Photosynthetic activities of spinach leaf protoplasts

Photosynthetic activities of protoplasts isolated from spinach leaf (Spinacia oleracea L.) were investigated. The protoplasts were stable up to 9 hr, without loss of the original activity of CO(2) fixation (33-75 mumoles CO(2)/mg Chl.hr) and light-dependent O(2) evolution (33-40 mumoles O(2)/mg Chl.hr), when stored in 0.8 m mannitol-0.05 m N-tris (hydroxymethyl)-methylglycine-NaOH buffer, pH 7, at 4 C in dark. The optimum pH of 8.5 for CO(2) fixation reaction carried out in the present experimental condition employed is about the same as that reported for intact spinach chloroplasts. The CO(2) concentration for half-maximal rate of CO(2) fixation by protoplasts. "Km (CO(2))," were determined to be 19.8 mum (pH 7) and 42 mum (pH 8.5) and are similar to those observed for intact spinach chloroplasts. Protoplasts showed postillumination CO(2) fixation. Over-all results indicate that spinach protoplasts are as active as the intact plant leaf tissues in their photosynthetic activities.     

Potato sucrose transporter expression in minor veins indicates a role in phloem loading.

The major transport form of assimilates in most plants is sucrose. Translocation from the mesophyll into the phloem for long-distance transport is assumed to be carrier mediated in many species. A sucrose transporter cDNA was isolated from potato by complementation of a yeast strain that is unable to grow on sucrose because of the absence of an endogenous sucrose uptake system and the lack of a secreted invertase. The deduced amino acid sequence of the potato sucrose transporter gene StSUT1 is highly hydrophobic and is 68% identical to the spinach sucrose transporter SoSUT1 (pS21). In yeast, the sensitivity of sucrose transport to protonophores and to an increase in pH is consistent with an active proton cotransport mechanism. Substrate specificity and inhibition by protein modifiers are similar to results obtained for sucrose transport into protoplasts and plasma membrane vesicles and for the spinach transporter, with the exception of a reduction in maltose affinity. RNA gel blot analysis shows that the StSUT1 gene is highly expressed in mature leaves, whereas stem and sink tissues, such as developing leaves, show only low expression. RNA in situ hybridization studies show that the transporter gene is expressed specifically in the phloem. Both the properties and the expression pattern are consistent with a function of the sucrose transporter protein in phloem loading.     

The contribution of fructose 2,6-bisphosphate to the regulation of sucrose synthesis during photosynthesis

This review discusses (a) how the concentration of fructose 2,6-bisphosphate is controlled in spinach leaves, (b) how fructose 2,6-bisphosphate and cytosolic metabolites control the cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11), and (c) how the activities of the fructose-1,6-bisphosphatase and of sucrose phosphate synthase (EC 2.3.1.14) are coordinated. These features provide the elements of a fine control network that regulates sucrose synthesis during photosynthesis. The rate of sucrose synthesis is coordinated with the supply of photosynthate, so that concentrations of metabolites and phosphate are maintained at a level in the chloroplast which allows rapid CO₂ fixation. The rate of sucrose synthesis can also be modified to alter the amount of photosynthate that remains in the chloroplast for conversion to starch.     

Comparison of the contents of sucrose and amino acids in the leaves, phloem sap and taproots of high and low sugar-producing hybrids of sugar beet (Beta vulgaris L.)

The contents of sucrose and amino acids in the leaves, phloemsap and taproots have been analysed in three experimental hybridsof sugar beet and compared with earlier analysed leaf and phloemsap contents in spinach and barley. The three hybrids accumulatedsucrose and amino acids to various extents in the mature rootsas well as in the young taproots (9–12 weeks). The differencesin the sucrose-to-amino acid ratios in the taproots were reflectedin the corresponding ratios in the phloem sap. The leaf contentsof sucrose and amino acids in the three hybrids were found tobe very similar to each other and also to those in spinach andbarley. In contrast, the phloem concentration of sucrose (1.3M) was much higher, and that of amino acids much lower thanin spinach and barley. In the taproots, the overall concentrationof sucrose was about half that in the phloem sap. From thesefindings it is con cluded that the decisive factor in the highsucrose accumulation in sugar beet roots is the very efficientprocess of phloem loading in the leaves. The patterns of theamino acids in the phloem sap and in the taproots resembledthose in the leaves, indicating that there is no special transportform for a-amino nitrogen from the leaves to the roots, butall amino acids which are present in the cytosol are translocated. Key words: Amino acids, Beta vulgaris L., phloem sap, sucrose, tap roots, transport     

Regulation of sucrose and starch synthesis in wheat (Triticum aestivum L.) leaves: role of fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate (F26BP) is a competitive inhibitor of the cytosolic fructose 1,6-bisphosphatase (cytFBPase, EC 3.1.3.11). In spinach (Spinacia oleracea L.) leaves it is a significant component of the complex regulatory network that co-ordinates rates of photosynthesis, sucrose synthesis and starch synthesis. However the role of F26BP has only been studied in plants that predominantly store starch in their leaves and its role in other species is not clear. This paper examines the significance of F26BP in the regulation of photosynthetic carbon metabolism in the intact leaves of wheat (Triticum aestivum L.), a plant that accumulates predominantly sucrose. The approach taken was to vary rates of photosynthesis and then correlate measurements of F26BP and a range of other metabolites with rates of carbohydrate synthesis obtained from (14)CO(2)-feeding experiments performed under physiological conditions. It was found that: (i) Amounts of 3-phosphoglycerate and fructose-6-phosphate are correlated with the amount of F26BP. (ii) F26BP is involved in inhibiting cytFBPase at low light and low CO(2), but other factors, for example triose-phosphate, must also be involved. (iii) Amounts of both F26BP and substrate are involved in co-ordinating rates of photosynthesis and sucrose synthesis, but the relative importance of these depends on the conditions. (iv) Amounts of F26BP do not correlate with the partitioning of fixed carbon between sucrose and starch. Together these data suggest that the amount of F26BP in wheat is regulated by mechanisms similar to those in spinach, and that the metabolite is one of the factors involved in co-ordinating sucrose synthesis and photosynthesis. However F26BP does not appear to be involved in regulating the partitioning of fixed carbon between sucrose and starch in wheat under the experimental conditions examined.     

Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species

Protoplasts from barley (Hordeum vulgare), pea (Pisum sativum), wheat (Triticum aestivum), and spinach (Spinacia oleracea) leaves were fractionated into chloroplast- and mitochondrion-enriched fractions. Pyruvate dehydrogenase complex capacities in mitochondria (mtPDC) and chloroplasts (cpPDC) were measured in appropriate fractions under conditions optimal for each isozyme. The total cellular capacity of PDC was similar in barley and pea but about 50% lower in wheat and spinach. In pea a distribution of 87% mtPDC and 13% cpPDC was found on a cellular basis. In barley, wheat, and spinach the subcellular distribution was the opposite, with about 15% mtPDC and 85% cpPDC. cpPDC activity was constant at about 0.1 nmol cell-1 h-1 in cells from different regions along the developing barley leaf and showed no correlation with developmental patterns of photosynthetic parameters, such as increasing Chl and NADP-glyceraldehyde-3-phosphate dehydrogenase activity. Similarly, the capacity of the mitochondrial isoform did not change during barley leaf development and had a developmental pattern similar to that of citrate synthase and fumarase. Differences in subcellular distribution of PDCs in barley and pea are proposed to be due to differences in regulation, not to changes in isozyme proportions during leaf development or to species-specific differences in phosphorylation state of mtPDC after organelle separation.     

The effect of sulphite on the regulation of photosynthetic sucrose synthesis in poplar leaves

Sulphite at concentrations from 0.05 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bart. ex Marsh) leaves via the transpiration stream. The rate of CO2 fixation and partitioning of newly fixed carbon between sucrose and starch were measured and compared with the contents of selected phosphorylated intermediates, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and the activation of sucrose-phosphate synthase (SPS). Supplying leaves with 0.5 mM sulphite led to an increase in the sucrose/starch partitioning ratio without altering the rate of ¹⁴CO2 fixation. The increase in sucrose synthesis compared to starch synthesis was accompanied by relatively small changes of 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate (Fru1,6BP), hexose phosphates (hexose-)), uridine 5'-diphosphoglucose (UDPGlc), an accumulation of triose phosphates (triose-P), an activation of SPS, and decreased Fru2,6BP contents. Supplying leaves with 1.0 mM sulphite decreased ¹⁴CO2 assimilation and increased partitioning of fixed carbon into starch. A selective inhibition of sucrose synthesis was accompanied by an accumulation of triose-P, Fru1,6BP, hexose-P, and a decrease of PGA contents. There was also a large increase of Fru2,6BP contents and a decline in the activation of SPS. It could be argued that sulphite affects the allocation of photosynthetic carbon to sucrose and that sulphite can inhibit photosynthesis via a selective inhibition of sucrose synthesis.     

Phosphoribulokinase from Nitrobacter winogradskyi: activation by reduced nicotinamide adenine dinucleotide and inhibition by pyridoxal phosphate.

CO2 fixation by particle-free extracts from Nitrobacter winogradskyi increased by addition of reduced nicotinamide adenine dinucleotide (NADH). Ribulose-1,5-diphosphate, however, increased CO2 fixation, even in the absence of NADH. Phosphoribulokinase (EC 2.7.1.19) was the enzyme of Nitrobacter extracts that was activated specifically by NADH. Pyridoxal-5-phosphate inhibited both CO2 fixation and NADH-activated phosphoribulokinase from Nitrobacter. However, it did not affect phosphoribulokinase from spinach leaves. Since the spinach enzyme had also no requirement for reduced pyridine nucleotides, it appears that pyridoxal phosphate interferes only with the binding of NADH and not with the binding of ribulose-5-phosphate and adenosine-5'-triphosphate. The regulation of phosphoribulokinase activity by NADH provided Nitrobacter with an energy-dependent control mechanism of CO2 assimilation.     

A simple method for the isolation of intact mesophyll protoplasts from cereal plants

Intact mesophyll protoplasts from cereal plants were easilyprepared by incubating leaves with the abaxial epidermis peeledoff at 20–25?C for 2–3 hr in 0.6 M mannitol containing1% cellulase at pH 5.6. From one gram (fresh weight) of leaves1.5–6?10⁶ protoplasts, more than 90% of which were morphologicallyintact, could be obtained. Protoplasts isolated from wheat,oat, corn and barley were efficiently infected with brome mosaicvirus (BMV), and supported viral multiplication. (Received June 21, 1977; )     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

Influence of the sucrose outflow from leaves of higher plants on delayed luminescence induction in photosynthesis

The effect of the transport of sucrose from leaves of higher plants on the width of the spectra of induction of delayed luminescence was studied. It was shown that the duration of the induction period decreases when the sucrose outflow from the leaves is limited by cooling the leaf petiole for two hours under light. It was concluded that the accumulation of sucrose in the conducting tissues of the leaf stimulates the increase in the CO2 fixation rate on rellumination after dark adaptation.     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.     

Intracellular site of sucrose synthesis in leaves

Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g. UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.     

A continuous spectrophotometric assay for sucrose phosphate synthetase

A continuous spectrophotometric assay for sucrose phosphate synthetase is described. In this assay, the production of UDP is coupled to NADH oxidation by the enzymes nucleoside-5′-diphosphate kinase, pyruvate kinase, and lactate dehydrogenase. The assay could not be used with crude extracts, but was found suitable for use with partially purified sucrose phosphate synthetase from the leaves of spinach, wheat, and maize. It has obvious advantages for kinetic studies.     

Expression of sucrose: fructan 6-fructosyltransferase (6-SFT) and myo -inositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves

Fructan is an important class of non-structural carbohydrates present in cool-season grasses. Sucrose: fructan 6-fructosyltransferase (6-SFT, EC 2.4.1.10), one of the enzymes thought to be involved in grass fructan biosynthesis, catalyzes the initiation and extension of 2,6-linked fructans.Myo-inositol is a central component in several metabolic pathways in higher plants.Myo-inositol 1-phosphate synthase (MIPS) (EC 5.5.1.4), the first enzyme in inositolde novo biosynthesis, catalyzes the formation ofmyo-inositol 1-phosphate (MIP) from glucose-6-phosphate. The expression of 6-SFT and MIPS genes is compared in barley (Hordeum vulgare L.) leaves under various conditions. In cool temperature treatments, both 6-SFT and MIPS mRNAs accumulate within two days and then decline after four days. Under warm temperatures and continuous illumination, the amount of 6-SFT and MIPS mRNA gradually accumulated in detached leaves and increased significantly by 8 h. In contrast, we observed no significant changes over time in attached (control) leaves. Treating detached leaves with glucose or sucrose in the dark resulted in accumulations of both 6-SFT and MIPS mRNA. Homologous expression patterns for 6-SFT and MIPS genes suggest that they may be similarly regulated in barley leaves. Although sucrose and glucose may play important roles in the expression of 6-SFT and MIPS genes, regulation likely involves multiple factors.     

Reversal of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition of carbon dioxide fixation in spinach chloroplasts and protoplasts by dicarboxylic acids

3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition of (14)CO(2) fixation in isolated intact spinach (Spinacia oleracea L.) chloroplasts was reversed (by about 34%) by l-malate but not by oxaloacetate (OAA). However, OAA reversed the DCMU inhibition in spinach protoplasts indicating an extrachloroplastic enzyme requirement. Extrachloroplastic OAA reduction was coupled with external dihydroxyacetone phosphate (DHAP) oxidation, and the malate formed from such coupling might then enter the chloroplasts. Evidence was presented using ruptured protoplasts that the export of recently formed 3-phosphoglyceric acid (PGA) out of chloroplasts in exchange for external DHAP was reversed by excess OAA. The PGA/DHAP shuttle across the chloroplast envelope was found to be regulated by the external concentrations of DHAP and OAA.     

The purification and properties of sucrose-phosphate synthetase from spinach leaves: the involvement of this enzyme and fructose bisphosphatase in the regulation of sucrose biosynthesis

The properties of spinach leaf sucrose-phosphate synthetase (EC 2.4.1.14) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) have been studied. These two enzymes have been considered to be important in the control of sucrose synthesis. Sucrose-phosphate synthetase from leaf tissue has not been studied in detail previously and we report a technique for purifying this enzyme 50-fold by chromatography on AH-Sepharose 4B. This method frees the enzyme from contaminants which interfere with assay procedures with little or no loss of activity. The partially purified enzyme has a K_m for UDP-glucose of 7.1 mm and for fructose 6-phosphate of 0.8 mm. Fructose 1,6-bisphosphate, inorganic phosphate and UDP are strong inhibitors. The inhibition patterns of these suggest that the enzyme operates either by an ordered bi-bi or a Theorell-Chance mechanism. Partially purified cytosolic fructose-1,6-bisphosphatase is not only inhibited by AMP as previously reported, but is also inhibited by fructose 6-phosphate and UDP. From our observations, we conclude that sucrose biosynthesis is indeed controlled through these two enzymes and it appears that the rate of sucrose synthesis is largely dependent upon the supply of triose phosphate and ATP from the chloroplast.     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose