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Is There a Metabolic Requirement for Photorespiratory Enzyme Activities in Heterotrophic Tissues?

2013; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês

10.1093/mp/sst111

1674-2052

Adriano Nunes‐Nesi, Alexandra Florian, Andrew J.M. Howden, Kathrin Jahnke, Stefan Timm, Hermann Bauwe, Lee Sweetlove, Alisdair R. Fernie,

Plant Stress Responses and Tolerance

Dear Editor, Photorespiration is an essential metabolic process in leaves that facilitates recovery of carbon lost by the oxygenase reaction of Rubisco and avoids the accumulation of the toxic product, 2-phosphoglycolate (2PG) of this reaction (Bauwe et al., 2012Bauwe H. Hagemann M. Kern R. Timm S Photorespiration has a dual origin and manifold links to central metabolism.Curr. Opin. Plant Biol. 2012; 15: 269-275Crossref PubMed Scopus (129) Google Scholar). However, there is also evidence to suggest that photorespiration has a more complex role during normal growth than the mere detoxification of 2PG and the recovery of 3-phosphoglycerate (3PGA) (Bauwe et al., 2012Bauwe H. Hagemann M. Kern R. Timm S Photorespiration has a dual origin and manifold links to central metabolism.Curr. Opin. Plant Biol. 2012; 15: 269-275Crossref PubMed Scopus (129) Google Scholar). The current view is that photorespiration is an essential mechanism to maintain optimal photosynthesis, normal growth, and development of oxygenic photosynthetic organisms in an oxygen-containing environment (Bauwe et al., 2012Bauwe H. Hagemann M. Kern R. Timm S Photorespiration has a dual origin and manifold links to central metabolism.Curr. Opin. Plant Biol. 2012; 15: 269-275Crossref PubMed Scopus (129) Google Scholar). Moreover, there is a hint that the photorespiratory enzymes may have roles in tissues other than leaves: analysis of expression of genes encoding photorespiratory enzymes using publicly available data through Bioarray Resource (BAR; www.bar.utoronto.ca; Toufighi et al., 2005Toufighi K. Brady S.M. Austin R. Ly E. Provart N.J The botany array resource: E-northerns, expression angling, and promoter analyses.Plant J. 2005; 43: 153-163Crossref PubMed Scopus (579) Google Scholar) revealed that transcripts encoding several enzymes associated with the photorespiratory process are present in roots and other heterotrophic tissues (Supplemental Table 1). High transcripts levels in these tissues are observed for glyoxylate reductase 1(GLYR1—At3g25530), glycolate oxidase (GOX—At4g18360), glutamate:glyoxylate aminotransferase 2 (GGAT2—At1g70580), and alanine:glyoxylate aminotransferase 2 (AGT2—At4G39660), with the isoform of glycolate oxidase being specifically expressed in roots. Similarly, comparison of the pep2pro proteome database (Baerenfaller et al., 2008Baerenfaller K. Grossmann J. Grobei M.A. Hull R. Hirsch-Hoffmann M. Yalovsky S. Zimmermann P. Grossniklaus U. Gruissem W. Baginsky S Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics.Science. 2008; 320: 938-941Crossref PubMed Scopus (414) Google Scholar; http://fgcz-pep2pro.uzh.ch/; Supplemental Table 2) revealed considerable abundance of the small subunit of Rubisco (RbcS—At1g67090), large subunit of Rubisco (RbcL—Atcg00490), glycolate oxidase (GOX—At4g18360), glutamate:glyoxylate aminotransferase 1 (GGAT 1—At1g23310), glutamate:glyoxylate aminotransferase 2 (GGAT2—At1g70580), alanine:glyoxylate aminotransferase 2 (AGT2—At4G39660), alanine:glyoxylate aminotransferase 3 (AGT3—At2G38400), and glyoxylate reductase 1 (GLYR1—At3g25530) (Supplemental Table 2). Interestingly, despite the lower amounts of these proteins in comparison with leaf tissues, significant amounts of RbcL, glycine decarboxylase (GDC) H-, T- and P-proteins, and glycerate kinase (GLYK) were detected in roots. Similarly, these proteins were found to be highly abundant in heterotrophic tissues under conditions of stress (reviewed in Jacoby et al., 2013Jacoby R.P. Millar A.H. Taylor N.L Application of selected reaction monitoring mass spectrometry to field-grown crop plants to allow dissection of the molecular mechanisms of abiotic stress tolerance.Front. Plant Sci. 2013; 4 (10.3389/fpls.2013.00020.): 20Crossref PubMed Scopus (19) Google Scholar), whilst SHM2 was demonstrated to be confined to vascular tissues (Engel et al., 2011Engel N. Ewald R. Gupta K.J. Zrenner R. Hagemann M. Bauwe H The presequence of Arabidopsis serine hydroxymethyltransferase SHM2 selectively prevents import into mesophyll mitochondria.Plant Physiol. 2011; 157: 1711-1720Crossref PubMed Scopus (47) Google Scholar). Despite the fact that these studies reveal the presence of several proteins of, or associated with, photorespiration in root tissues and, consequently, several of these enzymes are included in the recently published Arabidopsis root-specific genome-scale network (Mintz-Oron et al., 2012Mintz-Oron S. Meir S. Malitsky S. Ruppin E. Aharoni A. Shlomi T Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity.Proc. Natl Acad. Sci. U S A. 2012; 109: 339-344Crossref PubMed Scopus (202) Google Scholar), little is yet known concerning their functionality in this tissue. In support of these results, the presence of photorespiratory enzyme activities in heterotrophic tissues was previously documented (Lernmark et al., 1991Lernmark U. Henricson D. Wigge B. Gardestrom P Glycine oxidation in mitochondria isolated from light grown and etiolated plant tissue.Physiol. Plant. 1991; 82: 339-344Crossref Scopus (13) Google Scholar; Igamberdiev et al., 1997Igamberdiev A.U. Bykova N.V. Gardeström P Involvement of cyanide-resistant and rotenone-insensitive pathways of mitochondrial electron transport during oxidation of glycine in higher plants.FEBS Lett. 1997; 412: 265-269Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Interestingly, while glycine decarboxylase activity in heterotrophic tissues of dicots is quite low, in cereals, it may reach about 20% of its levels in photosynthetic tissues (Lernmark et al., 1991Lernmark U. Henricson D. Wigge B. Gardestrom P Glycine oxidation in mitochondria isolated from light grown and etiolated plant tissue.Physiol. Plant. 1991; 82: 339-344Crossref Scopus (13) Google Scholar). In addition, the activity of glycine decarboxylase in maize scutellum was shown (Igamberdiev et al., 1997Igamberdiev A.U. Bykova N.V. Gardeström P Involvement of cyanide-resistant and rotenone-insensitive pathways of mitochondrial electron transport during oxidation of glycine in higher plants.FEBS Lett. 1997; 412: 265-269Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In this storage tissue, GDC enzyme activity is probably related to oxidation of glycine formed from glyoxylate the production of which is considerable during operation of the glyoxylate cycle (Igamberdiev et al., 1997Igamberdiev A.U. Bykova N.V. Gardeström P Involvement of cyanide-resistant and rotenone-insensitive pathways of mitochondrial electron transport during oxidation of glycine in higher plants.FEBS Lett. 1997; 412: 265-269Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In order to obtain more direct experimental evidence that photorespiratory enzymes have a metabolic impact on root metabolism, we analyzed the metabolite profiles of roots from previously characterized photorespiratory mutants, as well as a knockout mutant for glycolate oxidase 3 which was first characterized here (Supplemental Figure 1). Plants were grown hydroponically with the root solution bubbled with normal air while the rosette tissues were simultaneously exposed to a high CO2-containing atmosphere (1%) which was necessary to allow normal growth. These mutants are either characterized by a clear but not intolerable reduction of the photorespiratory carbon flow (hpr1-1 and gox3-1) or in which essential steps of photorespiration are completely blocked (pglp1-1, glyk1-1, and shm1-2) and therefore display photorespiratory phenotypes under normal air conditions (Timm et al., 2013Timm S. Florian A. Wittmiß M. Jahnke K. Hagemann M. Fernie A. Bauwe H Serine acts as metabolic signal for the transcriptional control of photorespiration-related genes in Arabidopsis thaliana.Plant Physiol. 2013; 162: 379-389Crossref PubMed Scopus (84) Google Scholar). We evaluated the root content of selected metabolites in the individual glycolate oxidase, gox3-1 (see Supplemental Figure 1), NADH-dependent peroxisomal hydroxypyruvate reductase, hpr1-1, glycerate 3-kinase, glyk1-1, serine hydroxymethyltransferase 1, shm1-2, and 2PG-phosphatase pglp1-1 knockout plants (described in Timm et al., 2013Timm S. Florian A. Wittmiß M. Jahnke K. Hagemann M. Fernie A. Bauwe H Serine acts as metabolic signal for the transcriptional control of photorespiration-related genes in Arabidopsis thaliana.Plant Physiol. 2013; 162: 379-389Crossref PubMed Scopus (84) Google Scholar). This analysis revealed considerable differences between the root metabolite profiles of the photorespiratory mutants and wild-type (Figure 1 and Supplemental Figure 2). Surprisingly, although the photorespiratory intermediate glycolate was detected in the roots of all five genotypes, its content was essentially invariant. This result suggests that glycolate may have an alternative source in roots other than photorespiratory metabolism. Recently, it has been observed that glycolate can be considered as a major hypoxic metabolite (Narsai et al., 2009Narsai R. Howell K.A. Carroll A. Ivanova A. Millar A.H. Whelan J Defining core metabolic and transcriptomic responses to oxygen availability in rice embryos and young seedlings.Plant Physiol. 2009; 151: 306-322Crossref PubMed Scopus (127) Google Scholar). In light of the heavily perturbed tricarboxylic acid (TCA) cycle and GABA shunt found (described below), another possible source of glycolate in root tissue could be a suboptimal glyoxylate cycle. As a consequence of that, both NADH- and NADPH-dependent glyoxylate reductase could convert glyoxylate to glycolate. Therefore, the physiological sense of the glycolate production in roots may well be maintenance of the redox balance under these conditions. In comparison with the effects of other mutations in the photorespiratory pathway, we exclusively observed changes in glycerate (11-fold greater than the wild-type) glyk1-1, with the effect on this metabolite being considerable but less pronounced in the other four mutants. From this, we can conclude that GLYK is needed in roots to maintain functional glycerate phosphorylation. In addition, we observed elevated root glycine contents in the gox3-1, hpr1-1, shm1-2, and pglp1-1 knockout mutants. The serine contents increased in all genotypes, with the exception of glyk1-1. Interestingly, when we estimated the absolute concentration for glycine and serine and calculated the glycine-to-serine ratio, a clear reduction in this ratio is observed in roots from gox3-1, shm1-2, and pglp1-1 knockout mutants (Figure 1). Thus, these data indicate that all of the analyzed intermediates of leaf photorespiration are present in roots as well and demonstrate that the lack of photorespiratory pathway enzymes in this tissue leads to accumulation of photorespiratory intermediates. That said, we were, however, unable to detect the PGLP protein in root tissue (data not shown), clearly indicating that the photorespiratory cycle is not complete in roots. Apart from the effects on photorespiratory intermediates, we also uncovered substantial changes in the TCA cycle. That is, the amounts of citrate, succinate, and malate increased in gox3-1, hpr1-1, shm1-2, and pglp1-1. Fumarate increased in hpr1-1, shm1-2, pglp1-1, and GABA in gox3-1, shm1-2, and in the pglp1-1 mutant. Surprisingly, most of the analyzed metabolites did not show any significant changes in the glyk1-1 mutant, with the exception of alterations in the levels of pyruvate. These results demonstrate that deficiency in the majority of the photorespiratory enzymes results in a perturbation of the TCA cycle. On the other hand, the GLYK function appears not to play an important role in the other metabolic routes investigated here (see also below). Furthermore, the levels of the important metabolites of nitrate assimilation, glutamate, and glutamine were significantly increased in knockout mutants of GOX3, HPR1, SHM1, and PGLP1, with similar changes observed for aspartate (significantly for gox3-1, pglp1-1, and shm1-2) and asparagine (significantly for gox3-1 and pglp1-1) levels. Intriguingly, these changes are highly reminiscent, although much smaller in magnitude, of the changes observed in leaves of the mutants (see Timm et al., 2013Timm S. Florian A. Wittmiß M. Jahnke K. Hagemann M. Fernie A. Bauwe H Serine acts as metabolic signal for the transcriptional control of photorespiration-related genes in Arabidopsis thaliana.Plant Physiol. 2013; 162: 379-389Crossref PubMed Scopus (84) Google Scholar)—a fact that underlines the functionality of enzymes of the pathway in heterotrophic tissues. There were additionally notable changes in the contents of a range of other amino acids whereas, and by contrast, changes in sugar levels were relatively minor (Supplemental Figure 2). Namely, alanine, proline, threonine, tryptophan, and valine were all significantly increased in the gox3-1, hpr1-1, shm1-2, and pglp1-1 mutants. Beta-alanine which accumulated in the gox3-1, shm1-2, and pglp1-1 mutant, whereas isoleucine, lysine, and tyrosine were significantly increased in the gox3-1, hpr1-1, and shm1-2 mutants. The changes in amino acid levels are very similar to those observed following inhibition of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (Araújo et al., 2012Araújo W.L. Tohge T. Osorio S. Lohse M. Balbo I. Krahnert I. Sienkiewicz-Porzucek A. Usadel B. Nunes-Nesi A. Fernie A.R Antisense inhibition of the 2-oxoglutarate dehydrogenase complex in tomato demonstrates its importance for plant respiration and during leaf senescence and fruit maturation.Plant Cell. 2012; 24: 2328-2351Crossref PubMed Scopus (72) Google Scholar) and further support our contention that, in the root, the photorespiratory enzymes likely function in a manner different from their classical mode analogous to that previously described for the TCA cycle (Sweetlove et al., 2010Sweetlove L.J. Beard K.F.M. Nunes-Nesi A. Fernie A.R. Ratcliffe R.G Not just a circle: flux modes in the plant TCA cycle.Trends Plant Sci. 2010; 15: 462-470Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). The increase in GABA levels of up to 2.5-fold in gox3-1, shm1-2, and in the pglp1-1 mutant might suggest an up-regulation of GABA shunt. This increase in GABA shunt can be associated to the observed increase in succinate levels in the roots of photorespiratory mutants. Taken together, these results suggest that GABA shunt supplies the TCA cycle with carbon from glutamate (Studart-Guimarães et al., 2007Studart-Guimarães A. Fait A. Nunes-Nesi A. Carrari F. Usadel B. Fernie A.R Reduced expression of succinyl–coenzyme A ligase can be compensated for by up-regulation of the γ-aminobutyrate shunt in illuminated leaves.Plant Physiol. 2007; 145: 626-639Crossref PubMed Scopus (140) Google Scholar). In addition, the induction of GABA pathway associated with stress response may be linked to the use of glycine/glyoxylate in transamination reactions of the photorespiratory pathway. We have previously reported that pglp1-1 mutant has a very strong photorespiratory phenotype which could not be completely recovered even under very high CO2 concentrations (1%). As a consequence of that, a massive metabolic reprogramming was observed in leaves under these conditions and evidently alters root metabolism as well (this study), despite the fact that PGLP is not present in roots. Since we rather found glycerate and serine but not glycolate levels elevated in roots of pglp1-1 (and the other genotypes), these changes could well be due to an induced phosphoserine pathway down from 3PGA to serine as a consequence of a suppressed and blocked photorespiratory pathway in leaves. Indeed, we have observed that, in either pglp1-1 or gox3-1 mutants, both enzymes upstream of GDC cause an accumulation of glycine or serine. These changes are likely due to an induced downstream flow from 3PGA within the non-photorespiratory serine pathway as an alternative source of serine and glycine in heterotrophic tissues and due to the absence of the photorespiratory carbon flow. In summary, the data presented here complement those from transcriptomics and proteomics in highlighting a functional role for the photorespiratory enzymes in roots indicating an intimate linkage with the TCA cycle. It has been previously speculated that they may act to play a role in adaptation to oxidative stress (Jacoby et al., 2011Jacoby R.P. Taylor N.L. Millar A.H The role of mitochondrial respiration in salinity tolerance.Trends Plant Sci. 2011; 16: 614-623Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and the demonstration of their functionality provides further support for this hypothesis. Further experiments which target other auxiliary functions of the photorespiratory pathway such as C1 metabolism and redox metabolism should be carried out in order that we can comprehensively establish the roles of these enzymes in roots and other heterotrophic tissues whilst the role of Rubisco in non-photosynthetic tissues has been much researched—the toolkit is now there to carry out analogous studies on the other enzymes of the core C2 cycle. Supplementary Data are available at Molecular Plant Online. We acknowledge the DFG for funding support in the Framework of the PROMICS Research Unit. A.N.N. appreciates funding support from Max Planck Society and CNPq (grant no. 306355/2012-4).