Peroxisomal hydroxypyruvate reductase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release

Recycling of carbon by the photorespiratory pathway involves enzymatic steps in the chloroplast, mitochondria, and peroxisomes. Most of these reactions are essential for plants growing under ambient CO(2) concentrations. However, some disruptions of photorespiratory metabolism cause subtle phenotypes in plants grown in air. For example, Arabidopsis thaliana lacking both of the peroxisomal malate dehydrogenase genes (pmdh1pmdh2) or hydroxypyruvate reductase (hpr1) are viable in air and have rates of photosynthesis only slightly lower than wild-type plants. To investigate how disruption of the peroxisomal reduction of hydroxypyruvate to glycerate influences photorespiratory carbon metabolism we analyzed leaf gas exchange in A. thaliana plants lacking peroxisomal HPR1 expression. In addition, because the lack of HPR1 could be compensated for by other reactions within the peroxisomes using reductant supplied by PMDH a triple mutant lacking expression of both peroxisomal PMDH genes and HPR1 (pmdh1pmdh2hpr1) was analyzed. Rates of photosynthesis under photorespiratory conditions (ambient CO(2) and O(2) concentrations) were slightly reduced in the hpr1 and pmdh1pmdh2hpr1 plants indicating other reactions can help bypass this disruption in the photorespiratory pathway. However, the CO(2) compensation points (Γ) increased under photorespiratory conditions in both mutants indicating changes in photorespiratory carbon metabolism in these plants. Measurements of Γ*, the CO(2) compensation point in the absence of mitochondrial respiration, and the CO(2) released per Rubisco oxygenation reaction demonstrated that the increase in Γ in the hpr1 and pmdh1pmdh2hpr1 plants is not associated with changes in mitochondrial respiration but with an increase in the non-respiratory CO(2) released per Rubisco oxygenation reaction.     

The Hyper-Gene Conversion Hpr5-1 Mutation of Saccharomyces Cerevisiae Is an Allele of the Srs2/Radh Gene

The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion. This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway. The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase. The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X. The hpr5-1 allele contains missense mutation in the putative ATP binding domain. A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms. We propose that the HPR5 gene functions in the RAD6 repair pathway.     

Metabolism of Hydroxypyruvate in a Mutant of Barley Lacking NADH-Dependent Hydroxypyruvate Reductase, an Important Photorespiratory Enzyme Activity

A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO₂ fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O₂. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO₂ efflux and ¹⁴CO₂ release from l-[U-¹⁴C]serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either [¹⁴C]serine or ¹⁴CO₂, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied [¹⁴C]serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolise a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.     

Peroxisomal malate dehydrogenase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release

Peroxisomes are important for recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxypyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by peroxisomal malate dehydrogenase (PMDH). PMDH, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana), reduces NAD⁺ to NADH via the oxidation of malate supplied from the cytoplasm to oxaloacetate. A double mutant lacking the expression of both PMDH genes was viable in air and had rates of photosynthesis only slightly lower than in the wild type. This is in contrast to other photorespiratory mutants, which have severely reduced rates of photosynthesis and require high CO₂ to grow. The pmdh mutant had a higher O₂-dependent CO₂ compensation point than the wild type, implying that either Rubisco specificity had changed or that the rate of CO₂ released per Rubisco oxygenation was increased in the pmdh plants. Rates of gross O₂ evolution and uptake were similar in the pmdh and wild-type plants, indicating that chloroplast linear electron transport and photorespiratory O₂ uptake were similar between genotypes. The CO₂ postillumination burst and the rate of CO₂ released during photorespiration were both greater in the pmdh mutant compared with the wild type, suggesting that the ratio of photorespiratory CO₂ release to Rubisco oxygenation was altered in the pmdh mutant. Without PMDH in the peroxisome, the CO₂ released per Rubisco oxygenation reaction can be increased by over 50%. In summary, PMDH is essential for maintaining optimal rates of photorespiration in air; however, in its absence, significant rates of photorespiration are still possible, indicating that there are additional mechanisms for supplying reductant to the peroxisomal HPR reaction or that the HPR reaction is altogether circumvented.     

High glycolate oxidase activity is required for survival of maize in normal air

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%-5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.     

Isolation and Genetic Analysis of Extragenic Suppressors of the Hyper-Deletion Phenotype of the Saccharomyces Cerevisiae Hpr1δ Mutation

The HPR1 gene of Saccharomyces cerevisae is involved in maintaining low levels of deletions between DNA repeats. To understand how deletions initiate in the absence of the Hpr1 protein and the mechanisms of recombination leading to deletions in S. cerevisiae, we have isolated mutations as suppressors of the hyper-deletion phenotype of the hpr1δ mutation. The mutations defined five different genes called HRS for hyper-recombination suppression. They suppress the hyper-deletion phenotype of hpr1δ strains for three direct repeat systems tested. The mutations eliminated the hyper-deletion phenotype of hpr1δ strains either completely (hrs1-1 and hrs2-1) or significantly (hrs3-1, hrs4-1 and hrs5-1). None of the mutations has a clear effect on the levels of spontaneous and double-strand break-induced deletions. Among other characteristics we have found are the following: (1) one mutation, hrs1-1, reduces the frequency of deletions in rad52-1 strains 20-fold, suggesting that the HRS1 gene is involved in the formation of RAD52-independent deletions; (2) the hrs2-1 hpr1δ mutant is sensitive to methyl-methane-sulfonate and the single mutants hpr1δ and hrs2-1 are resistant, which suggests that the HPR1 and HRS2 proteins may have redundant DNA repair functions; (3) the hrs4-1 mutation confers a hyper-mutator phenotype and (4) the phenotype of lack of activation of gene expression observed in hpr1δ strains is only partially suppressed by the hrs2-1 mutation, which suggests that the possible functions of the Hpr1 protein in gene expression and recombination repair can be separated. We discuss the possible relationship between the HPR1 and the HRS genes and their involvement in initiation of the events responsible for deletion formation.     

The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end

Hydroxypyruvate (HP) is an intermediate of the photorespiratory pathway that originates in the oxygenase activity of the key enzyme of photosynthetic CO(2) assimilation, Rubisco. In course of this high-throughput pathway, a peroxisomal transamination reaction converts serine to HP, most of which is subsequently reduced to glycerate by the NADH-dependent peroxisomal enzyme HP reductase (HPR1). In addition, a NADPH-dependent cytosolic HPR2 provides an efficient extraperoxisomal bypass. The combined deletion of these two enzymes, however, does not result in a fully lethal photorespiratory phenotype, indicating even more redundancy in the photorespiratory HP-into-glycerate conversion. Here, we report on a third enzyme, HPR3 (At1g12550), in Arabidopsis (Arabidopsis thaliana), which also reduces HP to glycerate and shows even more activity with glyoxylate, a more upstream intermediate of the photorespiratory cycle. The deletion of HPR3 by T-DNA insertion mutagenesis results in slightly altered leaf concentrations of the photorespiratory intermediates HP, glycerate, and glycine, indicating a disrupted photorespiratory flux, but not in visible alteration of the phenotype. On the other hand, the combined deletion of HPR1, HPR2, and HPR3 causes increased growth retardation, decreased photochemical efficiency, and reduced oxygen-dependent gas exchange in comparison with the hpr1xhpr2 double mutant. Since in silico analysis and proteomic studies from other groups indicate targeting of HPR3 to the chloroplast, this enzyme could provide a compensatory bypass for the reduction of HP and glyoxylate within this compartment.     

Genetic Control of Intrachromosomal Recombination in Saccharomyces Cerevisiae. I. Isolation and Genetic Characterization of Hyper-Recombination Mutations

Eight complementation groups have been defined for recessive mutations conferring an increased mitotic intrachromosomal recombination phenotype (hpr genes) in Saccharomyces cerevisiae. Some of the mutations preferentially increase intrachromosomal gene conversion (hpr4, hpr5 and hpr8) between repeated sequences, some increase loss of a marker between duplicated genes (hpr1 and hpr6), and some increase both types of events (hpr2, hpr3 and hpr7). New alleles of the CDC2 and CDC17 genes were recovered among these mutants. The mutants were also characterized for sensitivity to DNA damaging agents and for mutator activity. Among the more interesting mutants are hpr5, which shows a biased gene conversion in a leu2-112::URA3::leu2-k duplication; and hpr1, which has a much weaker effect on interchromosomal mitotic recombination than on intrachromosomal mitotic recombination. These analyses suggest that gene conversion and reciprocal exchange can be separated mutationally. Further studies are required to show whether different recombination pathways or different outcomes of the same recombination pathway are controlled by the genes identified in this study.     

Characterization of Mutations That Suppress the Temperature-Sensitive Growth of the Hpr1Δ Mutant of Saccharomyces Cerevisiae

The hpr1Δ3 mutant of Saccharomyces cerevisiae is temperature-sensitive for growth at 37° and has a 1000-fold increase in deletion of tandem direct repeats. The hyperrecombination phenotype, measured by deletion of a leu2 direct repeat, is partially dependent on the RAD1 and RAD52 gene products, but mutations in these RAD genes do not suppress the temperature-sensitive growth phenotype. Extragenic suppressors of the temperature-sensitive growth have been isolated and characterized. The 14 soh (suppressor of hpr1) mutants recovered represent eight complementation groups, with both dominant and recessive soh alleles. Some of the soh mutants suppress hpr1 hyperrecombination and are distinct from the rad mutants that suppress hpr1 hyperrecombination. Comparisons between the SOH genes and the RAD genes are presented as well as the requirement of RAD genes for the Soh phenotypes. Double soh mutants have been analyzed and reveal three classes of interactions: epistatic suppression of hpr1 hyperrecombination, synergistic suppression of hpr1 hyperrecombination and synthetic lethality. The SOH1 gene has been cloned and sequenced. The null allele is 10-fold increased for recombination as measured by deletion of a leu2 direct repeat.     

Fatty acid beta-oxidation in germinating Arabidopsis seeds is supported by peroxisomal hydroxypyruvate reductase when malate dehydrogenase is absent

Peroxisomal malate dehydrogenase (PMDH) oxidises NADH produced by fatty acid beta-oxidation during seed germination and seedling growth. Arabidopsis thaliana beta-oxidation mutants exhibit seed dormancy or impaired seed germination and failure of seedlings to degrade triacylglycerol (TAG), but the pmdh1 pmdh2 null mutant germinates readily and degrades TAG slowly during seedling growth. We reasoned that in the pmdh1 pmdh2 mutant an alternative means of oxidising NADH operates to allow a slow rate of beta-oxidation, such as NADH and NAD⁺ transport across the peroxisomal membrane or activity of another peroxisomal oxido-reductase. Here we show that peroxisomal hydroxypyruvate reductase (HPR) is present in germinating seeds and although knocking out HPR has little effect on germination and early seedling growth, when knocked out in combination with PMDH it exacerbates the pmdh1 pmdh2 phenotype. It greatly increases the proportion of dormant seeds and reduces the rate of seed germination. Seedlings have increased sucrose dependence and resistance to 2,4-dichlorophenoxybutyric acid (2,4-DB), and slower rate of TAG breakdown. When PMDH is absent, malate is lower in amount in germinating seeds and when HPR is also absent, serine (the immediate precursor of hydroxypyruvate) is much higher. These results indicate that HPR can oxidise NADH at sufficient rate in the absence of PMDH to support beta-oxidation and hence seed germination. We conclude that while HPR normally plays little role in seed germination our results support the growing body of evidence that peroxisomal NADH cannot be exported to the cytosol for oxidation but is oxidised by resident oxido-reductases.     

A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis

Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis.     

HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene.

The HPR1 gene has been cloned by complementation of the hyperrecombination phenotype of hpr1-1 strains by using a color assay system. HPR1 is a gene that is in single copy on chromosome IV of Saccharomyces cerevisiae, closely linked to ARO1, and it codes for a putative protein of 752 amino acids (molecular mass, 88 kilodaltons). Computer searches revealed homology (48.8% conserved homology; 24.8% identity) with the S. cerevisiae TOP1 gene in an alpha-helical stretch of 129 amino acids near the carboxy-terminal region of both proteins. The ethyl methanesulfonate-induced hpr1-1 mutation is a single-base change that produces a stop codon at amino acid 559 coding for a protein that lacks the carboxy-terminal TOP1 homologous region. Haploid strains carrying deletions of the HPR1 gene show a slightly reduced mitotic growth rate and extremely high rates of intrachromosomal excision recombination (frequency, 10 to 15%) but have a undetectable effect on rDNA recombination. Double-null mutants hpr1 top1 grow very poorly. We conclude that Hpr1 is a novel eucaryotic protein, mutation of which causes an increase in mitotic intrachromosomal excision recombination, and that it may be functionally related to an activity of the topoisomerase I protein.     

Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme,NADH-dependent hydroxypyruvate reductase

The syringolide receptor P34 mediates avrD-Rpg4 gene-for-gene complementarity in soybean. However, the mechanism underlying P34 signal transmission after syringolide binding is unknown. In an effort to identify a second messenger for P34, soybean leaf proteins were run though a P34-affinity column. A 42-kDa protein which specifically bound to the column was identified as a putative plant NADH-dependent hydroxypyruvate reductase (HPR) by N-terminal peptide sequencing. HPR is an important enzyme involved in the plant photorespiration system. Screening of a soybean cDNA library yielded two distinct HPR clones that encoded proteins with 97% identity (P42-1 and P42-2). Surprisingly, only P42-2 displayed good binding with P34 in a yeast two-hybrid assay, indicating that P42-2, but not P42-1, is a potential second messenger for P34. Glycerate and its analogs, which are utilized in the photorespiration system, were tested for their inhibitory effect on syringolide-induced hypersensitive response (HR) to evaluate the biological significance of P42-2. Interestingly, the downstream products of HPR (glycerate and 3-phosphoglycerate) inhibited HR but the upstream compounds (hydroxypyruvate or serine) did not have a significant effect on HR. These results suggest that P42-2 is a primary target for a P34/syringolide complex and that P42-2 binding with the complex probably induces HR by inhibiting one or more HPR functions in soybean.