175-1190Crossref PubMed Scopus (61) Google Scholar,38.Schreier T.B. et al.Plastidial NAD-dependent malate dehydrogenase: a moonlighting protein involved in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex.Plant Cell. 2018; 30: 1745-1769Crossref PubMed Scopus (34) Google Scholar,40.Smith S.M. Moonlighting NAD+ malate dehydrogenase is essential for chloroplast biogenesis.Plant Cell. 2018; 30: 1663-1664Crossref PubMed Scopus (3) Google Scholar]. Analysis of such leaves showed that chloroplast development is impaired in the absence of plNAD-MDH. Remarkably, Arabidopsis NAD-dependent MDH enzymes from the peroxisome, mitochondrion or cytosol could not complement the plnad-mdh mutant when they were targeted to the plastid, implying that NAD-MDH activity per se was not important. This conclusion was confirmed when enzymatically-inactive variants of plNAD-MDH were produced by mutagenesis and found to complement the plnad-mdh mutant [38.Schreier T.B. et al.Plastidial NAD-dependent malate dehydrogenase: a moonlighting protein involved in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex.Plant Cell. 2018; 30: 1745-1769Crossref PubMed Scopus (34) Google Scholar]. Therefore, the plNAD-MDH protein, not its enzyme activity, is required for plastid development. Further experiments showed that plNAD-MDH forms a complex with 'Filamentous temperature sensitive' (Fts) protein FtsH12, which is a subunit of FtsH12-FtsHi complex associated with the chloroplast inner envelope membrane [38.Schreier T.B. et al.Plastidial NAD-dependent malate dehydrogenase: a moonlighting protein involved in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex.Plant Cell. 2018; 30: 1745-1769Crossref PubMed Scopus (34) Google Scholar]. This complex is proposed to act as the ATP-driven import motor that functions with the 'Translocon of the Inner membrane of Chloroplasts' (TIC) complex, to draw proteins into the plastid. The plNAD-MDH protein also interacts physically with enzymes of starch metabolism, suggesting that it has additional roles in plastid function [38.Schreier T.B. et al.Plastidial NAD-dependent malate dehydrogenase: a moonlighting protein involved in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex.Plant Cell. 2018; 30: 1745-1769Crossref PubMed Scopus (34) Google Scholar]. These results indicate that plNAD-MDH has important roles in both plastid biogenesis and in the CTM malate pathway. These stud