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Redox regulation of photosynthetic enzymes has been a preferred research topic in recent years. In this area chloroplast fructose-1,6-bisphosphatase is probably the most extensively studied target enzyme of the CO2assimilation pathway. This review analyzes the structure, biosynthesis, phylogeny, action mechanism, regulation and kinetics of fructose-1,6-bisphosphatase in the light of recent findings on structure–function relationship, and from a molecular biology viewpoint.

A cDNA clone encoding for pea (Pisum sativum L.) cytosolic fructose-1,6-bisphosphatase (E.C. 3.1.3.11) has been isolated by reverse transcription-polymerase chain reaction of the total mRNA. The sequence analysis displayed a 341-amino acid protein of about 37300 Da molecular mass, corresponding to the subunit of this homotetrameric enzyme; it showed about 80% homology with the other ten higher plant cytosolic FBPases sequenced so far. The enzyme displayed a strong transcriptional expression in green organs (sessile and petioled leaves, stem, pod and grain), and poor expression in root and senescent basal leaves. It is noteworthy the high FBPase transcriptional expression in pod, which displays up to 4-fold higher content of FBPase-specific mRNA than that of root. The mRNA related to cytosolic FBPase was detected after 24 h continuous illumination of 24-h-dark-grown seedlings; this light-induced transcriptional expression is slower than that of chloroplast FBPase, which appears soon after 2 h light. In both cases the corresponding mRNAs disappeared when the light was turned off. The translational expression was also manifested, both as FBPase protein and activity, after 24 h illumination. This delay in the expression of cytosolic FBPase with respect to that of the plastidic enzyme can be interpreted as an indirect effect induced by a metabolite of the photosynthetic carbon pathway, rather than a direct effect of light on the DNA-expression mechanism. Pea cytosolic FBPase was not activated by dithiothreitol, with or without coupling to thioredoxins f or m. The enzyme showed a half-life of 6 h. Keywords: cloning, cytosol, fructose-1,6-bisphosphatase, pea, sequencing.

Abstract.: In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequenced Arabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently published sequence of Chlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases. This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.

The alignment of the six higher plant photosynthetic fructose-1,6-bisphosphatases (FBPases) so far sequenced shows a lack of homology in the region which just precedes the cluster engaged in light modulation. Earlier experiments suggested that this region is the docking point in FBPase-thioredoxin (Trx) binding, and could be responsible for the interspecific differences in the enzyme-Trx interaction and Trx ability for FBPase activation. Using a pea chloroplast FBPase-coding cDNA, we have prepared two chimeric clones for FBPase. One of them (pDELFBP) shows a deletion of the 17 amino acids (Leu154 to Glu170) coding sequence, whereas in the second (pPFBPW) the above sequence was substituted by the corresponding one of the wheat enzyme. After Escherichia coli overexpression in pET-3d and later purification, both modified FBPases showed FBPase activity when determined under non-reducing conditions. However, only DELFBP lost the Trx f modulatory effect, indicating the important role played by this fragment in FBPase-Trx interaction and activity. Under these conditions the substituted PFBPW enzyme retains FBPase activity, even though clearly diminished. Superose 12 filtration experiments after preincubating the wild-type and modified FBPases with Trx f, showed the existence of an enzyme-Trx f binding with the wild-type and the substituted PFBPW, but not with the deleted DELFBP protein. Similarly, gradient PAGE under native conditions, followed by Western blot and developing with FBPase and Trx f antibodies, indicated the existence of such a binding between the wild-type and PFBPW, on the one hand, and both Trxs f and m, on the other, although never with the deleted DELFBP enzyme. These results show the central role played by the regulatory site preceding fragment of chloroplast FBPase in its binding with Trx. Computer-aided tridimensional models for the wild-type and modified FBPases are proposed.

Chloroplastic fructose‐1,6‐bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys49, Cys153, Cys173, Cys178and Cys190) have been modified individually into serine residues by site‐directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild‐type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin hsystem). Finally, the C49S mutant is essentially identical to the wild‐type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299–4306].

Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys 49, Cys 153, Cys 173, Cys 178and Cys 190) have been modified individually into serine residues by site-directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin hsystem). Finally, the C49S mutant is essentially identical to the wild-type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299–4306].

When we compare the primary structures of the six chloroplast fructose-1,6-bisphosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceding the redox regulatory cysteines cluster can be observed. This region is a good candidate for binding of FBPase to its physiological modulator thioredoxin (Td), as this association shows clear differences between species. Using a cDNA clone for pea chloroplast FBPase as template, we have amplified by PCR a DNA insert coding for a 19 amino acid fragment (149Pro-167Gly), which was expressed in pGEMEX-1 as a fusion protein. This protein strongly interacts with pea Td m, as shown by ELISA and Superose 12 gel filtration, depending on pH of the medium. Preliminary assays have shown inhibition of FBPase activity in the presence of specific IgG against the 19 amino acid insert. Surprisingly the fusion protein enhances the FBPase activation in competitive inhibition experiments carried out with FBPase and Td. These results show the fundamental role played by this domain in FBPase-Td binding, not only as docking point for Td, but also by inducing some structural modification in the Td molecule. Taking as model the structural data recently published for spinach photosynthetic FBPase [29], this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.

An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in crude extracts of leaves is proposed. It is based on the ELISA technique, and offers two modifications. A non-competitive technique has a higher sensitivity and is the right option for samples of low fructose-1,6-bisphosphatase content. However, this method is not sufficiently specific when the total protein is higher than 5 μg/cm3; so, despite its lower sensitivity, in these circumstances a competitive technique is more suitable. Thus photosynthetic fructose-1,6-bisphosphatase can be measured without interferences from the gluconeogenic cytosolic enzyme of the photosynthetic cell or from a non-specific phosphatase present in the chloroplast.

During the last decade, plant thioredoxins (TRX) h-type have been shown to be implicated in several new roles like the protection against the oxidative stress by their ability to reduce some antioxidant proteins as peroxiredoxins (PRX) or methionine-sulphoxide-reductases (MSR). However, the concept of the oxidative stress is changing and this fact raises the question of the TRX roles in this new context. In the January issue of Plant Physiology, we have presented two TRXsh from Pisum sativum differently involved in the control of the redox status. PsTRXh1 is an h-type TRX that acts by reducing classical antioxidant proteins. PsTRXh2 seems to be also involved in redox control, however it could act contrary to its counterpart h1. Both proteins may play antagonistic roles in pea in order to lead a better control of the redox status.