Structural basis for the isotype-specific interactions of ferredoxin and ferredoxin: NADP+ oxidoreductase: an evolutionary switch between photosynthetic and heterotrophic assimilation.

In higher plants, ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) are each present as distinct isoproteins of photosynthetic type (leaf type) and non-photosynthetic type (root type). Root-type Fd and FNR are considered to facilitate the electron transfer from NADPH to Fd in the direction opposite to that occurring in the photosynthetic processes. We previously reported the crystal structure of the electron transfer complex between maize leaf FNR and Fd (leaf FNR:Fd complex), providing insights into the molecular interactions of the two proteins. Here we show the 2.49 Å crystal structure of the maize root FNR:Fd complex, which reveals that the orientation of FNR and Fd remarkably varies from that of the leaf FNR:Fd complex, giving a structural basis for reversing the redox path. Root FNR was previously shown to interact preferentially with root Fd over leaf Fd, while leaf FNR retains similar affinity for these two types of Fds. The structural basis for such differential interaction was investigated using site-directed mutagenesis of the isotype-specific amino acid residues on the interface of Fd and FNR, based on the crystal structures of the FNR:Fd complexes from maize leaves and roots. Kinetic and physical binding analyses of the resulting mutants lead to the conclusion that the rearrangement of the charged amino acid residues on the Fd-binding surface of FNR confers isotype-specific interaction with Fd, which brings about the evolutional switch between photosynthetic and heterotrophic redox cascades.

FUM2, a Cytosolic Fumarase, Is Essential for Acclimation to Low Temperature in Arabidopsis thaliana.

Although cold acclimation is a key process in plants from temperate climates, the mechanisms sensing low temperature remain obscure. Here, we show that the accumulation of the organic acid fumaric acid, mediated by the cytosolic fumarase FUM2, is essential for cold acclimation of metabolism in the cold-tolerant model species Arabidopsis (Arabidopsis thaliana). A nontargeted metabolomic approach, using gas chromatography-mass spectrometry, identifies fumarate as a key component of the cold response in this species. Plants of T-DNA insertion mutants, lacking FUM2, show marked differences in their response to cold, with contrasting responses both in terms of metabolite concentrations and gene expression. The fum2 plants accumulated higher concentrations of phosphorylated sugar intermediates and of starch and malate. Transcripts for proteins involved in photosynthesis were markedly down-regulated in fum2.2 but not in wild-type Columbia-0. Plants of fum2 show a complete loss of the ability to acclimate photosynthesis to low temperature. We conclude that fumarate accumulation plays an essential role in low temperature sensing in Arabidopsis, either indirectly modulating metabolic or redox signals or possibly being itself directly involved in cold sensing.

Profiling of spatial metabolite distributions in wheat leaves under normal and nitrate limiting conditions.

The control and interaction between nitrogen and carbon assimilatory pathways is essential in both photosynthetic and non-photosynthetic tissue in order to support metabolic processes without compromising growth. Physiological differences between the basal and mature region of wheat (Triticum aestivum) primary leaves confirmed that there was a change from heterotrophic to autotrophic metabolism. Fourier Transform Infrared (FT-IR) spectroscopy confirmed the suitability and phenotypic reproducibility of the leaf growth conditions. Principal Component-Discriminant Function Analysis (PC-DFA) revealed distinct clustering between base, and tip sections of the developing wheat leaf, and from plants grown in the presence or absence of nitrate. Gas Chromatography-Time of Flight/Mass Spectrometry (GC-TOF/MS) combined with multivariate and univariate analyses, and Bayesian network (BN) analysis, distinguished different tissues and confirmed the physiological switch from high rates of respiration to photosynthesis along the leaf. The operation of nitrogen metabolism impacted on the levels and distribution of amino acids, organic acids and carbohydrates within the wheat leaf. In plants grown in the presence of nitrate there was reduced levels of a number of sugar metabolites in the leaf base and an increase in maltose levels, possibly reflecting an increase in starch turnover. The value of using this combined metabolomics analysis for further functional investigations in the future are discussed.

Acclimation of metabolism to light in Arabidopsis thaliana: the glucose 6-phosphate/phosphate translocator GPT2 directs metabolic acclimation.

Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In Arabidopsis thaliana, this dynamic acclimation requires expression of GPT2, a glucose 6-phosphate/phosphate translocator. Here, we examine the impact of GPT2 on leaf metabolism and photosynthesis. Plants of wild type and of a GPT2 knockout (gpt2.2) grown under low light achieved the same photosynthetic rate despite having different metabolic and transcriptomic strategies. Immediately upon transfer to high light, gpt2.2 plants showed a higher rate of photosynthesis than wild-type plants (35%); however, over subsequent days, wild-type plants acclimated photosynthetic capacity, increasing the photosynthesis rate by 100% after 7 d. Wild-type plants accumulated more starch than gpt2.2 plants throughout acclimation. We suggest that GPT2 activity results in the net import of glucose 6-phosphate from cytosol to chloroplast, increasing starch synthesis. There was clear acclimation of metabolism, with short-term changes typically being reversed as plants acclimated. Distinct responses to light were observed in wild-type and gpt2.2 leaves. Significantly higher levels of sugar phosphates were observed in gpt2.2. We suggest that GPT2 alters the distribution of metabolites between compartments and that this plays an essential role in allowing the cell to interpret environmental signals.

The physiological importance of photosynthetic ferredoxin NADP+ oxidoreductase (FNR) isoforms in wheat.

Ferredoxin NADP(+) oxidoreductase (FNR) enzymes catalyse electron transfer between ferredoxin and NADPH. In plants, a photosynthetic FNR (pFNR) transfers electrons from reduced ferredoxin to NADPH for the final step of linear electron flow, providing reductant for carbon fixation. pFNR is also thought to play important roles in two different mechanisms of cyclic electron flow around photosystem I; and photosynthetic reductant is itself partitioned between competing linear, cyclic, and alternative electron flow pathways. Four pFNR protein isoforms in wheat that display distinct reaction kinetics with leaf-type ferredoxin have previously been identified. It has been suggested that these isoforms may be crucial to the regulation of reductant partition between carbon fixation and other metabolic pathways. Here the 12 cm primary wheat leaf has been used to show that the alternative N-terminal pFNRI and pFNRII protein isoforms have statistically significant differences in response to the physiological parameters of chloroplast maturity, nitrogen regime, and oxidative stress. More specifically, the results obtained suggest that the alternative N-terminal forms of pFNRI have distinct roles in the partitioning of photosynthetic reductant. The role of alternative N-terminal processing of pFNRI is also discussed in terms of its importance for thylakoid targeting. The results suggest that the four pFNR protein isoforms are each present in the chloroplast in phosphorylated and non-phosphorylated states. pFNR isoforms vary in putative phosphorylation responses to physiological parameters, but the physiological significance requires further investigation.

Subcellular distribution of tail-anchored proteins in Arabidopsis.

Tail-anchored (TA) proteins function in key cellular processes in eukaryotic cells, such as vesicle trafficking, protein translocation and regulation of transcription. They anchor to internal cell membranes by a C-terminal transmembrane domain, which also serves as a targeting sequence. Targeting occurs post-translationally, via pathways that are specific to the precursor, which makes TA proteins a model system for investigating post-translational protein targeting. Bioinformatics approaches have previously been used to identify potential TA proteins in yeast and humans, yet little is known about TA proteins in plants. The identification of plant TA proteins is important for extending the post-translational model system to plastids, in addition to general proteome characterization, and the identification of functional homologues characterized in other organisms. We identified 454 loci that potentially encode TA proteins in Arabidopsis, and combined published data with new localization experiments to assign localizations to 130 proteins, including 29 associated with plastids. By analysing the tail anchor sequences of characterized proteins, we have developed a tool for predicting localization and estimate that 138 TA proteins are localized to plastids.

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm.

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an alpha-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.

Characterization of ADP-glucose transport across the cereal endosperm amyloplast envelope.

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[alpha-32P]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid.

The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthesis.

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.

Differential uptake of photosynthetic and non-photosynthetic proteins by pea root plastids.

The photosynthetic proteins RuBiSCO, ferredoxin I and ferredoxin NADP(+)-oxidoreductase (pFNR) were efficiently imported into isolated pea chloroplasts but not into pea root plastids. By contrast non-photosynthetic ferredoxin III and heterotrophic FNR (hFNR) were efficiently imported into both isolated chloroplasts and root plastids. Chimeric ferredoxin I/III (transit peptide of ferredoxin I attached to the mature region of ferredoxin III) only imported into chloroplasts. Ferredoxin III/I (transit peptide of ferredoxin III attached to the mature region of ferredoxin I) imported into both chloroplasts and root plastids. This suggests that import depends on specific interactions between the transit peptide and the translocon apparatus.

Altered activity of the P2 isoform of plastidic glucose 6-phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves.

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions.

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.

Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L. cv. Axona).

Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid. The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with K(a) values of 4.5 micro M and 3.8 micro M for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis-Menten kinetics with glucose 1-phosphate as the substrate with K(m) values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively. A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM. The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources. PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm. Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis. Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development. The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.

Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform.

The intracellular location of ADPglucose pyrophosphorylase (AGPase) in wheat during endosperm development was investigated by analysis of the recovery of marker enzymes from amyloplast preparations. Amyloplast preparations contained 20-28% of the total endosperm activity of two plastidial marker enzymes and less than 0.8% of the total endosperm activity of two cytosolic marker enzymes. Amylo plasts prepared at various stages of development, from 8-30 d post anthesis, contained between 2% and 10% of the total AGPase activity; this implies that between 7% and 40% of the AGPase in wheat endosperm is plastidial during this period of development. Two proteins were recognized by antibodies to both the large and small subunits of wheat AGPase. The larger of the two AGPases was the major form of the enzyme in whole cell extracts, and the smaller, less abundant, form of AGPase was enriched in plastid preparations. The results are consistent with data from other graminaceous endosperms, suggesting that there are distinct plastidial and cytosolic isoforms of AGPase composed of different subunits. The plastidial isoform of AGPase from wheat endosperm is relatively insensitive to the allosteric regulators 3-phosphoglycerate and inorganic orthophos phate compared with plastidial AGPase from other species. Amyloplast AGPase showed no sensitivity to physiological concentrations of inorganic orthophosphate. 15 mM 3-phosphoglycerate caused no stimulation of the pyrophosphorolytic reaction, and only 2-fold stimulation of the ADPglucose synthesizing reaction.