Caroline G. Bowsher

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 γ-32P-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 32P-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 γ-32P-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.

The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening.

A membrane-associated lipoxygenase from breaker-stage fruit of tomato (Lycopersicon esculentum Mill.) was purified and partially sequenced. Using degenerate oligonucleotides corresponding to portions of this sequence, a cDNA was amplified by PCR and used to screen a breaker fruit cDNA library. Two clones, tomloxA and tomloxB, were isolated and one of these (tomloxA) corresponded to the isolated protein. Genomic clones were isolated and sequence data from these were used to obtain the 5[prime] ends of the cDNAs. The 2.8-kb cDNAs encode proteins that are similar in size and sequence to each other and to other plant lipoxygenases. DNA blot analysis indicated that tomato contains three or more genes that encode lipoxygenase. RNA blot analysis showed that tomloxA is expressed in germinating seeds as well as in ripening fruit, where it reached its peak during breaker stage. tomloxB appears to be fruit specific and is at its highest level in ripe fruit.

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.

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.

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.

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.

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

Abstract Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In A rabidopsis 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.

Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol

ABSTRACT Plastids are diverse organelles that differ in form and function depending on their location within a plant. Their evolutionary origin, as free-living cyanobacteria, has left remnants of autonomy, and whereas the majority of the genetic control now lies within the nucleus, in terms of metabolism the plastid is fundamental to the life of the cell. This chapter describes the involvement of the plastid in carbon and nitrogen metabolism, in particular nitrate and ammonium assimilation, the Calvin cycle, oxidative pentose-phosphate pathway, glycolysis, and terpenoid biosynthesis. We have selected these pathways because they provide an opportunity to describe the metabolic interchange between plastids and cytosol and show duplication of some or all of the reactions in these two subcellular compartments. We discuss current knowledge of the likely ancestry of the genes encoding these pathways and consider how this has contributed to the compartmentation of nitrogen and carbon metabolism within the cell.

Identification of N-terminal regions of wheat leaf ferredoxin NADP+ oxidoreductase important for interactions with ferredoxin.

Wheat leaves contain two isoproteins of the photosynthetic ferredoxin:NADP(+) reductase (pFNRI and pFNRII). Truncated forms of both enzymes have been detected in vivo, but only pFNRII displays N-terminal length-dependent changes in activity. To investigate the impact of N-terminal truncation on interaction with ferredoxin (Fd), recombinant pFNRII proteins, differing by deletions of up to 25 amino acids, were generated. During purification of the isoproteins found in vivo, the longer forms of pFNRII bound more strongly to a Fd affinity column than did the shorter forms, pFNRII(ISKK) and pFNRII[N-2](KKQD). Further truncation of the N-termini resulted in a pFNRII protein which failed to bind to a Fd column. Similar k(cat) values (104-140 s(-1)) for cytochrome c reduction were measured for all but the most truncated pFNRII[N-5](DEGV), which had a k(cat) of 38 s(-1). Stopped-flow kinetic studies, examining the impact of truncation on electron flow between mutant pFNRII proteins and Fd, showed there was a variation in k(obs) from 76 to 265 s(-1) dependent on the pFNRII partner. To analyze the sites which contribute to Fd binding at the pFNRII N-terminal, three mutants were generated, in which a single or double lysine residue was changed to glutamine within the in vivo N-terminal truncation region. The mutations affected binding of pFNRII to the Fd column. Based on activity measurements, the double lysine residue change resulted in a pFNRII enzyme with decreased Fd affinity. The results highlight the importance of this flexible N-terminal region of the pFNRII protein in binding the Fd partner.

Phosphoglucomutase Activity During Development of Wheat Grains

Summary Phosphoglucomutase (PGM) activity was measured during the development of endosperm of spring wheat ( Triticum aestivum L. cv. Axona) in crude extracts and in isolated amyloplasts. Results show a 60 % reduction in activity of plastidial PGM activity between 5 and 15 days post anthesis. Measurements of the concentrations of the substrates and co-factors of the PGM reaction in the tissue during this period indicate that the enzyme should be saturated in vivo , suggesting that the reduction in activity of PGM during endosperm development is a function of the amount of PGM protein. The implications of this for starch synthesis are discussed.