Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes

The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea ( Pisum sativum ) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO₂ (0.1 m M NaHCO₃) than that at optimal CO₂ (1.0 m M NaHCO₃). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.     

Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of ¹⁴C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO₂ fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO₂ assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.Abbreviations FBP Fructose-1,6-bisphosphate - HMP Hexose Monophosphates - PGA 3-phosphoglycerate - PMP Pentose Monophosphates - RBP Ribulose-1,5-bisphosphate - SBP Sedoheptulose-1,7-bisphosphate - TP Triose Phosphates     

The acclimation of photosynthesis and respiration to temperature in the C3–C4 intermediate Salsola divaricata: induction of high respiratory CO2 release under low temperature

Photosynthesis in C₃–C₄ intermediates reduces carbon loss by photorespiration through refixing photorespired CO₂ within bundle sheath cells. This is beneficial under warm temperatures where rates of photorespiration are high; however, it is unknown how photosynthesis in C₃–C₄ plants acclimates to growth under cold conditions. Therefore, the cold tolerance of the C₃–C₄ Salsola divaricata was tested to determine whether it reverts to C₃ photosynthesis when grown under low temperatures. Plants were grown under cold (15/10 °C), moderate (25/18 °C) or hot (35/25 °C) day/night temperatures and analysed to determine how photosynthesis, respiration and C₃–C₄ features acclimate to these growth conditions. The CO₂ compensation point and net rates of CO₂ assimilation in cold‐grown plants changed dramatically when measured in response to temperature. However, this was not due to the loss of C₃–C₄ intermediacy, but rather to a large increase in mitochondrial respiration supported primarily by the non‐phosphorylating alternative oxidative pathway (AOP) and, to a lesser degree, the cytochrome oxidative pathway (COP). The increase in respiration and AOP capacity in cold‐grown plants likely protects against reactive oxygen species (ROS) in mitochondria and photodamage in chloroplasts by consuming excess reductant via the alternative mitochondrial respiratory electron transport chain.     

Apoptosis-resistant phenotype in HL-60-derived cells HCW-2 is related to changes in expression of stress-induced proteins that impact on redox status and mitochondrial metabolism

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.     

Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.     

Thematic Review Series: Fat-Soluble Vitamins: Vitamin K: Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.     

Photosynthesis and phage: early studies on phosphorus metabolism in photosynthetic microorganisms with 32P,and how they led to the serendipic discovery of 32P-decay suicide of bacteriophage

During my PhD thesis research (1946–1949), I explored the effects of light on the uptake of ³²P-labeled inorganic phosphate (P_i) by cells of photosynthetic bacteria and microalgae, and the dynamics of P turnover between low and high molecular weight cell constituents. The results were interpreted as evidence for the conversion of light energy to the chemical energy of phosphorylated compounds. The experimental results also suggested to me that the precursors of the P in DNA bacteriophages of Escherichia coli must be low molecular weight phosphorylated compounds present within the host cells and led to the design of an experiment to determine the conservation of ³²P of an infecting phage particle in its numerous progeny. The experiment envisaged was never conducted because phage labeled with ³²P of high specific activity showed unexpected loss of viability. Thus, by serendipity, ‘suicide’ of phage due to ³²P-β decay was discovered. ³²P-decay ‘suicide’ provided a technique that was useful for analysis of phage genetic structure and replication. This memoir describes the unusual circumstances leading to the decisive role of serendipity in revealing an extraordinary phenomenon. This revised version was published online in June 2006 with corrections to the Cover Date.