Roles of malic enzymes in plant development and stress responses

ABSTRACT

Malic enzyme (ME) comprises a family of proteins with multiple isoforms located in different compartments of eukaryotic cells. It is a key enzyme regulating malic acid metabolism and can catalyze the reversible reaction of oxidative decarboxylation of malic acid. And it is also one of the important enzymes in plant metabolism and is involved in multiple metabolic processes. ME is widely present in plants and mainly discovered in cytoplasmic stroma, mitochondria, chloroplasts. It is involved in plant growth, development, and stress response. Plants are stressed by various environmental factors such as drought, high salt, and high temperature during plant growth, and the mechanisms of plant response to various environmental stresses are synergistic. Numerous studies have shown that ME participates in the process of coping with the above environmental factors by increasing water use efficiency, improving photosynthesis of plants, providing reducing power, and so on. In this review, we discuss the important role of ME in plant development and plant stress response, and prospects for its application. It provides a theoretical basis for the future use of ME gene for molecular resistance breeding.

Malic enzyme (ME) is a key enzyme regulating malic acid metabolism. It can combine the coenzyme to catalyze the reversible reaction of malate oxidative decarboxylation, produce pyruvic acid and CO₂, and accompany the production of NAD(P)H. Depending on the cofactors, plant MEs can be divided into NAD⁺ dependent malic enzymes (NAD-ME; EC 1.1.1.38 and EC 1.1.1.39) and NADP⁺ dependent malic enzymes (NADP-ME; EC 1.1.1.40).¹ And they are widely found in nature, including bacteria, archaea, fungi, animals and plants. Both types of MEs contain multiple members that are distributed in different organelles within the cell and perform their respective functions. NADP-ME acts as a coenzyme mainly in chloroplasts and cytosol, which can catalyze the oxidative decarboxylation of malic acid and NADP⁺ to produce pyruvic acid, CO₂ and NADPH. Secondly, it can produce NADPH as a reducing agent for other anabolic reactions, and as a key enzyme in carbon metabolism, playing an important role in plant photosynthesis; while NAD-ME decarboxylates malic acid in the mitochondria to produce pyruvic acid, which controls the exchange of metabolites such as carbon in the mitochondria to maintain the normal TCA cycle.^2–5 In addition to this, NADP is an important reducing agent that provides NADPH for the synthesis of defensive substances such as flavonoids and lignin. In addition, it is also a substance essential for the reactive oxygen species (ROS) metabolic system. In contrast, NAD-ME acts primarily as an oxidant in catabolism, which produces energy (ATP) through oxidation.⁶ In other words, NADP-ME and NAD-ME may play different roles in the metabolism and synthesis of substances. For example, one of the important roles of NADP-malic enzymes is the regeneration of NADPH as a reducing agent for other anabolic reactions. Even the same type of ME does not function exactly in plants of different photosynthetic types (C3, C4 or CAM).

In advanced plants, Rothemel and Nelson first report the full-length cDNA sequence of NADP-ME gene in maize.⁷ There are some reports of NADP-ME genes in barley,⁸ corn,⁹ sorghum,¹⁰ rice¹¹ and Arabidopsis.^12,13 In addition, the key enzyme genes MdNADP-ME1, 2, 3 of malate metabolism in apples are studied by real-time quantitative PCR and bioinformatics analysis. The results show that the above three genes all contain five conserved amino acid regions (motif I-V, which are typical of NADP-ME in plants). MdNADP-ME1-3 belong to the plant NADP-ME family, which is highly conserved (Figure 1).¹⁴ The above evidence indicates that the NADP-ME family is highly conserved in many species, and because malic acid is one of the intermediates in the metabolic process, the enzyme maintains an acid-base and sub-equilibrium, providing a stable carbon source for photosynthesis and playing an irreplaceable role in the process of constructing cellular components.

Figure 1.

Schematic diagram of five conserved amino acid structures of MdNAD-ME1, 2, 3¹⁴ I-V represents five conserved amino acid regions. The two functional domains are malic and NAD_bind_1_malic_enz.

Plants are often affected by environmental factors during growth and development. Studies have shown that the ME in plants plays an important role not only in metabolic pathways such as photosynthesis and respiration, but also in the fertility and defense functions of plants.

Relationship between ME and plant development

The growth and development of organisms are regulated by various factors, such as genes, phospholipases, illumination time, and certain exogenous substances.^15–22 With the continuous development of technology, some studies have found that ME is also involved in the regulation of plant growth and development. ME regulates the growth and development of organisms. It has been found in cancer cells to regulate cell metabolism and proliferation by inhibiting ME and p53.²³ Simultaneously, ME exists in the process of plant growth and development, and participates in multiple metabolic processes in plants. It is one of the important enzymes in the life activities of organisms. In recent years, studies have shown that ME participates in the development of plants.

Citric acid is the main organic acid present in the whole fruit development of melon. After pollination of melon, the activity of ME is positively correlated with the concentration of citric acid and increases with time, which indicates that ME may be related to the accumulation of organic acids during fruit development.²⁴ Similarly, during the development of peaches, the involvement and changes of NADP-ME were also found, and both NADP-ME1 and NADP-ME2 increased with fruit development. And interestingly, increasing the NADP-ME activity during the hardening phase of the fruit provides a large amount of NADPH for the synthesis of lignin, phenylpropanoids and flavonoids. However, no significant changes in NAD-ME were observed during this process.²⁵ The expression analysis shows that the relative expression of MdNAD-ME1 is elevated in different developmental stages of apple; the relative expression of MdNAD-ME2 is decreased, indicating that NAD-ME1 and NAD-ME2 may play different roles in plant growth and development. Their specific mechanism of action needs further research to confirm.¹⁴ In addition, ME also participates in the development of maize embryos²⁶ and plays a key role.

In addition, the accumulation of lipids is a crucial link in the growth and development of oil crops. NADP-ME plays an irreplaceable role in this process. The enzyme performs irreversible decarboxylation of malic acid and pyruvic acid to produce NADPH for biosynthesis of fatty acids. This effect of NADP-ME was confirmed in canola. Kang et al. confirmed that in the developmental embryos of rapeseed, NADP-ME and its reaction products pyruvate and NADPH may contribute to the synthesis of fatty acids.²⁷ Coincidentally, when malic acid and pyruvic acid are used as precursors of synthetic fatty acids, the synthesis rate of malate and pyruvic acid is the highest, and no exogenous reducing agent is needed.²⁸ These data indicate that not only NADP-ME acts as one of the key enzymes in the process of plastid synthesis and accumulation of fatty acids, but also its products serve as a good carbon source in the body, which increases the efficiency of the reaction optimizing the benefits of the fatty acid production and accumulation process. NADP-ME is not only involved in the lipid synthesis process, it also affects the development of chloroplasts. Takeuchi et al. also showed that NADP-ME activity levels are inversely correlated with chlorophyll content and photosystem II activity, and that there is no thylakoid accumulation in chloroplasts in transgenic rice. Therefore, they speculate that the high activity of NADP-ME will cause abnormal chloroplast development.²⁹ The above data indicate that the content of ME varies at various stages of plant development, which may indicate that ME plays different roles depending on the stage of plant development. And the specific regulation network needs further research and summary to lay a theoretical foundation for future molecular breeding using ME genes.

Relationship between ME and plant stress resistance

Environmental factors can seriously affect the normal growth and development of plants. In order to survive in adverse environments, plants resist abiotic stress through various enzymes, gene overexpression, CO₂ and other substances.^30–40 With the further development of many biosciences such as bioinformatics, many studies have shown that ME plays important role in plant stress resistance.

The malic acid reservoir of soybean root tip maintains aluminum-induced citric acid outflow. GmME 1 encodes a cytoplasmic ME that helps to increase the concentrations and efflux of malate and citric acid in the body, thereby improving aluminum resistance in plants and helping them to adapt better to the conditions in which they live.⁴¹ Moreover, NADP-ME is also an important player in plant-based defense in Arabidopsis. After pathogen-associated molecular patterns (PAMPs) treatment and pathogen infection, total NADP-ME activity and NADP-ME2 transcript levels are enhanced, and NADP-ME2 loss-of-function mutants (nadp-me2) shows enhanced susceptibility.⁴² Overexpression of rice NAD-ME1 (OsNAD-ME1) can increase the activity of NAD-ME in Arabidopsis thaliana, and also increase its resistance to various abiotic stresses such as NaCl, NaHCO₃, mannitol and H₂O₂.⁴³ This evidence gives us reason to believe that ME not only balances the concentration of malic acid in the cells, but also reduces the production of ROS, thereby reducing the oxidative damage caused by ROS.

The role of ME in response to temperature stress

Low temperature is a global natural disaster that affects the yield and distribution of crops. The persistent low temperature is unfavorable to the development of crops, which reduces photosynthesis and inhibits the metabolism of roots, stems and leaves, thus affecting the growth and development of plants. Numerous studies have shown that plants can respond to low temperature stress through protein kinases, acyl carrier proteins, certain genes, and the like.^44–50 In recent years, some studies have shown that ME participates in the response process of low temperature stress and plays an active role.

Studies have shown that long-term low temperature induces an increase in malate content or NADP-ME activity in Secale cereale L. When the low temperature stress is removed, both malic acid content and enzyme activity decrease. The above results indicate that Secale cereale L. not only regulates the malic acid content as a good osmotic adjustment substance, but also responds to the osmotic stress caused by low temperature by regulating NADP-malic enzyme activity.

It also regulates the increase in the risk of reactive oxygen species (ROS) caused by low temperatures and exacerbates photooxidative damage to protect plants from cold damage.⁵¹ NADP-ME can not only be a good osmotic adjustment substance by participating in some enzymatic reactions, but also balance the higher solute concentration in cells caused by low temperature. Moreover, malic acid can be used as an additional carbon source to store carbon dioxide in response to damage to the photosystem caused by low temperature stress. Wheeler et al. identified that the expression level of TaNADP-ME2 in the hexaploid wheat “Shijiazhuang 4185” species reached the maximum at 12 h under low temperature stress, but the expression of TaNADP-ME1 decreased (Figure 2).⁵² And they discovered that TaNADP-ME1 and TaNADP-ME2 genes are photo-responsive genes. However, the effect of low temperature stress on the expression of NADP-ME gene in Aloe vera is not obvious.⁵³ There is evidence that NADP-ME does respond to low temperature stress, but it plays different roles in different species.

Figure 2.

Changes of ME in plants under low temperature stress. NADP-ME and TaNADP-ME₂ were induced under cold stress, and TaNADP-ME₁ was inhibited under cold stress.

NADP-ME is mainly located on the plasma membrane of the cell, and hypothermia induces the expression of NADP-ME gene, which may be used to maintain the membrane fluidity and ensure the normal metabolic activities.^54–56 In addition, NADP-ME is a hydrophilic protein,⁵⁶ at low temperature, its large amount of production can make more free water into bound water, so as to reduce intracellular water icing, improve the ability to protect against the cold, which may be one of the ways that NADP-ME play a role in the cold.

In summary, ME does not show significant changes in all plants after exposure to low temperature stress. In other words, ME may only participate in low temperature stress responses in some plants. It is speculated that only NADP-ME located on the membrane system (such as the plastid membrane) can respond to low temperature stress, while the expression of other types of NADP-ME is not induced by low temperature.

ME is not only related to low temperature stress, it also plays an important role in high temperature stress. The NAD-ME activity in C3 plant rice increased with increasing temperature within a certain temperature range.⁵⁷ Under the condition of temperature difference in a certain temperature range, plants can adapt to the change of temperature through their own regulation. In the range of 22–55 ℃, the activities of NADP-ME increased with the increase of temperature. These results indicated that NADP-ME was a heat-resistant enzyme, and CAM plants were more tolerant to high temperature. High temperature can regulate NADP-ME activity and then regulate CAM activity.^58,59

Relationship between ME and drought tolerance

For plant growth, drought is a growth limiting factor. Drought causes physiological toxic effects such as osmotic stress, free radical accumulation, membrane lipid peroxidation and nitric acid accumulation in plants, causing metabolic disorders. Studies in plants have shown that phytochrome B,⁶⁰ glycosyltransferase QUA1⁶¹ and certain genes⁶² can regulate the drought tolerance of plants. However, with the development of biological technologies such as genetic engineering, bioinformatics and molecular biology, more and more evidence indicates that ME is also involved in drought stress response.

Under the drought stress, a new ~122kDa NAD-ME subtype was formed in the mitochondria of the bundled sheath (BS) cells of the amaranth. After the watering was resumed, the isomer (~122kDa) disappeared again. And due to the emergence of new isomers, the activity of NAD-ME in mitochondria of BS cells increased, which seems to indicate that the new NAD-ME subtype is related to plant drought resistance.⁶³ However, its specific mechanism of action requires further research. Similarly, by quantitative RT-PCR and immunochemical methods, the specific activity of NADP-ME in tobacco leaves significantly increases under drought stress, and the de novo synthesis of NADP-ME is found to be enhanced. However, during the recovery period, the activity of the test enzyme recovers to near its basal level.⁶⁴ Changes in enzyme activity and content provide indirect evidence for the involvement of NADP-ME in the response to drought stress, and its specific regulatory network needs further improvement. In a well-watered environment, the average water use efficiency (WUE) of NAD-ME and NADP-ME C4 grasses is similar, but under drought conditions, the WUE of C4 grasses increases, and the NAD-ME grasses are more drought-prone than NADP-ME increases water use efficiency to a greater extent.⁶⁵

NADP-ME may be involved in drought stress response by catalyzing the release of CO₂ in photosynthesis II.⁶⁶ Under drought stress, the plant closes in the stomata and leads to a decrease in CO₂ absorption. At this time, the content of NADP-ME can be increased to compensate for the deficiency of CO₂, thereby maintaining the photosynthesis II reaction. This theory can explain the enhancement of the C4 enzyme system in C3 plants under drought stress,⁶⁷ while the protein of NADP-ME is a hydrophilic protein that can be protected by increasing cell osmotic pressure and reducing water loss (Figure 3).⁶⁸ The above studies showed that after drought stress, ME changed its specific activity to form isomers, as a biosynthesis reactant for osmotically active compounds, and improved water utilization and other ways to resist drought stress. This provides a target for studying the response mechanism of ME against drought.

Figure 3.

NADP-ME is involved in drought stress responses. Drought stress causes stomatal closure, decreases intracellular CO₂ concentration, blocks photosynthesis, increases NADP-ME content and catalyzes the release of CO₂ to maintain photosystem II.

Research progress of ME in plant salt response

Like other abiotic stresses, salt stress can also have certain adverse effects on plant growth, metabolism, and reproduction, such as photosynthesis,^69,70 ion homeostasis,^71,72 seed germination,^72–84 membrane permeability,^72,85–87 and so on.^88–90 In the long-term evolution, the halophyte itself forms a special structure such as salt glands and salt sacs to help plants resist salt stress.^91–100 In addition, certain hormones, genes, ions, and foreign substances, etc., participate in the plant salt stress response process through various pathways.^101–116

In recent years, it has been found that salt stress can also induce the expression of ME gene and the accumulation of ME protein in various plants. Under salt stress, anions such as malic acid and Cl⁻ accumulate in the cells to balance the excess Na⁺ in the plant. At this time, the ME synthesized in the cells is likely to be involved in the metabolism of malic acid.¹¹⁷ In addition, only three and four NADP-ME genes were found in the genomes of Zea mays and Arabidopsis, respectively, but five NADP-ME genes were found in the genome of Populus euphratica (PtNADP-ME1- 5).¹¹⁸ It is speculated that the members of NADP-ME and their physiological functions in woody plants may be more complex than herbaceous plants.

The sorghum-overexpressing NADP-ME gene in the germination stage has better salt tolerance, and the gene expression is found to be the highest under 100 mM NaCl treatment. In addition, the expression of this gene can also improve the photosynthetic efficiency of Arabidopsis under salt stress,¹¹⁹ This may provides energy for a series of metabolic processes in which plants resist salt stress. High salt and dehydration induce the expression of NADP-ME gene (AvME) in aloe, and further studies find that the expression of AvME protein increases significantly after 48 hours of high salt stress induction, and with the prolongation of treatment time, its level is gradually rising.⁵³ In addition to this, salt stress causes osmotic stress and is accompanied by excessive ROS, thereby increasing the oxidative stress of the plant. However, NADP-ME catalyzes the oxidative decarboxylation of L-malic acid to produce NADPH, providing the reductive capacity needed to scavenge ROS. Liu et al. confirmed that carbonate induces the expression of NADP-ME2 mRNA in roots, and that NADP-ME2 transcript increases during 72 hours of exposure to NaHCO₃, NaCl and PEG stress. These indicate that rice NADP-ME2 is involved in salt and osmotic stress responses.¹²⁰

In addition, overexpression of cytoNADP-ME in rice enhances the salt tolerance of transgenic Arabidopsis seedlings. In rice (Oryza sativa L.) seedlings, salt stress induces the expression of the cytoNADP-ME gene. Moreover, the NADP-ME activity in leaves and roots also increases with the increase of NaCl concentration.¹²¹ Similarly, the activity of NAD-ME and NADP-ME in the leaves of eucalyptus increased under salt stress, which provided a reducing power for the cells to resist salt stress.¹²² Based on the above-mentioned numerous evidences, we speculate that ME is mainly used in three ways after being induced by salt stress. First, the increased activity of ME will produce more NADPH to scavenge ROS to reduce the damage caused by salt stress. Secondly, NADP can be used to synthesize osmotic adjustment substances. Due to the increased activity of NADP-ME, the content of malic acid, a product of the catalytic reaction of NADP-ME, increases the infiltration stress caused by salt stress. Finally, NADP-ME can balance the pH of cytosol by synthesizing malate.

In summary, ME may participate in salt stress response by increasing plant photosynthesis, providing reducing power, etc., to protect plants from salt stress and survive. However, the specific ME regulation process is still in need of further in-depth research and summary.

Conclusion and perspectives

ME is a key enzyme regulating malic acid metabolism, and NADP-ME is one of the key enzymes in the process of photosynthesis. There are many substances closely related to it, and the change of ME will cause changes in the content of other substances and then cause other anti-stress. The activity of NADP-ME shows a large change under temperature, drought and salt stress, which indicate that the NADP-ME gene is a non-specific induction gene, which is closely related to drought, salt and temperature stress.

In addition to regulating the function of photosynthetic systems, ME can not only produce reducing power NADPH to scavenge ROS, but also reduce damage caused by oxidative stress. Moreover, the product can also be used to synthesize osmotic adjustment substances such as malate, regulate the osmotic pressure and stomatal movement of guard cells, participate in response to osmotic stress, and maintain the pH and ion balance of cells. In addition, it can also carry out fatty acid biosynthesis by providing carbon and NADPH and organic acid synthesis, participate in membrane lipid regeneration, and even promote fruit development. Therefore, it is helpful to analyze the role of ME in plant development and resisting various adversities, which is helpful to analyze its role in adversity defense. If we can study the ME gene in response to different stresses, and select excellent anti-reverse genes, and use genetically modified technology to transfer them back to the applied plants, it will provide a certain theoretical basis for the future use of ME gene for molecular resistance breeding, which is conducive to the development of resistance breeding.

Author Contributions

Xi Sun, Guoliang Han and Zhe Meng wrote the manuscript. Na Sui and Lin Lin modified the article. All authors read and approved the final manuscript.

Acknowledgments

We are grateful for financial support from Shandong Provincial Natural Science Foundation (ZR2016JL028), Major Program of Shandong Provincial Natural Science Foundation (2017C03), Shandong Provincial Natural Science Foundation (ZR2015EM007).

Conflict of interests

The authors declare that they have no competing interests.