Sorbitol metabolism by apple seedlings

The aims of this work were to compare the roles of sorbitol and sucrose in seedlings of Malus domestica, to discover which tissues synthesize sorbitol and which break it down, and to examine these tissues for enzymes of sorbitol metabolism. The detailed distribution of label was determined after supplying intact seedlings with ¹⁴CO₂, and excised parts of seedlings with [U-¹⁴C]fructose and [U-¹⁴C]sorbitol. The results showed that appreciable synthesis of sorbitol occurred only in the leaves but did not depend directly on photosynthesis. All tissues examined metabolized sorbitol but metabolism was extensive only in root apices, and in leaves which had been kept in the dark. The above experiments suggest that sorbitol supplements but does not replace sucrose. Extracts of apple leaves showed no trace of either a polyol or a polyol phosphate dehydrogenase but did exhibit sorbitol-6-phosphate phosphatase activity. A limited number of experiments with extracts of the blades of Laminaria digitata indicated that they contained mannitol-1-phosphate phosphatase and mannitol dehydrogenase.     

Lipid metabolism in green leaves of developing monocotyledons

Lipid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivum L.) leaves. The endogenous levels of acyl lipids and their constituent fatty acids from the same leaf sections were also analysed. The principle chloroplast acyl lipids showed a relative increase in amount with the age of the leaf section. Their content of -linolenic acid also increased whereas there was little change in the amount of this acid in phosphatidylcholine and phosphatidylethanolamine, which are primarily non-chloroplastic. The content of trans-3-hexadecenoic acid in phosphatidylglycerol increased approximately 20-fold between the youngest (basal) and oldest (distal) leaf sections.The incorporation of [¹⁴C]acetate was always high into monogalactosyldiacylglycerol, phosphatidylcholine and the neutral lipid (mainly pigments) fractions. With increasing age, the neutral lipids were less well labelled. In three of the plant species but not in barley, phosphatidylglycerol was heavily labelled. Monogalactosyldiacylglycerol usually contained the highest amount of radioactivity in the middle leaf sections. Apart from these generalisations, each plant type had its own specific pattern of radiolabelling.     

Sorbitol metabolism in Aerobacter aerogenes

Sorbitol (d-glucitol) metabolism in Aerobacter aerogenes PRL-R3 is shown to proceed via the pathway: sorbitol --> sorbitol 6-phosphate --> d-fructose 6-phosphate. Sorbitol phosphorylation is mediated by a phosphoenolpyruvate (PEP):sorbitol 6-phosphotransferase system, and sorbitol 6-phosphate oxidation by a pyridine-nucleotide-linked dehydrogenase. Mutants deficient in sorbitol 6-phosphate dehydrogenase or a component (enzyme I) of the phosphotransferase system did not grow on sorbitol, whereas revertants which had regained these enzymatic activities grew normally. Extracts of the enzyme I-deficient mutant failed to catalyze the phosphorylation of sorbitol in the presence of PEP, and adenosine 5'-triphosphate could not replace the PEP requirement for sorbitol phosphorylation in extracts of the wild-type strain.     

Polyphenol metabolism of developing apple skin of a scab resistant and a susceptible apple cultivar

During fruit development, the concentration of main polyphenols (flavonols, flavanols, dihydrochalcones, hydroxycinnamic acids, anthocyanins) and the activities of related enzymes (phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, peroxidase) were monitored in apple (Malus domestica Borkh.). The seasonal survey was performed at five different sampling dates and included the healthy peel of the resistant cultivar ‘Florina’ and healthy peel, scab symptomatic spot and the tissue around the infected spot of the susceptible cultivar ‘Golden Delicious’. From all enzymes tested, chalcone synthase/chalcone isomerase had the highest activity in both cultivars, while phenylalanine ammonia lyase had the lowest. The healthy peels of the susceptible and the resistant cultivar did not show differences in the accumulation of the main polyphenol groups present in the apple skin. However, in the resistant cultivar ‘Florina’, an increase of polyphenol enzyme activities could be observed in late stages of fruit development, which seems to be related to the anthocyanin accumulation in ripe fruits. Significant differences in the polyphenol metabolism were observed in the three different tissues of the susceptible cultivar ‘Golden Delicious’. Increased concentrations of hydroxycinnamic acids, dihydrochalcones and flavan-3-ols were found in the scab symptomatic spots and surrounding tissues. Phenylalanine ammonia-lyase, dihydroflavonol 4-reductase, flavanone 3-hydroxylase and peroxidase showed higher activities in the scab symptomatic spot compared to other analysed tissues, whereas the activities of other enzymes remained unchanged. Highest induction of polyphenol accumulation after scab infection was observed in early developmental stages, whereas enzyme activities were increased in later stages.     

Oleate metabolism in microsomes from developing leaves ofPisum sativum L.

Microsomes from young leaves of pea,Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA:lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties.Abbreviations FA unesterified fatty acid (s) - PC phosphatidylcholines - 18:1 oleoyl moieties - 18:2 lmoleoyl moieties Dedicated to Professor Helmut K. Mangold, Bundesanstalt für Fettforschung, Münster, on his 60th birthday     

Nitrogen nutrition and metabolic interconversions of nitrogenous solutes in developing cowpea fruits

Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticeably deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, serine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development.     

Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves

In order to study the regulation of carbohydrate metabolism in leaf tissue the activity of fructose-6-phosphate,2-kinase was determined in individual sections of developing primary leaves of barley. Activity was about 25-fold higher in the leaf tip than in the leaf sheath when measured on a fresh weight basis. There was a gradual increase in enzyme activity from the leaf base to the leaf tip. The higher activity of fructose-6-phosphate,2-kinase in the apical parts of the leaf was associated with higher levels of fructose-2,6-bisphosphate. This was especially pronounced when isolated leaf segments were treated with vanadate and kept in darkness. As compared to the kinase, little difference was observed in the fructose-2,6-bisphospatase activity among leaf sections. The significance of these patterns for regulation of carbohydrate metabolism in different tissues is discussed.     

Protein metabolism in leaves and developing grains of rices differing in grain protein content

Four semi-dwarf rices (Oryza sativa L.) differing in percentage of grain protein, grown in a flooded field receiving basal N fertilization, had a peak activity of root glutamate dehydrogenase 4 weeks after transplanting. A lower peak occurred during panicle formation 10 weeks after transplanting. The percentage of N of the active leaf blades was also highest 4 weeks after transplanting. The activity of nitrate reductase in the leaf blades was low and decreased after transplanting.     

Phenolic compounds in apple leaves after infection with apple scab

Leaves of the scab-susceptible apple (Malus domestica) cultivar Golden Delicious were harvested from May to August 2008 and 2009. Some leaves were healthy and some infected with fungus Venturia inaequalis. The phenolic compounds were analysed in healthy leaves, infected leaves and in the scab spot tissue. In comparison to healthy leaves, the infected leaves showed higher contents of hydroxycinnamic acid, flavanols and phloridzin, while lower contents on procyanidins, quercetins and phloretin. The total amount of phenolic compounds in the infected tissue was 10 to 20 % higher than in the healthy leaves. Accumulation of phenolic compounds is a post-infection response, and probably their further transformation is a prerequisite for plant resistance.     

Sorbitol and other carbohydrates in dormant apple shoots as influenced by controlled temperatures

The influence of controlled temperatures on levels of sorbitol and other carbohydrates was determined to provide further information on dormancy of apple trees (Malus sylvestris Mill.). For 3 years, 2-year-old “Red Delicious” apple shoots were collected from mature trees in an orchard at intervals during the autumn and winter, and shoots were stored for 6 hr to 1 week at temperatures from 18 to ?60 °C. Sorbitol and other carbohydrates were estimated in the sap or wood by gas chromatography.Controlled temperatures had a marked influence on the carbohydrate content of excised 2-year-old apple shoots. Levels of sorbitol in the sap were maximum at ?0.6 °C. The increase was greatest at the earliest sampling before complete hardening had occurred in each of the 3 years tested. Total sorbitol in the wood was less influenced by storage at various controlled temperatures than sorbitol in the sap. Levels of fructose, glucose, and sucrose in the wood were higher at temperatures below ?0.6 °C than at warmer temperatures. Levels of starch were usually inversely related to soluble sugars.     

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves

Summary The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of theArabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 m/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.     

Stomata of apple leaves cultured in vitro

Examination by scanning electron microscopy showed abaxial stomata on in vitro cultured apple (Malus pumila Mill.) leaves. With leaf ontogeny, most of these stomata appeared to lose their regulatory ability while developing wide vestibules of up to 20 m in diameter. It is proposed that these deformed stomata may be a possible cause for the excessive transpirational water loss and consequent dehydration associated with transferring plants regenerated in vitro from culture.     

Starch metabolism in leaves

Starch is the most abundant storage carbohydrate produced in plants. The initiation of transitory starch synthesis and degradation in plastids depends mainly on diurnal cycle, post-translational regulation of enzyme activity and starch phosphorylation. For the proper structure of starch granule the activities of all starch synthase isoenzymes, branching enzymes and debranching enzymes are needed. The intensity of starch biosynthesis depends mainly on the activity of AGPase (adenosine 5'-diphosphate glucose pyrophosphorylase). The key enzymes in starch degradation are beta-amylase, isoamylase 3 and disproportionating enzyme. However, it should be underlined that there are some crucial differences in starch metabolism between heterotrophic and autotrophic tissues, e.g. is the ability to build multiprotein complexes responsible for biosynthesis and degradation of starch granules in chloroplasts. The observed huge progress in understanding of starch metabolism was possible mainly due to analyses of the complete Arabidopsis and rice genomes and of numerous mutants with altered starch metabolism in leaves. The aim of this paper is to review current knowledge on transient starch metabolism in higher plants.     

Comparison of the effect of rapidly and gradually developing water-stress on carbohydrate metabolism in spinach leaves

Abstract. The effect of water-stress on photosynthetic carbon metabolism in spinach ( Spinacia oleracea L.) has been studied in experiments in which water-stress was induced rapidly by floating leaf discs on sorbitol solutions or wilting detached leaves, and in experiments in which water-stress was allowed to develop gradually in whole plants as the soil dried out. In both short- and long-term water stress, the rate of photosynthesis in saturating CO₂ did not decrease until leaf water potential decreased below -1.0 MPa. However, at smaller water deficits there was already an inhibition of starch synthesis, while sucrose synthesis remained constant or increased. This change in partitioning was accompanied by an increase in activation of sucrose-phosphate synthase (revealed as an increase in activity assayed in the presence of low hexose-phosphate and inorganic phosphate, while the activity assayed with saturating hexosephosphates remained unaltered). Water-stressed leaves had a two- to three-fold higher sucrose content at the end of the night, and contained less starch than non-stressed leaves. When leaves were held in the dark, sucrose was mobilized initially, while starch was not mobilized until the sucrose had decreased to a low level; in water-stressed leaves, starch mobilization commenced at a two-fold higher sucrose content. It is concluded that water-stressed leaves maintain higher sucrose and lower starch levels than non-stressed leaves. This response is found in rapid and long-term stress, and represents an inherent response to water deficits.     

The occurrence of nitrate reductase in apple leaves

Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH₂) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10⁻³ to 10⁻⁵m with no effect upon nitrate reductase activity.     

Polysomal changes in developing wheat leaves

Summary When leaf sections of 7-day old dark grown wheat leaves were incubated in white light, they unrolled and greened. Gibberellic acid was able to replace the light requirement and abscisic acid (ABA) inhibited the response to light. The percentage of ribosomes occurring as polysomes increased in response to light but not in response to GA₃ treatment. Although ABA inhibited the unrolling and greening in light, it did not cause a preferential decrease or inhibition of polysome formation.     

Starch depletion and sugars in developing cotton leaves

Cotton plants (cv. Coker 100) were exposed to a 14-hour dark period. Starch degradation occurred with no accumulation of sugars due mainly to translocation. Considerable amounts of starch degradation products however were detected from leaves after phloem transport was blocked. A minor component (10 to 25% of total starch) with a linear structure, amylose, was preferentially degraded, whereas the major multiple-branched component (about 80%), amylopectin, showed an increasing resistance to degradation with leaf age. This relationship was also shown by the decreasing iodine-binding capacity of unit starch with increasing leaf age. The structural resistance of amylopectin to enzymic dark degradation was one of the barriers to starch dissolution in cotton.