The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

(14)C-Labelled octulose phosphates were formed during photosynthetic (14)CO(2) fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero-d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though (14)C-labelled d-glycero-d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. (14)CO(2) predominantly labelled positions 5 and 6 of d-glycero-d-ido-octulose 1,8-P(2) consistent with labelling predictions of the modified scheme. The kinetics of (14)CO(2) incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to (12)CO(2) rapidly displaced the (14)C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO(2).) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO(2) assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO(2) fixation at rates in excess of 120 μmol h(−1) mg(−1) Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero-d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that (14)C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO(2) fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction

The major chloroplast envelope polypeptide is the phosphate translocator and not the protein import receptor

DURING photosynthetic CO2 fixation, fixed carbon is exported from the chloroplasts in the form of triose phosphate by the chloroplast phosphate translocator, which is the principal polypeptide (E29) from spinach chloroplast envelopes1. We have sequenced this nuclear-coded envelope membrane protein from both spinach and pea chloroplasts2,3. An envelope membrane protein, E30, has been identified as a possible receptor for protein import into pea chloroplasts using an anti-idiotypic antibody approach4–6; antibodies raised against purified E30 inhibited binding and import of proteins into chloroplasts7. The amino-acid sequence of E30 deduced from its complementary DNA7 turned out to be highly homologous to that of E29, assigned by us as the spinach phosphate translocator2, and was identical to the corresponding polypeptide from pea chloroplasts3. Differences in the binding properties to hydroxylapatite of £30 and the phosphate translocator suggested that E30 was not responsible for the chloroplast phosphate-transport activity but was the chloroplast import receptor7. Here we present evidence that argues against this and which identifies E30 as the chloroplast phosphate translocator

Photosynthesis-dependent H₂O₂ transfer from chloroplasts to nuclei provides a high-light signalling mechanism

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression

Isolation of Inner and Outer Membranes of the Chloroplast Envelope from Spinach and Pea

International audienceThe outer membrane and the inner membrane of the chloroplast envelope, also called OEM and IEM, have distinct functions connected with chloroplast biogenesis and chloroplast communication with the rest of the cell. Here we describe a method for the isolation of these membranes starting from intact chloroplast preparations, with two alternative procedures based on the starting material. One was developed from spinach leaves, the other one from pea leaves. The two procedures differ by the means that are used to isolate and rupture chloroplasts and to liberate each membrane