Import of Sucrose and its Partitioning in the Synthesis of Galactomannan and Raffinose-oligosaccharides in the Developing Guar (Cyamopsis tetragonolobus) Seed

Import of sucrose and its transformation to galactomannan andraffinose-oligosaccharides have been studied in the developingguar seed. The amount of galactomannan gradually increased withthe ageing of the seed. During the entire period of pod development,sucrose constituted the major portion of the free sugars inthe seed (both endosperm and cotyledons) as well as in the podwall. Besides myo-inositol, the free sugars detected in thedeveloping endosperm and cotyledons were glucose, fructose,raffinose and stachyose. Some compounds, possibly glycosides(RG values higher than that of fructose), were also detectedin the endosperm. In the later stages of seed development, therelative proportion of raffinose in the free sugars increased,reaching 50% of the total free sugars in 77-d-old cotyledons.With pod maturity, the activities of soluble acid and boundacid invertases in the pod wall increased manifold with a concomitantdecline in the non-reducing sugar content. These enzymes seemto be involved in the mobilization of sucrose from this fruitingstructure into the seed. An increased synthesis of raffinose-oligosaccharidesboth in the endosperm and cotyledons was associated with highactivities of soluble acid invertase (pH 4.8) and sucrose-UDPglucosyl transferase in these tissues. Feeding uniformly labelled¹⁴C-sugars to the detached intact pods as well as to the isolatedendosperm and cotyledons resulted in labelling of all endogenousfree sugars and galactomannan. The uptake and incorporationinto galactomannan of ¹⁴C was stimulated by Co²⁺, Mn²⁺ and Mg²⁺.Except for mannose, a major proportion of the ¹⁴C from glucose,fructose and sucrose appeared in sucrose in both endosperm andcotyledons indicating a fast reconstitution of sucrose in situ.Based on the present results, a possible mode of transformationof sucrose to galactomannan and raffinose-oligosaccharides hasbeen proposed. Key words: Sucrose, galactomannan, raffinose-oligosaccharides, invertase, sucrose-UDP glucosyl transferase, ¹⁴C-incorporation, guar seed     

Fluoride-induced alteration of carbon and nitrogen metabolism in developing wheat grains

Effect of fluoride (10 and 50 mM) on the activities of sucrose metabolizing enzymes, alkaline inorganic pyrophosphatase, and transaminases in relation to the accumulation of free sugars, starch, and soluble protein was studied in detached ears of wheat cultured in a liquid medium. Culturing for 5 d in the presence of fluoride reduced the amount of grain starch whereas contents of total free sugars, particularly sucrose, and soluble protein increased. Fluoride inhibited the activities of soluble acid and neutral invertases, as well as sucrose synthase acting in the cleavage direction. Uptake of uniformly labelled ¹⁴C-sucrose or fructose was also drastically reduced by fluoride. Glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities also increased with fluoride addition in correspondence with an increase in soluble protein. Apparently, the wheat grain responds to fluoride-mediated disruption of carbon metabolism by a compensatory effect on nitrogen metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Response of sugar interconversion, starch and protein accumulation to supplied metabolites during pod filling in chickpea

Detached chickpea inflorescences bearing pods at 20 days after flowering (DAF) were cultured for 5 days in complete liquid medium supplemented separately with asparate, myo-inositol, alpha-ketoglutarate and phytic acid. Effect of these metabolites on sugar interconvestion and starch and protein accumulation in developing pods was studied. Substituting asparate (62.5 mM) for glutamine in culture medium decreased relative proportion of sucrose in all pod tissues but increased the level of sugars, starch and protein in pod wall and cotyledons. In cotyledons, whereas myo-inositol (75 mM) reduced the accumulation of starch without affecting protein level, alpha-ketoglutarate (44 mM) increased both starch and protein accumulation. Both myo-inositol and alpha-ketoglutarate increased relative proportion of sucrose in cotyledons. Phytic acid (1 mM) decreased in cotyledons 14C incorporation from glucose into EtOH extract (principally constituted by sugars), amino acids and proteins but increased the same into starch. In cotyledons, phytic acid also increased 14C incorporation from glutamate into amino acids but this increase was negatively correlated with protein synthesis. Phytic acid decreased the relative distribution of 14C from glucose and glutamate into sucrose from pod wall but enhanced the same into EtOH extract from embryo. Based on the results, it is suggested that mode of metabolic response to exogenously supplied metabolites widely differs in pod tissues of chickpea.     

Relationships between Respiration Rate and Adenylate and Carbohydrate Pools of the Soybean Fruit

Relationships between respiration rate and adenylate and carbohydrate pools of the soybean (Glycine max L. Merrill) fruit during rapid seed growth were evaluated. Plants at mid pod-fill were subjected to different concentrations of CO(2) to alter the amount of photosynthate produced and, thus, available to the fruit. Respiration rate of the intact fruits was measured, along with glucose, sucrose, and starch concentrations, adenylate energy charge (AEC), and total adenylate pool (SigmaAdN) in the pod wall, seed coat, and cotyledons. The concentration of sucrose remained relatively constant in the pod wall (1.0 milligram per 100 milligrams dry weight), seed coat (6.5 milligrams per 100 milligrams dry weight), and cotyledons (4.5 milligrams per 100 milligrams dry weight) at moderate and high respiration rates. Furthermore, AEC remained relatively constant in the pod wall (0.55), seed coat (0.24), and cotyledons (0.44) during changes in respiration rate. This suggests that the amount of assimilate transported to the fruit, and its flux through the sucrose pools of the fruit parts, were important in the regulation of the respiration rate of the fruit. The average SigmaAdN in the seed coat (1300 picomoles per milligram dry weight) was significantly greater than in the cotyledons (750 picomoles per milligram dry weight) and pod wall (300 picomoles per milligram dry weight). In addition, the SigmaAdN in the seed coat and cotyledons increased with increasing respiration rate of the fruit. The high SigmaAdN in the seed coat and its increase with increases in respiration rate of the fruit suggest that an energy-requiring process is involved in the movement of sucrose through the seed coat.     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

Inter-conversion of free sugars and accumulation of galactomannan in response to supplied metabolic mediators during pod filling in guar

Detached inflorescences of guar (Cyamopsis tetragonoloba), each bearing 4 uniformly-developing pods at 42 days post anthesis (DPA), were cultured for 6 days in complete liquid medium manipulated with a fixed concentration of mannose and varying concentration of myo-inositol. Such inflorescences, but with 2 pods, were also maintained in the solutions of (i) glucose(U-14C) containing myo-inositol or phytohormones, and (ii) mannose(U-14C) containing galactose for 36 hr. Effect of such exogenously supplied metabolic mediators on interconversion of free sugars in pod wall, endosperm and cotyledons and galactomannan accumulation in endosperm was studied. Myo-inositol decreased, over control, the relative proportion of invert sugars in pod wall, endosperm and cotyledons and at lower concentration (27.75 mM) it decreased the level of free sugars in pod wall and galactomannan in endosperm. In all pod tissues, 14C from both glucose and mannose got incorporated into myo-inositol as well as various sugars and maximum incorporation occurred in sucrose. High concentration of total free sugars and their 14C activity in pod wall indicated that this pod tissue was a potent accumulator of free sugars. With myoinositol, the relative proportion of 14C from glucose into raffinose sugars of pod wall and endosperm increased with a simultaneous decrease in this incorporation into galactomannan of the latter. Accompanying this, relative proportion of 14C into hexoses and myo-inositol decreased in pod tissues. Galactose increased 14C incorporation from mannose into total free sugars, sucrose and galactomannan with a concomitant decline in the labelling of hexoses. IAA and ABA enhanced 14C incorporation from glucose into total free sugars and this enhancement was much higher with IAA than ABA. The latter inhibited 14C incorporation into galactomannan. Based on these results, it was suggested that myo-inositol at lower concentration was inadequate to mediate the metabolism of sugars and, thereby, galactomannan synthesis. Galactose and mannose exhibited a mutual beneficial effect on their transportation to pods. Phytohormones stimulated the accumulation of sucrose in pod wall for its obligatory unloading into the seed.     

Interconversion and translocation of free sugars during galactomannan utilization in germinating guar (Cyamopsis tetragonoloba) seed

With the onset of the degradation of galactomannan, the galactose and mannose levels increased in the endosperm. The hydrolysis of galactomannan was more or less complete within the first 3 days of germination. In the cotyledons, sucrose was the predominant free sugar during the period of rapid galactomannan hydrolysis and reducing sugars (glucose + fructose) were present in only 10–20% proportion. The level of soluble acid invertase activity was in the order of embryonic axis > endosperm > cotyledons. On the basis of (a) absence of galactose and mannose, (b) high proportion of sucrose, (c) very fast conversion of [¹⁴C]glucose and [¹⁴C]mannose to [¹⁴C]sucrose and (d) very low levels of both soluble and bound invertases in cotyledons, we conclude that there is an active synthesis of sucrose in this tissue where disaccharide seems to be least hydrolysed during the period of galactomannan mobilization. A rapid hydrolysis of galactomannan in endosperm during early germination resulted in the synthesis of some starch, as a temporary reserve, in cotyledons. When the cotyledons entered the phase of first leaf formation, cotyledonary sucrose was hydrolysed giving rise to invert sugars. In the embryonic axis, the increase in the ratio of reducing sugars to sucrose coupled with a higher level of invertase, compared with sucrose-UDP glucosyl transferase, indicated that free sugars from the cotyledons are translocated to the embryonic axis as sucrose.     

Seed growth rate and carbohydrate pool sizes of the soybean fruit

The relationships between various carbohydrate pools of the soybean (Glycine max [L.] Merrill) fruit and growth rate of seeds were evaluated. Plants during midpod-fill were subjected to various CO₂ concentrations or light intensities for 7 days to generate different rates of seed growth. Dry matter accumulation rates of seeds and pod wall, along with glucose, sucrose, and starch concentrations in the pod wall, seed coat, and embryo were measured in three-seeded fruits located from nodes six through ten. Seed growth rates ranged from 4 to 37 milligrams·day⁻¹·fruit⁻¹. When seed growth rates were greater than 12 milligrams·day⁻¹·fruit⁻¹, sucrose concentration remained relatively constant in the pod wall (1.5 milligrams·100 milligrams dry weight⁻¹), seed coat (8.5 milligrams·100 milligrams dry weight⁻¹), and embryo (5.0 milligrams·100 milligrams dry weight⁻¹). However, sucrose concentrations decreased in all three parts of the fruit as growth rate of the seeds fell below 12 milligrams·day⁻¹·fruit⁻¹. This relationship suggests that at high seed growth rates, flux of sucrose through the sucrose pools of the fruit was more important than pool size for growth. Starch concentration in the pod wall remained relatively constant (2 milligrams·100 milligrams dry weight⁻¹) at higher rates of seed growth but decreased as seed growth rates fell below 12 milligrams·day⁻¹·fruit⁻¹. This suggests that pod wall starch may buffer seed growth under conditions of limiting assimilate availability. There was no indication that carbohydrate pools of the fruit were a limitation to transport or growth processes of the soybean fruit.     

Modulation of Reserve Mobilization by Sucrose,Glutamine, and Abscisic Acid During Seedling Establishment in Sunflower

We carried out in vitro feeding experiments using sunflower as a model to differentiate the modulatory effects of metabolites (sucrose and glutamine) and hormones (gibberellic acid and abscisic acid) on reserve mobilization, metabolite partitioning, and key enzyme activities. Exogenous sucrose negatively not only modulated the mobilization of carbon reserves (oils and starch), but it also delayed the degradation of nitrogen reserves (storage proteins) in the cotyledons. Similarly, exogenous glutamine negatively not only modulated storage protein hydrolysis, but it also retarded oil and starch degradation. Different from the metabolites, exogenous abscisic acid affected only the mobilization of oils and storage proteins. Sucrose and glutamine caused non-reducing sugar accumulation in the cotyledons and axis, but abscisic acid did not change the content of these compounds in both seedling parts. Curiously, glutamine failed to cause amino acid accumulation in the cotyledons and abscisic acid increased the amino acid content in both cotyledons and axis. Gibberellic acid did not stimulate reserve mobilization and metabolite consumption. Although the mobilization of oils, storage proteins, and starch has been delayed by sucrose and glutamine, these metabolites augmented the activity of isocitrate lyase, acid proteases, and amylases. Only abscisic acid reduced amylase activity and increased glutamine synthetase activity. Accordingly, sucrose and glutamine exert a “crossed effect” on reserve mobilization, that is, sucrose delays storage protein hydrolysis and glutamine retards oil and starch degradation. These effects may be mediated by non-reducing sugars and they are, at least in part, different from those exerted by abscisic acid.     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

Ontogenic changes in enzymes of carbon metabolism in relation to carbohydrate status in developing mungbean reproductive structures

The content of free sugars and the activities of enzymes involved in carbon metabolism-sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were determined during seed development in mungbean pods. A decrease in carbohydrate content of pod wall from 10 to 25 days after flowering (DAF) and a concomitant increase in the seed till 20 DAF was observed. Sucrose remained the dominant soluble sugar in the pod wall and seed. In the branch of inflorescence and pod wall, the activities of sucrose metabolizing enzymes, viz. acid and alkaline invertase, sucrose synthase (synthesis and cleavage) and sucrose phosphate synthase were higher at 5-10 DAF, whereas in seed the maximum activities of these enzymes were observed at the time of maximum seed filling stage (10-20 DAF). High activities of sucrose synthase at the time of rapid seed filling can be correlated to its sink strength. Higher activities of phosphoenol pyruvate carboxylase in the branch of inflorescence and pod wall than in seed may indicate the involvement of the fruiting structure for recapturing respired CO2. High activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could provide NADPH and carbon skeletons required for the synthesis of various seed reserves.     

Metabolism of Phloem-borne Amino Acids in Maternal Tissues2Pisum sativum L.)

The amino acid composition of the EDTA-induced phloem exudatereaching the fruit and the seed, and of the solutes releasedby the seed coat during fruit development were determined inglasshouse-grown pea (Pisum sativum L. cv. Finale) suppliedeither with nitrate-free nutrients (nodulated plants) or withcomplete medium (non-nodulated plants). The EDTA-promoted exudationtechnique was used supposedly to collect phloem sap and theempty seed technique supposedly to collect the solutes secretedby the seed coat to the embryo sac cavity. In young seeds embryosac liquid was sampled directly from the embryo sac. The maincarbohydrate transported and secreted was sucrose. The mainamino acids reaching the fruit were asparagine, glutamine, andhomoserine. Their proportions were steady during a day-nightcycle and throughout fruit development. Amino acid compositionchanges occurred first in the pathway from fruit stalk to seedfunicle, due to the formation of threonine (probably from homoserine)and in the seed coat due to production of glutamine, alanineand valine which, together with threonine were the main secretedamino acids. The temporary nitrogen reserves of the pod walland seed coat were remobilized as asparagine during senescence.Phloem exudate of nodulated plants showed a higher (about twice)proportion of asparagine but lower proportions of homoserineand glutamine than in EDTA-induced phloem exudate of nitrate-fedplants. The two types of nitrogen nutrition also produced somechanges in relative proportions of threonine and homoserinesecreted by the seed coat. Key words: Pisum sativum, phloem, amino acids, pod wall, seed coat     

Turgor-dependent unloading of assimilates from coats of developing legume seed. Assessment of the significance of the phenomenon in the whole plant

The in vivo significance of turgor-dependent unloading was evaluated by examining assimilate transport to and within intact developing seeds of Phaseolus vulgaris (cv. Redland Pioneer) and Vicia faba (cv. Coles Prolific). The osmotic potentials of the seed apoplast were low. As a result, the osmotic gradients to the seed coat symplast were relatively small (i.e. 0.1 to 0.3 MPa). Sap concentrations of sucrose and potassium in the seed apoplast and coat symplast accounted for some 45 to 60% of the osmotic potentials of these compartments. Estimated turnover times of potassium and sucrose in the seed apoplast of < 1 h were some 5 to 13 times faster than the respective turnover times in the coat symplast pools. The small osmotic gradient between the seed apoplast and coat symplast combined with the relatively rapid turnover of solutes in the apoplast pool, confers the potential for a small change in assimilate uptake by the cotyledons to be rapidly translated into an amplified shift in the cell turgor of the seed coat. Observed adjustments in the osmotic potentials of solutions infused between the coat and cotyledons of intact seed were consistent with the in vivo operation of turgor-dependent unloading of solutes from the coat. Homeostatic regulation of turgor-dependent unloading was indicated by the maintenance of apoplast osmotic potentials of intact seeds when assimilate balance was manipulated by partial defoliation or elevating pod temperature. In contrast, osmotic potentials of the coat symplast adjusted upward to new steady values over a 2 to 4 h period. The resultant downward shift in coat cell turgor could serve to integrate phloem import into the seed coat with the new rates of efflux to the seed apoplast. Circumstantial evidence for this linkage was suggested by the approximate coincidence of the turgor changes with those in stem levels of ³²P used to monitor phloem transport. The results obtained provide qualified support for the in vivo operation of a turgor homeostat mechanism. It is proposed that the homeostat functions to integrate assimilate demand by the cotyledons with efflux from and phloem import into the coats of developing legume seed.     

Cessation of assimilate uptake in maturing soybean seeds

In vitro assimilate uptake and metabolism were evaluated in embryos of known age isolated from seeds at mid-podfilling through physiological maturity. The capacity of isolated Wye soybean embryos to take up exogenous [¹⁴C]sucrose dropped nearly 4-fold in less than 1 week at incipient cotyledon yellowing. This drop in rate of sucrose uptake coincided with cessation of seed growth as well as rapid decline in leaf photosynthetic rate that preceded leaf yellowing. Conversely, the rate of [³H]glutamine uptake by cotyledons increased as they yellowed. Yellow cotyledons also rapidly converted exogenous [³H]glutamine to ethanolinsoluble components, but converted little exogenous [¹⁴C]sucrose to ethanol-insoluble components, primarily because of greatly reduced sucrose uptake. Sustained import and metabolism of amino acids remobilized from senescing leaves may prolong seed growth beyond loss of photosynthetic competency and sucrose availability.     

Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.)

The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.

In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.

We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles.

     

Enzymes of carbohydrate metabolism in the developing rice grain

The levels of reducing and nonreducing sugars, starch, soluble protein, and selected enzymes involved in the metabolism of sucrose, glucose-1-P, and glucose nucleotides were assayed in dehulled developing rice grains (Oryza sativa L. line IR1541-76-3) during the first 3 weeks after flowering. The level of reducing sugars in the grain was highest 5 to 6 days after flowering. The level of nonreducing sugars and the rate of starch accumulation were maximum 11 to 12 days after flowering, when the level of soluble protein was also the highest. The activities of bound and free invertase, sucrose-UDP and sucrose-ADP glucosyltransferases, hexokinase, phosphoglucomutase, nucleoside diphosphokinase, and UDP-glucose and ADP-glucose pyrophosphorylases were high throughout starch deposition, and were maximum, except for nucleoside diphosphokinase which did not increase in activity, between 8 and 18 days after flowering. Soluble primed phosphorylase and ADP glucose-α-glucosyltransferase (starch synthetase) were both present during starch accumulation. Phosphorylase activity was at least 2-fold that of soluble starch synthetase but the synthetase followed more closely the rate of starch accumulation in the grain. The activity of starch synthetase bound to the starch granule also increased progressively with increased starch content of the grain.     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.     

The cellular pathway of sucrose transport in developing cotyledons ofVicia faba L. andPhaseolus vulgaris L.: a physiological assessment

The cellular pathway of sugar uptake in developing cotyledons of Vicia faba L. and Phaseolus vulgaris L. seed was evaluated using a physiological approach. The cotyledon interface with the seed coat is characterised by a specialised dermal cell complex. In the case of Vicia faba cotyledons, the epidermal component of the dermal cell complex is composed of transfer cells. Sucrose is the major sugar presented to the outer surface of both cotyledons and it is taken up from the apoplasm unaltered. Estimated sucrose concentrations within the apparent free space of Vicia and Phaseolus cotyledons were 105 and 113 mM respectively. Rates of in-vitro uptake of [¹⁴C]sucrose by cotyledon segments or by whole cotyledons following physical removal or porter inactivation of the outer cells demonstrated that, for both Vicia and Phaseolus cotyledons, the dermal cell complexes are the most intense sites of sucrose uptake. Accumulation of [¹⁴C]sucrose in the storage parenchyma of whole cotyledons was directly affected by experimental manipulation of uptake by the outer cell layers and plasmolytic disruption of the interconnecting plasmodesmata. These findings indicated that sucrose accumulated by the dermal cell complexes is transported symplasmically to the storage parenchyma. Overall, it is concluded that the dermal cell complexes of the developing legume embryo, irrespective of the presence or absence of wall ingrowths, are the major sites for the uptake of sucrose released from the maternal tissues to the seed apoplasm. Thereafter, the accumulated sucrose is transported radially inward through the symplast to the storage parenchyma.Abbreviations AFS apparent free space - CF 5-(6)-carboxyfluorescein - CFDA 5-(6)-carboxyfluorescein diacetate - Mes 2-(N-morpholino)ethanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SRG sulphorhodamine G The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are grateful to Stella Savoury for preparing the photomicrographs.     

Manipulation Through Liquid Culture in the Contents of Sucrose and Glutamine and Their Transformation to Starch and Protein in Wheat Grain

Detached ears of wheat were cultured in liquid medium manipulated for sucrose and glutamine contents, and the accumulation of starch and protein in relation to the activities of sucrose cleaving—, ammonia assimilating—, and transaminating enzymes was studied in the grain. With an increase in the concentrations of sucrose from 44 to 176 mM and glutamine from 6.4 to 25.7 mM (keeping their ratio at a constant value of 7:1), the contents of starch and protein increased in the grains. However, when the grains were cultured in the medium containing 8.5 to 34 mM glutamine and a fixed concentration of 117 mM sucrose, there was a gradual increase in protein and decrease in starch content in the grain. By such manipulation in the liquid medium, the content of free amino acids also increased in the grain up to 12 days culturing. Amongst sucrose cleaving enzymes, the activities of sucrose-UDP glucosyl transferase and soluble alkaline invertase were much lower than the activity of soluble acid invertase. At high concentration (34 mM) of glutamine in the medium, containing 117 mM sucrose, there was drastic decrease in the activities of soluble acid invertase and UDPG pyrophosphorylase but the activities of ADPG pyrophosphorylase, alkaline inorganic pyrophosphatase, glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase increased in the grain with increase in glutamine concentration in the culture medium. Evidently, an increase in the level of amino nitrogen, coupled with an optimum sucrose concentration in the grain raised through liquid culturing enhances the conversion of sucrose to protein at the cost of starch accumulation in wheat.     

Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue

Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6-bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.

Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient.