Two redundant octanoyltransferases and one obligatory lipoyl synthase provide protein-lipoylation autonomy to plastids of Arabidopsis.

Octanoyltransferases (LIP2) are important for the lipoylation of several α-ketoacid decarboxylases and glycine decarboxylase, all of which are essential multienzyme complexes of central metabolism, by attaching de novo-synthesised octanoyl moieties to the respective target subunits. Lipoyl synthase (LIP1) then inserts two sulphur atoms each into the protein-bound octanoyl chains to generate the functional lipoamide arms. In plants, most of the above multienzyme complexes occur only in mitochondria. Pyruvate dehydrogenase is an exception, since it also occurs in plastids. Plastidial LIP1 and LIP2 are known, but it is not clear how essential these enzymes are. Here, we report that not just one but two redundant LIP2 isoforms, LIP2p and LIP2p2, operate in plastids of Arabidopsis. The combined deletion of the two isoenzymes is embryo-lethal. Deletion of the plastidial lipoyl synthase LIP1p is also embryo-lethal, indicating that all plastidial LIP1 activity is due to LIP1p. These features suggest that protein lipoylation is based on an autonomous and partially redundant de novo lipoylation pathway in plastids.

Does the Cyanophora paradoxa genome revise our view on the evolution of photorespiratory enzymes?

In the present-day O2 -rich atmosphere, the photorespiratory pathway is essential for organisms performing oxygenic photosynthesis; i.e. cyanobacteria, algae and land plants. The presence of enzymes for the plant-like 2-phosphoglycolate cycle in cyanobacteria indicates that, together with oxygenic photosynthesis, genes for photorespiratory enzymes were endosymbiotically conveyed from ancient cyanobacteria to photosynthetic eukaryotes. The genome information for Cyanophora paradoxa, a member of the Glaucophyta representing the first branching group of primary endosymbionts, and for many other eukaryotic algae was used to shed light on the evolutionary relationship of photorespiratory enzymes among oxygenic phototrophs. For example, it became possible to analyse the phylogenies of 2-phosphoglycolate phosphatase, serine:glyoxylate aminotransferase and hydroxypyruvate reductase. Analysis of the Cyanophora genome provided clear evidence that some photorespiratory enzymes originally acquired from cyanobacteria were lost, e.g. glycerate 3-kinase, while others were replaced by the corresponding enzymes from the α-proteobacterial endosymbiont, e.g. serine:glyoxylate aminotransferase. Generally, our analysis supports the view that many C2 cycle enzymes in eukaryotic phototrophs were obtained from the cyanobacterial endosymbiont, but during the subsequent evolution of algae and land plants multiple losses and replacements occurred, which resulted in a reticulate provenance of photorespiratory enzymes with different origins in different cellular compartments.

Perspectives on plant photorespiratory metabolism.

Being intimately intertwined with (C3) photosynthesis, photorespiration is an incredibly high flux-bearing pathway. Traditionally, the photorespiratory cycle was viewed as closed pathway to refill the Calvin-Benson cycle with organic carbon. However, given the network nature of metabolism, it hence follows that photorespiration will interact with many other pathways. In this article, we review current understanding of these interactions and attempt to define key priorities for future research, which will allow us greater fundamental comprehension of general metabolic and developmental consequences of perturbation of this crucial metabolic process.

The variety of photorespiratory phenotypes - employing the current status for future research directions on photorespiration.

Mutations of genes encoding for proteins within the photorespiratory core cycle and associated processes are characterised by lethality under normal air but viability under elevated CO2 conditions. This feature has been described as 'the photorespiratory phenotype' and assumed to be distinctly equal for all of these mutants. In recent years a broad collection of photorespiratory mutants has been isolated, which has allowed a comparative analysis. Distinct phenotypic features were observed when Arabidopsis thaliana mutants defective in photorespiratory enzymes were compared, and during shifts from elevated to ambient CO2 conditions. The exact reasons for the mutant-specific photorespiratory phenotypes are mostly unknown, but they indicate even more plasticity of photorespiratory metabolism. Moreover, a growing body of evidence was obtained that mutant features could be modulated by alterations of several factors, such as CO2 :O2 ratios, photoperiod, light intensity, organic carbon supply and pathogens. Hence, systematic analyses of the responses to these factors appear to be crucial to unravel mechanisms how photorespiration adapts and interacts with the whole cellular metabolism. Here we review current knowledge regarding photorespiratory mutants and propose a new level of phenotypic sub-classification. Finally, we present further questions that should be addressed in the field of photorespiration.

Evolution of the biochemistry of the photorespiratory C2 cycle.

Oxygenic photosynthesis would not be possible without photorespiration in the present day O2 -rich atmosphere. It is now generally accepted that cyanobacteria-like prokaryotes first evolved oxygenic photosynthesis, which was later conveyed via endosymbiosis into a eukaryotic host, which then gave rise to the different groups of algae and streptophytes. For photosynthetic CO2 fixation, all these organisms use RubisCO, which catalyses both the carboxylation and the oxygenation of ribulose 1,5-bisphosphate. One of the reaction products of the oxygenase reaction, 2-phosphoglycolate (2PG), represents the starting point of the photorespiratory C2 cycle, which is considered largely responsible for recapturing organic carbon via conversion to the Calvin-Benson cycle (CBC) intermediate 3-phosphoglycerate, thereby detoxifying critical intermediates. Here we discuss possible scenarios for the evolution of this process toward the well-defined 2PG metabolism in extant plants. While the origin of the C2 cycle core enzymes can be clearly dated back towards the different endosymbiotic events, the evolutionary scenario that allowed the compartmentalised high flux photorespiratory cycle is uncertain, but probably occurred early during the algal radiation. The change in atmospheric CO2 /O2 ratios promoting the acquisition of different modes for inorganic carbon concentration mechanisms, as well as the evolutionary specialisation of peroxisomes, clearly had a dramatic impact on further aspects of land plant photorespiration.

The glycine decarboxylase complex is not essential for the cyanobacterium Synechocystis sp. strain PCC 6803.

In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.

Metabolic response of potato plants to an antisense reduction of the P-protein of glycine decarboxylase.

Potato (Solanum tuberosum L. cv. Desiré) plants with reduced amounts of P-protein, one of the subunits of glycine decarboxylase (GDC), have been generated by introduction of an antisense transgene. Two transgenic lines, containing about 60-70% less P-protein in the leaves compared to wild-type potato, were analysed in more detail. The reduction in P-protein amount led to a decrease in the ability of leaf mitochondria to decarboxylate glycine. Photosynthetic and growth rates were reduced but the plants were viable under ambient air and produced tubers. Glycine concentrations within the leaves were elevated up to about 100-fold during illumination. Effects on other amino acids and on sucrose and hexoses were minor. Nearly all of the glycine accumulated during the day was metabolised during the following night. The data suggest that the GDC operates far below substrate saturation under normal conditions thus allowing a flexible and fast response to changes in the environment.

Genetic transformation of the C3-C4 intermediate plant, Flaveria pubescens (Asteraceae).

The dicot genus Flaveria (Asteraceae), besides species with C3 or C4 photosynthesis, contains taxa with a broad range of different states of transition between the two major photosynthetic types. We have developed a reproducible and efficient Agrobacterium-mediated method for the stable genetic transformation of the C3-C4 intermediate species F. pubescens. Fusion constructs of the reporter gene ß-glucuronidase (uidA, GUS) to several plant promoters, mainly derived from genes encoding subunits of the glycine cleavage system (gdcs), have been used to confirm the reproducibility and efficiency of the method. The stable integration of the foreign DNA has been examined by Southern analysis, kanamycin resistance, GUS enzyme activities and histochemical staining. Transformed shoots can be routinely obtained within 8-10 weeks after co-cultivation with A. tumefaciens.

H-protein of the glycine cleavage system in Flaveria: alternative splicing of the pre-mRNA occurs exclusively in advanced C4 species of the genus.

A recent paper reports on the occurrence of alternative splicing of H-protein pre-mRNA in the C4 species, Flaveria trinervia, that is organ-specifically regulated. The analysis of 11 other species of the genus, F. cronquistii and F. pringlei (C3), F. anomala, F. chloraefolia, F. floridana, F. linearis and F. pubescens (C3-C4 intermediate), F. brownii (C4-like), F. palmeri, F. bidentis and F. australasica (C4), revealed that this post-transcriptional effect is not specific for F. trinervia. It occurs in all the examined C4 species of the genus Flaveria except the less advanced C4-like species, F. brownii. Both the position and the direct effect of alternative splicing, the addition of two alanine residues near to the N-terminus of the derived mature H-protein, are invariant. A quantification of the relative amounts of both transcripts revealed that, as in F. trinervia, the alternative mRNA strongly dominates in leaves. In sharp contrast, none of the C3, C3-C4 intermediate, or C4-like species showed alternative splicing. By Western analysis both H-isoproteins have been detected in F. trinervia and their ratio approximately corresponds to the measured transcript levels. It is concluded that alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system in all advanced Flaveria C4 species.

Chloroplast pentose-5-phosphate 3-epimerase from potato: cloning, cDNA sequence, and tissue-specific enzyme accumulation.

A cDNA clone encoding the chloroplast enzyme pentose-5-phosphate 3-epimerase (EC 5.1.3.1) in potato (Solanum tuberosum) was isolated and sequenced. The deduced sequence of 235 amino acids is similar to protein sequences of bacterial epimerases. Northern blot analysis showed the highest level of epimerase mRNA expression in potato leaves, whereas it was low in roots, tubers, and stems. Epimerase protein is mulated only in plant tissues possessing chloroplasts, i.e. in land to a lesser extent in stem. In contrast, transketolase, a sequential enzyme of epimerase in the reductive and oxidative pentose phosphate cycle, is accumulated in all plant tissues.

H-protein of glycine decarboxylase is encoded by multigene families in Flaveria pringlei and F. cronquistii (Asteraceae).

In Flaveria pringlei and F. cronquistii unlike other plants, H-protein of the glycine cleavage system is encoded by small multigene families. From leaf cDNA libraries and by reverse transcription of mRNA with subsequent polymerase chain reaction (PCR) amplification, we have obtained three different H-protein cDNA clones from each species. The relative levels of total H-protein mRNA, as well as of different H-protein transcripts, have been determined in leaves, stems, and roots of F. pringlei. Stems, with a total of 22% relative to leaves, contain substantial amounts of H-protein transcripts. The corresponding level in roots is relatively low (2.3% relative to leaves) but easily detectable. One of the transcripts occurs only in leaves (HFP20) and another one (HFP13) is present exclusively in photosynthesizing organs. Only one of the H-protein transcripts (HFP4) was found in all three organs, in leaves, stems, and roots of F. pringlei.

Structure and expression analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-cleavage system from Flaveria pringlei.

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.

Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia.

Alternative splicing is a well-known post-transcriptional regulatory mechanism in eukaryotic organisms but there are only very few reports on alternative splicing in plants. The analysis of cDNAs encoding H-protein of the glycine decarboxylase multi-enzyme complex from the C4 species Flaveria trinervia revealed the presence of two transcript populations that differ in the length of their coding regions by six nucleotides. Otherwise, including their 3' nontranslated region, they are identical. From a genomic Southern analysis and from the sequencing of several independent cDNA clones it is evident that both types of transcript are derived from a single-copy gene. This gene, FTgdcsH, has been cloned and sequenced. It comprises four short exons. The two alternative splice sites are located at the end of intron 1. The shorter transcript closely corresponds to published H-protein mRNA sequences from other organisms. The longer transcript encodes two additional alanine residues very close to the N-terminus of the mature H-protein. A quantification of the relative amounts of both transcripts in different organs revealed that, with 80-90% of the total H-protein mRNA, the alternative mRNA dominates in leaves whereas roots contain more of the 'correctly' spliced transcript. It is concluded that, in F. trinervia and with a distinct organ preference, alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system.

T-protein of the glycine decarboxylase multienzyme complex: evidence for partial similarity to formyltetrahydrofolate synthetase.

We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum. The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins. Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes. This suggests that the T-protein sequence is not as unique as previously thought.

In vitro plant regeneration from leaf explants of C3 and C 3-C 4-intermediate Flaveria species.

A procedure is described for the invitro regeneration of whole plants of Flaveria cronquistii (C3 species) F. pubescens and F. chloraefolia (both C3-C4 intermediate species) using different concentrations of 6-benzylaminopurine and alpha-napnthalenic acid.