Neutral invertase, hexokinase and mitochondrial ROS homeostasis

Abstract

Alkaline/neutral invertases (A/N-Invs) are unique to plants and photosynthetic bacteria. Although considerable advances have been made in our understanding of sucrose metabolic enzymes in plants, the function of A/N-Invs remained puzzling. In a recent study, we have analyzed the subcellullar localization of a cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) Arabidopsis A/N-Inv. Unexpectedly, At-A/N-InvA knockout plants showed a more severe growth defect than At-A/N-InvG knockout plants and a link between the two A/N-Invs and oxidative stress defense was found. Overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts reduced the activity of the ascorbate peroxidase 2 (APX2) promoter, that was stimulated by hydrogen peroxide and abscisic acid. It is discussed here how sugars and ascorbate might contribute to mitochondrial reactive oxygen species homeostasis. We hypothesize that both mitochondrial and cytosolic A/N-Invs and mitochondria-associated hexokinases are key mediators, integrating metabolic and sugar signaling processes.

A/N-Inv Localization in Arabidopsis thaliana Protoplasts

It is well-accepted that acid-type plant invertases are responsible for the hydrolysis of sucrose in the intercellular space (apoplast or cell wall), and in vacuoles. These invertases play a role in controlling sucrose allocation and overall plant development, in response to environmental stimuli. Recent studies indicated that A/N-Invs not only localize in the cytosol, but also in other subcellular locations, such as chloroplasts,¹ mitochondria² and plastids,² suggesting that these A/N-Inv isoforms fulfil novel physiological roles.^3,4 We demonstrated that At-A/N-InvG is localized in the cytosol, while At-A/N-InvA is present in (or attached to) mitochondria, with the mitochondrial sorting signal present in the first 31 amino acids of its N terminus. How mitochondrial invertase function is integrated in intermediary metabolism and whether it represents a functional link between respiration and cytosolic sucrose metabolism is unknown.

The Enzymatic Activities of A/N-Invs

Generally, A/N-Invs are classified based to their pH optimum, either close to 6.5–7.0 or to 7.8–8.0, and accordingly named neutral-Invs or alkaline-Invs, respectively. We detected that At-A/N-InvA shows a broad activity range with an optimum at pH 7.5. By contrast, At-A/N-InvG was found to be an alkaline-Inv with an activity optimum at pH 9.5. The estimated K_m's are in the same range as other A/N-Invs.^1,5 The inhibitory effect of Tris on At-A/N-InvA was comparable to that on other A/N-Invs.⁵ Surprisingly, AtA/N-Inv-G activity increased at lower Tris concentrations.

Analysis of atinva and atinvg Knock-Out Plants

A growing body of evidence suggests that cytosolic A/N-Invs are indispensable for normal plant growth and development, with increasing enzyme activities under stress.^6–11 Hexokinase (HXK) is found to be mainly associated with the outer mitochondrial membrane. Together with the presence of A/N-Invs in mitochondria, this suggests relationships between mitochondrial A/N-Invs, cytosolic carbohydrate metabolism, HXK-mediated catalytic activity and/or signaling, and stress defense reactions. Our results showed that atinva plants have a severe root growth defect and a weak leaf growth defect. An even more prominent leaf and root growth defect, however, was observed for atinvg plants, reminiscent of plants suffering from oxidative stress, limited nitrate availability or impaired signaling.^9,12–14 We found that the total A/N-Inv activity was reduced by more that 30% in both knockout lines. Considering the presence of 9 A/N-Invs in Arabidopsis thaliana, this suggests that At-A/N-InvA and At-A/N-InvG activity contribute significantly to total A/N-Inv activity. Alternatively, the At-A/N-InvA and At-A/N-InvG proteins themselves (independently of their catalytic activities) might play a regulatory role in controlling overall A/N-Inv activity and plant growth and development as suggested before for At-A/N-InvG.⁸

The Role of At-A/N-InvA and At-A/N-InvG in Oxidative Stress Defense

Recent data showed that exogenous sucrose additions can help plants to protect themselves against oxidative stress prior to or upon exposure.^15–17 Moreover, a connection was reported between mitochondria-associated HXK (mtHXK) activity and mitochondrial ROS levels, possibly linked to ROS signaling pathways leading to antioxidant defense reponses.^18,19 Now, cytosolic A/N-Invs are emerging as important regulators of plant growth and development, possibly also involved in metabolic signaling processes, especially under stress conditions.^6,10 Thus, the importance of A/N-Inv activity may reside in regulating the cytosolic and/or organellar Suc concentration, or in the production of Glc which could be sensed by mitochondria-associated HXKs^19–21 or serve as a substrate for HXK to control mitochondrial ROS production.¹⁸ Ascorbate peroxidases (APXs) use ascorbate (AsA) as substrate; these enzymes play crucial roles in H₂O₂ scavenging processes, in concert with the different enzymes of the so-called Halliwell-Asada pathway representing one of the most important antioxidant systems in the cytosol,²² chloroplasts,²³ and mitochondria.²⁴ Quantitative RT-PCR of an array of genes involved in antioxidant defense showed that superoxide dismutase (CSD1, FSD1, MSD1), catalase (CAT2) and APX2 as well as At-A/N-Invs and HXK1 transcription levels significantly increased upon application of exogenous H₂O₂ (Fig. 1), suggesting that A/N-Invs are part of the antioxidant system involved in cellular ROS homeostasis. Consistent with the hypothesis that A/N-Invs counteract oxidative stress, untreated atinva and atinvg knockout lines also showed increased expression levels of APX2, CAT2, CSD1, FSD1 and MSD1 (Fig. 1). Thus, the two A/N-Invs isoenzymes under study appear to play a dual role in sugar metabolism and antioxidant defense. Overexpression of At-A/N-InvA and At-A/N-InvG in Arabidopsis leaf mesophyll protoplasts and the use of a promoter APX2-luciferase reporter (responding to cytosolic ROS levels) indeed provided further evidence for this point of view (Fig. 2). Both H₂O₂ and ABA activated the APX2 promoter, but to a much lesser extent in the At-A/N-InvA and At-A/N-InvG overexpressing protoplasts. Both metabolizable sugars and AsA downregulated APX2 promoter activity in the light and in the dark. DCMU, an inhibitor of photosystem II, abolishes the production of chloroplastic H₂O₂ and subsequently the induction of APX2 in Arabidopsis leaves in the light but not in the dark (Fig. 2).²⁵ In the dark, cytosolic H₂O₂ levels are more likely depending on the level of mitochondrial ROS production. APX2 promoter activity is around six times higher in the light than in the dark.

Figure 1.

qRT-PCR data of antioxidative gene expression for wt and knockout plants, with (+H) or without H₂O₂ treatment. Actin2 (At3g18780) and UBQ10 (At4g05320) were used as reference genes. The values are fold changes in transcript levels normalized to the two reference genes with respect to the wt control. Means ± SE (n = 3).

Figure 2.

APX2 promoter luciferase assay of protoplasts derived from wt plants with or without overexpression of At-A/N-InvA or At-A/N-InvG under different treatments (30 mM Glc, 30 mM Suc, 30 mM AsA, 30 mM Mtl, 200 µM H₂O₂, 10 µM DCMU and 10 µM ABA). The experiment was executed with (L) or without (D) light. The values are fold changes normalized to the wt control. Means ± SE (n = 3).

Sugars, Ascorbate and Mitochondrial ROS Homeostasis

It has been demonstrated that cytosolic HXK isoforms are associated with the mitochondrial outer membrane²⁶ and sugar supplementation or enhanced Glc production might help to increase the number of mitochondria, the respiration rate and ATP generation. We propose that a continuous and efficient supply of Glc to mtHXK is necessary to maintain its activity at a rather constant level, in order to control the flux through the mitochondrial electron transport chain (ETC), influencing mitochondrial ROS production¹⁸ (Fig. 3). In particular, the tightly bound outer-membrane mtHXK could provide a basal level of ADP for ATP synthesis, preventing ROS accumulation. This mechanism would be particularly useful when other sources of ADP become limiting for oxidative phosphorylation. One candidate to act as substrate for mtHXK is the cytosolic Glc generated by cytosolic A/N-Invs or other enzymes. Another possibility is that cytosolic sucrose passes through the porins in the outer mitochondrial outer membrane (Fig. 3). Subsequently, it may be transported by a specific inner membrane sucrose transporter and hydrolyzed by one or more A/N-Invs residing in the mitochondrial matrix (Fig. 3).²⁷ The generated Glc may then be pumped back into the intermembrane space (IMS) by a specific Glc transporter, diffusing through the pores, and serving as a substrate for the mitochondrial outer membrane-associated HXK. Such mechanism might help to control the Glc concentration in the vicinity of mtHXK, even when the cytosolic Glc concentration is very low. The reported specific inhibition of Glc backflow into the IMS by AsA²⁷ and the fact that A/N-Invs are inhibited by their own hexose products⁵ make up efficient feedback mechanisms to control the activities of A/N-Invs and mtHXK, linked to mitochondrial ROS production. If mitochondrial AsA contents become too low to counteract ROS accumulation, Glc outflow and mtHXK activity would be promoted, stimulating ADP recycling and avoiding ATP synthesis-related limitation of respiration and subsequent H₂O₂ release.¹⁸ Such a fine regulatory mechanism would be particularly useful when other sources of Glc or ADP become limited.

Figure 3.

Model showing the putative role of A/N-Invs and mtHXKs in oxidative defense related processes in plant mitochondria. Cytosolic Suc can serve as a substrate for cytosolic A/N-Invs (cA/N-Invs). However, Suc can also enter the matrix via a Suc transporter in the inner membrane (IM). Glc and Fru are subsequently produced by mtA/N-Inv. Glc is transported back into the intermembrane space (IMS) through a Glc transporter that is regulated by AsA and through the outer membrane (OM) to serve as a substrate for HXK bound to the OM (termed mtHXK). mtHXK contributes to a steady-state ADP recycling via voltage dependent anion channels (VDAC) and adenine nucleotide transporters (ANT) to regulate H₂O₂ formation in the electron transport chain (ETC) on the IM. mtHXK activity is inhibited by ADP. It is not known whether methyl jasmonate and G6P can induce the detachement of mtHXK from the OM, as observed in animals. The G6P generated by mtHXK (as well as chloroplastic triose phosphates), can enter glycolysis and the pyruvate (Pyr) produced crosses the IM via Pyr carboxylase (PC) for entering the Krebs (TCA) cycle. Alternatively, G6P can be used to produce UDPGlc for the resynthesis of carbohydrates (e.g., Suc, cellulose). Fru resulting from mtA/N-Inv or cA/N-Inv activity can be transformed into F6P by fructokinase (FK), which can enter glycolysis or can be used for Suc synthesis by sucrose phosphate synthetase (SPS) and sucrose phosphate phosphatase (SPP). Mitochondrial SOD and glutathione peroxidase (GPX) can assist in ROS scavenging processes within the matrix. AsA is synthesized witin the IMS, and can serve as a substrate for APX, to produce dehydroxyascorbate (DHA), which can be imported into the mitochondrial matrix by a transporter in the IM which also can function as a Glc transporter, regulated by AsA. Acetyl CoA, acetyl coenzyme A; Cytc, cytochrome c; DHAP, dihydroxyacetone phosphate; F1,6-bP, fructose 1,6-biphosphate; PGA, phosphoglycolic acid; Q, plastiquinon pool; TCA, tricarboxylic acid cycle.

Methyl jasmonate (MeJa), a well-known stress hormone in plants, induces the release of HXK from the outer mitochondrial membrane in animal (cancer) cells²⁸ (Fig. 3). Similar mechanisms might be operating in plants, although further experimental verification is necessary. In accordance with our model (Fig. 3), and with experimental data,^29,30 MeJa-dependent (transient or partial) release of HXK from the outer mitochondrial membrane would slow down glycolysis and respiration (by disruption of the glycolytic metabolon²⁶), resulting in extra ROS and ROS signaling. MeJa also upregulates several AsA and GSH biosynthetic genes^31–33 as well as other antioxidant enzyme genes. Typically, dehydroascorbate (DHA) reductases (DHRs) and glutathione reductases (GRs) are also stimulated under such conditions, in order to regenerate AsA from DHA and GSH from GSSG.³⁴ We speculate that glucosamine could work via similar mechanisms, since it inhibits AtHXK1-mediated effects such as hypocotyl elongation.³⁵

While de novo GSH biosynthesis is catalyzed by two enzymes (GSH1 and GSH2³⁶), de novo AsA synthesis is very complex with at least four possible pathways,³⁷ all requiring the input of G6P.³⁸ Figure 4 shows two of the pathways used in plants, as compared to the pathway leading to AsA synthesis in animals. There is little doubt that the Man/Gal pathway (also termed Smirnoff-Wheeler pathway) is the most prominent pathway in plants,³⁷ although it involves no less than nine enzymes to transform G6P into AsA. Key intermediates in this pathway also deliver cell wall precursors, linking AsA and cell wall biosynthesis. Lately, the considerably shorter myo-inositol (MI) pathway (six enzymes; red in Fig. 4) gained more attention.^39,40 This pathway partly resembles the animal pathway (Fig. 4). Myo-inositol oxygenase (MIOX; four genes in Arabidopsis) links MI to AsA metabolism. The last step leading to AsA is catalyzed by a L-gulono-1,4-lactone dehydrogenase/oxidase (GulLO).⁴⁰ Seven putative GulLO genes are present in the Arabidopsis genome.⁴⁰ Overexpression of these genes in tobacco BY2 cells pointed out that AtGulLO2, 3 and 5 encode functional enzymes.⁴⁰ The AtGulLO2 (At2g46750) and AtGulO5 (At2g46740) genes are highly expressed and the derived proteins are predicted to be located in mitochondria,⁴⁰ similar to L-galactono-1,4-lactone dehydrogenase (GLDH), catalyzing the last step in AsA synthesis by the Man/Gal pathway³⁷ (Fig. 4).

Figure 4.

Biosynthetic pathways of L-ascorbate in plants and animals. The Man/Gal pathway is highlighted in yellow and the animal pathway is highlighted in green. The myo-inositol pathway is in red. Enzymes catalyzing the numbered reactions include: 1, phosphoglucose isomerase; 2, phosphomannose isomerase; 3, phosphomannomutase; 4, GDP-D-mannose pyrophosphorylase; 5, GDP-D-mannose 3′,5′-epimerase; 6, GDP-L-galactose phosphorylase; 7, L-galactose-1-P phosphatase; 8, L-galactose dehydrogenase; 9, L-galactono-1,4-lactone dehydrogenase; 10, phosphoglucomutase; 11, UDP-Glc pyrophosphorylase; 12, UDP-Glc dehydrogenase; 13, glucuronate-1-phosphate uridylyltransferase; 14, glucuronokinase; 15, glucuronate reductase; 16, aldono lactonase; 17, guluno-1,4-lactone dehydrogenase; 18, D-myo-inositol 3-phosphate synthase; 19, D-myo-inositol monophosphatase; 20, D-myo-inositol oxygenase.

Slowing down glycolysis, respiration and growth also occurs under more general (and longer term) sugar starvation conditions (as compared to a more short term one as discussed above for MeJa). Trehalose 6-phosphate (T6P; an inhibitor of SnRK1 activity⁴¹), the bZIP11 TF,⁴² 5Ptase13 ⁴³ and the SnRK1 protein kinases⁴⁴ function in a regulatory circuit operating under sugar starvation. Focusing on the genes involved in the MI pathway to AsA synthesis, it was recently reported that bZIP11 stimulates expression of MIOX2 (At2g19800) and MIOX4 (At5g56640).⁴² The MIOX2 gene is also induced after an extended night in Arabidopsis leaves,⁴⁵ and, accordingly, is repressed by glucose.^46,47 Intriguingly, both the AtGulLO2 and AtGulO5 genes are upregulated under potassium⁴⁸ and iron deficiency (Genevestigator data; www.genevestigator.com) in Arabidopsis roots. AtGulLO2 is apparently also repressed by sugar (Genevestigator). Moreover, a GulLO homolog of tobacco is upregulated by MeJa.³³ Taken together, these observations suggest that the shorter MI (or animal-like) pathway leading to AsA synthesis might become relatively more important under metabolic stress conditions and this is an interesting area for future research. So far, most of the research focused on photosynthetically active source leaves, since AsA synthesis is stimulated by light.⁴⁹ More studies are needed to explore AsA metabolism and import in sink tissues (fruits, roots, seeds, tubers, etc.,), which might be subjected even more frequently to sugar starvation. One recent study showed that shading experiments on kiwi vines affected kiwi fruit sugar levels much more strongly than it affected AsA levels.⁵⁰ This suggests that plants should have mechanisms to keep their AsA levels above a certain threshold level, even under (dark-induced) sugar starvation conditions. Increasing the flux through the MI pathway might be part of the metabolic reprogramming under carbon starvation. AsA and GSH homeostasis seem to be vital for normal plant growth and development. Recent insights suggest that GSH itself could act as a signal, while AsA is indispensable as a cofactor in many enzymes, including some involved in phytohormone synthesis. Moreover, via redox regulation both GSH and AsA are also intimately connected with various signaling pathways in plants.⁴⁹

In conclusion, the interplay between neutral A/N-Invs, HXKs and MeJa could be at the core of a mechanism enabling plant cells to sense different proportions of Suc and hexoses. Moreover, this seems to be intimately linked to (mitochondrial) AsA metabolism. The metabolic and compartmental fluxes in our hypothetical scheme (Fig. 3) appear to act in accordance with sugar signaling networks. Together, these mechanisms seem to contribute to mitochondrial ROS homeostasis, and perhaps, also to total cellular ROS homeostasis, especially in the dark.

Addendum to: Xiang L, Le Roy K, Bolouri-Moghaddam MR, Vanhaecke M, Lammens W, Rolland F, Van den Ende W. Exploring the neutral invertase-oxidative stress defense connection in Arabidopsis thaliana. J Exp Bot. 2011;62:3849–3862. doi: 10.1093/jxb/err069.