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.     

Distribution of enzymes in mesophyll and parenchyma-sheath chloroplasts of maize leaves in relation to the C4-dicarboxylic acid pathway of photosynthesis

1. Mesophyll and parenchyma-sheath chloroplasts of maize leaves were separated by density fractionation in non-aqueous media. 2. An investigation of the distribution of photosynthetic enzymes indicated that the mesophyll chloroplasts probably contain the entire leaf complement of pyruvate,P(i) dikinase, NADP-specific malate dehydrogenase, glycerate kinase and nitrite reductase and most of the adenylate kinase and pyrophosphatase. The fractionation pattern of phosphopyruvate carboxylase suggested that this enzyme may be associated with the bounding membrane of mesophyll chloroplasts. 3. Ribulose diphosphate carboxylase, ribose phosphate isomerase, phosphoribulokinase, fructose diphosphate aldolase, alkaline fructose diphosphatase and NADP-specific ;malic' enzyme appear to be wholly localized in the parenchyma-sheath chloroplasts. Phosphoglycerate kinase and NADP-specific glyceraldehyde phosphate dehydrogenase, on the other hand, are distributed approximately equally between the two types of chloroplast. 4. After exposure of illuminated leaves to (14)CO(2) for 25sec., labelled malate, aspartate and 3-phosphoglycerate had similar fractionation patterns, and a large proportion of each was isolated with mesophyll chloroplasts. Labelled fructose phosphates and ribulose phosphates were mainly isolated in fractions containing parenchyma-sheath chloroplasts, and dihydroxyacetone phosphate had a fractionation pattern intermediate between those of C(4) dicarboxylic acids and sugar phosphates. 6. These results indicate that the mesophyll and parenchyma-sheath chloroplasts have a co-operative function in the operation of the C(4)-dicarboxylic acid pathway. Possible routes for the transfer of carbon from C(4) dicarboxylic acids to sugars are discussed.     

Studies on Cellulose Synthesis by a Cell-free Oat Coleoptile Enzyme System: Inactivation by Airborne Oxidants

Particulate cell wall polysaccharide synthetase from oat coleoptiles could use either guanosine diphosphate glucose or uridine diphosphate glucose; the latter was a much more effective glucose donor. The neutral polymer derived from uridine diphosphate glucose utilization yielded, after cellulase digestion, mostly cellobiose and to a lesser extent a substance tentatively identified as a mixed-linkage β1,4 = β1,3-trisaccharide; only cellobiose was found after guanosine diphosphate glucose utilization. The uridine diphosphate glucose utilizing system was inactivated by peroxyacetyl nitrate treatment of intact tissue and to a lesser extent by ozone treatment suggesting that this system is a possible site of interference with cellulose and non-cellulosic glucan biosynthesis in vivo. Direct treatment of the enzyme in vitro by peroxyacetyl nitrate, iodoacetamide or p-chloromercuribenzoate also inactivated the enzyme, indicating that the mechanism of inactivation possibly involves reaction with sulfhydryl groups.     

Influence of exogenous sucrose on the greening of oat

The greening of roots and leaves has been studied in whole oat seedlings grown on White's medium either with or without 2% sucrose. The added nutrient promotes chlorophyll synthesis and chloroplast differentiation in the roots. Yet it manifests a negative effect in the foliar tissues where it accelerates the decline in chlorophyll as well as the chloroplast ultrastructural alterations usually associated with senescence. The negative effect of the nutrient in the leaves is probably a consequence of the addition of exogenous sucrose to the endogenous sugars produced by photosynthesis. The foliar tissues would therefore be in the presence of high sucrose concentrations, which are known to be harmful for the photosynthetic apparatus. SDS-PAGE analysis of thylakoid polypeptides from root and leaf chloroplasts has revealed organ-specific differences in the electrophoretic patterns.     

Red Light-Independent Instability of Oat Phytochrome mRNA in Vivo

Phytochrome A (phyA) mRNA abundance decreased rapidly in total RNA samples isolated from 4-day-old etiolated oat seedlings following a red light pulse. Putative in vivo phyA mRNA degradation products were detectable both before and after red light treatment. Cordycepin-treated coleoptiles were unable to accumulate the chlorophyll a/b-binding protein mRNA in response to red light, indicating that cordycepin effectively inhibited mRNA synthesis. In cordycepin-treated coleoptiles, phyA mRNA rapidly decreased in abundance, consistent with the hypothesis that phyA mRNA is inherently unstable, rather than being destabilized after red light treatment of etiolated oat seedlings.     

Studies on ferrochelatase. The enzymic formation of haem in proplastids, chloroplasts and plant mitochondria

1. Ferrochelatase was demonstrated in the chloroplasts and proplastids isolated from the primary leaves of beans (a dicotyledon) and oats (a monocotyledon). It was also detected in chloroplasts from etiolated bean seedlings made green by illumination before being harvested. The specific activities of the three types of bean organelles are similar, as are the specific activities of the oat proplastids and chloroplasts. 2. Chloroplasts from young spinach leaves also contain ferrochelatase; these chloroplasts were tested for their ability to form magnesium tetrapyrroles and found unable to catalyse the insertion of Mg(2+) into mesoporphyrin IX. 3. Ferrochelatase was also detected in potato tuber mitochondria. 4. Ferrochelatase activity in these plant preparations is much less stable on storage than similar preparations from bacteria and animal tissues. 5. Temperature affects the activities of spinach chloroplast ferrochelatase and rat liver ferrochelatase differently. Activity of the chloroplast enzyme increases as the temperature rises from 20.6 degrees to 26 degrees , but becomes increasingly inactivated as the temperature rises further to 38 degrees . The initial velocity of the mammalian enzyme, however, increases as the temperature rises from 25.8 degrees to 65 degrees , but the enzyme is inactivated after several minutes at 65 degrees .     

Photoreversible calcium fluxes induced by phytochrome in oat coleoptile cells

The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible ΔA(725 - 800 nm) of >0.001 A centimeters⁻¹, recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells.     

Four isoforms of glutamine synthetase in light-grown soybeans

Glutamine synthetase (GS) activity recovered from linear sucrose gradients was associated with the cytosol of cells isolated from etiolated soybean hypocotyls whereas light-grown tissue contained increased GS activity localized in both the cytosol and chloroplasts. DEAE-cellulose chromatography indicated two GS isoforms in etiolated hypocotyls whereas light-grown hypocotyls and primary leaves contained four isoforms. Only one GS isoform was recovered from both etiolated and light-grown cotyledons.     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.     

Localization of ferredoxin isoproteins in mesophyll and bundle sheath cells in maize leaf

Four ferredoxin isoproteins were identified in the C₄ plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etiolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of etiolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C₄ plant.     

Carbonic anhydrase of the cotton plant

Nonaqueous fractionation of leaves of the cotton plant suggested that carbonic anhydrase was associated with the chloroplasts. Activity of this enzyme in aqueous extracts prepared in media containing no reductants was stable at 4°. Response to sulfhydryl reagents varied. The results indicated that thiol groups, necessary for the activity of the enzyme, were partially protected from oxidation.     

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm³ and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm³. During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm³. However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm³ and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm³ was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.     

Iron deficiency and the structure and physiology of maize chloroplasts

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.     

Acetylcholinesterase from oat Seedlings. I. Preliminary biochemical characterization of the enzyme

The activity of acetylcholinesterase (AChE) isolated from coleoptiles of etiolated oat seedlings is strongly inhibited by neostigmine and less so by eserine. The optimum of the enzyme activity occurs at pH 7.2 and a temperature of + 36 °C. The enzyme Michaelis constant is 280 μM. Choline within the range of concentration from 0.001 to 10 mM does not affect the enzyme activity. Calcium ions at 5 mM concentration cause inhibition, while magnesium and manganese ions do not affect the enzyme activity. AChE isolated from oat seedlings differs in a number of properties from AChE occurring in the tissues of other plants. This research was supported in part by grant CPBP 05.02.4.07.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

Polyamine oxidase from barley and oats

The pH optimum for the stability of the barley leaf polyamine oxidase is 4.8, which is also the pH optimum for its activity with spermine as substrate. Zonal centrifugation indicates that the enzyme is associated with a particle which is slightly more dense than chloroplasts, and the peak of activity corresponds with the peak of nucleic acid. Neither DNase nor RNase released the enzyme from the particles, despite the hydrolysis of more than 50% of the nucleic acid. The enzyme from the leaves of oat seedlings grown in the dark was purified 900-fold. Mg²⁺ and Ca²⁺ inhibited both barley and oat enzymes by ca 50% at 50 mM. The optimum pH for both spermine and spermidine oxidation by the oat enzyme was 6.5. The MW of the enzyme from both sources determined by gel chromatography was ca 85 000.     

Intracellular location of nitrate reductase and nitrite reductase. II. Wheat roots

Approximately 15% of the total nitrite reductase of crude homogenates of wheat roots applied to sucrose gradients was separated with an organelle whose isopycnic density was about 1.22 g·cm⁻³. The activity recovered in the supernatant was thought to be particulate in origin, because similar ratios of activity of isoenzyme 1 and 2 of nitrite reductase were found in both particulate and supernatant fractions. The particle with nitrite reductase activity also contained glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triose phosphate isomerase and NADPH diaphorase. This root particle and whole chloroplasts from leaves had a similar isopycnic density as well as these enzymes, and thus the data suggest that the root particle may be a proplastid.

Nitrate reductase was found only in the supernatant and it was not associated with any of the root organelles.

Mitochondria from wheat roots had an equilibrium density of 1.18 g·cm₋₃ and contained both NAD and NADP glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triosephosphate isomerase and NADPH diaphorase but not nitrite reductase. Microbodies of wheat roots had an equilibrium density of about 1.20 g·cm⁻³ on the sucrose gradient and contained catalase and glycollate oxidase.     

Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells

In frost-hardy and partially dehardened leaves of Brassica oleracea L. var. sabellica L. the distribution of cryoprotective sugars and of chloride between chloroplasts and the nonchloroplast part of leaf cells was investigated using the nonaqueous isolation technique as a means of cell fractionation. In chloroplasts of frost-hardy leaves high concentrations of sucrose and raffinose and comparatively low concentrations of chloride have been found. The ratios between sugars and chloride were so as to ascertain complete protection of the frost-sensitive thylakoid membranes during freezing. During dehardening, sugars decreased especially in the chloroplasts. There was a conversion of sucrose and raffinose into monosaccharides. This led to a large increase in the concentration of glucose and fructose in the nonchloroplast parts of the cells. There is evidence that the sugar concentration in the vacuole increased at the expense of sugars located in chloroplasts and cytoplasm. The quantity of sugars that remained in the chloroplasts did not appear to be sufficient for complete membrane protection at very low freezing temperatures.     

Intracellular location of nitrate reductase and nitrite reductase. I. Spinach and tobacco leaves

The intracellular location of nitrate and nitrite reductase was determined by extraction and isolation of organelles from spinach and tobacco leaves using sucrose based extraction media and isopycnic sucrose density gradient centrifugation. Nitrite reductase was located in the chloroplasts and nitrate reductase in the cytosol. With certain extraction media, nitrate reductase was found to be associated with all organelles but especially with the broken chloroplasts. This scattered and variable distribution was attributed to indiscriminate adsorption of nitrate reductase by all organelles, since bovine serum albumin eliminated this phenomenon. A low activity of nitrate reductase in crude homogenates or the supernatant fraction of tobacco leaves was due to a heat-stable, small molecular weight inhibitor. Neither soluble or insoluble polyvinylpyrollidone nor sulfhydryl reagents protected nitrate reductase from the inhibitor.