Ewald Komor

Sucrose Accumulation in the Sugarcane Stem Is Regulated by the Difference between the Activities of Soluble Acid Invertase and Sucrose Phosphate Synthase.

To assess the relative importance of morphological and biochemical factors in the regulation of sucrose (Suc) accumulation in the sugarcane (Saccharum spp. hybrids) stem, we investigated morphological and biochemical correlates of Suc accumulation among parents and progeny of a family segregating for differences. In contrast to the parents, no relationship was observed between morphology and the level of Suc accumulation among the progeny. The level and timing of Suc accumulation in the whole stalk and within individual internodes was correlated with the down-regulation of soluble acid invertase (SAI) activity. High SAI activity prevented most, but not all, Suc accumulation. There was a critical threshold of SAI activity above which high concentrations of Suc did not accumulate. This low level of SAI activity was always exceeded in the internodes of the lower-Suc-storing genotypes. However, low activity of SAI was not sufficient by itself to account for the Suc accumulation in the higher-Suc-storing genotypes. Major differences in Suc accumulation among the population were attributed to the difference between activities of SAI and Suc phosphate synthase, provided SAI is below the critical threshold concentration. This result is not unexpected, since the pathway of Suc transport for storage involves Suc hydrolysis and resynthesis.

Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics, mechanism, and regulation.

Cotyledons of Ricinus communis take up externally supplied sucrose at a rate of up to 150 μmol/h/g fresh weight, which is very high when compared with other sugar transport systems of higher plants. The uptake of sucrose is catalysed with a Km of 25 mmol l−1; at high sucrose concentrations a linear (diffusion) component becomes obvious. Other mono-, di-, or trisaccharides do not compete for sucrose uptake. Sucrose is accumulated by the cotyledons up to 100-fold, whereby most of the transported, externally supplied sucrose mixes with sucrose present in the tissue. At low sucrose concentrations, however; a small unexchangeable internal pool of sucrose becomes evident. Poisons of energy metabolism such as FCCP inhibit uptake and accumulation of sucrose. The transport of sucrose induces an increase of respiration, from which an energy requirement of 1.4 ATP/sucrose taken up can be calculated. Sucrose is taken up together with protons at an apparent stoichiometry of 0.3 protons/sucrose. Other sugars do not cause proton uptake. The Km for sucrose induced proton uptake is 5 mmol l−1; the discrepancy to the Km for sucrose uptake as well as the low proton: sucrose stoichiometry might possibly be caused by a large contribution of diffusion barriers. The estimated proton-motive potential difference would by sufficient to explain an electrogenic sucrose accumulation. The rate of uptake of sucrose is subject to feedback inhibition by internal sucrose. It is also regulated during growth of the seedlings since it develops rapidly during the first days of germination and declines again after the 4th day of germination, though no substantial increase of passive permeability resistance was observed.

A non-invasive measurement of phloem and xylem water flow in castor bean seedlings by nuclear magnetic resonance microimaging

A flow-sensitive nuclear magnetic resonance (NMR) microimaging technique was applied to measure directly the in-vivo water flow in 6-d-old castor bean seedlings. The achieved in-plane resolution of the technique allowed discrimination between xylem and phloem water flow. Both the xylem- and the phloem-average flow velocities in the intact seedling could be quantified. Furthermore, the total conductive cross-sectional area of the xylem vessels and the phloem sieve elements could be determined using the non-invasive and non-destructive NMR microimaging technique. Hence, it was possible to calculate the in-vivo volume flow rates for both xylem and phloem water flow. Our non-destructive technique showed that previously used methods to measure phloem water flow affected the flow rate itself. In the intact seedlings we found values of 16.6 μl·h−1, two fold lower than those previously estimated from phloem exudation rates. Finally, our results demonstrate for the first time that water is internally circulated between phloem and xylem, and that water flow within the xylem is maintained by this internally circulated water, even in the absence of any significant transpiration or evaporation.

A proton-cotransport system in a higher plant: Sucrose transport in Ricinus communis

Abstract Sucrose uptake into cotyledons of castor beans (Ricinus communis) proceeds partly by an active transport system and partly by passive permeation; α-methylglucoside (α-MG) is almost exclusively transported by the passive route. The Km for active sucrose uptake is 18 mM and the Vmax 4–5 μmoles/100 mg fresh weight per 1 h. The tissue is able to accumulate sucrose more than 100-fold. Sucrose but not α-MG induces a respiratory increase, which shows a similar sucrose concentration dependence as active sucrose transport. The addition of saturating amounts of sucrose to the incubation medium leads to a transient alkalinization of the medium. A second addition does not show this effect, neither does α-MG at any time. In the presence of 100 mM K+, which depolarizes the membrane potential, the uptake of sucrose is strongly inhibited. The results suggest that active sucrose transport in this tissue is mediated by an electrogenic proton cotransport system.

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

Abstract. The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants.

Sucrose storage in cell suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation

We have investigated the regulation of sucrose storage in cell-suspension cultures of sugarcane. When grown in batch culture, sucrose accumulation commences after about 5 d, when the nitrogen supply is exhausted. Sucrose storage is also induced by decreasing the nitrogen supply to cells growing in a chemostat. The measured activity of sucrose-phosphate synthase is high enough to account for the rate of sucrose accumulation, provided precautions are taken to avoid the hydrolysis of UDP during the assay. The cells contained high sucrose-synthase activity but pulsing experiments with [14C]glucose and unlabelled fructose indicated that this enzyme did not contribute substantially to the synthesis of sucrose, because the glucosyl and fructosyl moieties of sucrose were equally labelled. Several lines of evidence demonstrate the presence of a cycle in which sucrose is synthesized and degraded simultaneously; sucrosephosphate-synthase activity doubles during the phase when the cells are actively storing sucrose but activity is also high after storage has ceased, or when the sucrose is being remobilised; pulse experiments with [14C]fructose also showed that sucrose synthesis occurs not only during the storage phase, but also after storage has stopped and during the rapid mobilisation of sucrose; the cells contain high activities of sucrose synthase and alkaline invertase and these are both at a maximum when sucrose storage is occurring; even during the storage phase. [14C]fructose pulses lead to labelling of free glucose which is evidence for rapid synthesis and degradation of sucrose. It is proposed that the rate and extent of sucrose storage is regulated by this cycle of synthesis and degradation. Measurements of enzyme activities and metabolite levels are presented, and it is discussed which factors could contribute to the regulation of these two opposing fluxes and, hence, the rate of net sucrose storage and mobilisation.

Sieve-tube exudate from Ricinus communis L. seedlings contains ubiquitin and chaperones

The cut hypocotyl of Ricinus communis L. seedlings exudes phloem sap which contains a characteristic set of proteins (Sakuth et al. 1993, Planta 191, 207–213). These sieve-tube exudate proteins were probed with antibodies to highly conserved proteins, namely ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), Rubisco subunit-binding protein, heat-shock protein (HSP 70), chaperonin GroEL and ubiquitin. Homologous proteins in the sieve-tube exudate were identified with antisera to HSP 70, Rubisco-subunit-binding protein and ubiquitin. Ribulose-1,5-bisphosphate carboxylase-oxygenase, which was present in the tissue, was not detected. Of all the cross-reactive proteins detected, ubiquitin was special because the ubiquitin-to-protein ratio in the sieve-tube exudate was higher than in both the surrounding hypocotyl and in the cotyledonary tissues. Therefore, ubiquitin features properties which favour its transfer into the sieve tubes and which might rely on efficient transport through plasmodesmata. It is assumed that chaperones and ubiquitin are needed for the maintenance of sieve-tube function, e.g. to ensure correct folding of proteins. Their possible involvement in protein translocation through plasmodesmata from companion cells to sieve tubes is discussed.

The mechanism of sugar uptake by sugarcane suspension cells

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. — (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). — (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the Km value was higher (70 μM) than for net influx (24 μM); the exchange efflux is proposed to be mediated by the same transport system with a Km value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. — (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H+ per sugar; during the onset of hexose transport there was a K+ exit of 0.94 K+ per sugar for charge compensation. (It was assumed that the “real” stoichiometries are 1 H+ and 1 K+ per sugar.) The Km values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. — (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. — (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.

Specific proteins in the sieve-tube exudate of Ricinus communis L. seedlings: separation, characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml−1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [35S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.

Sucrose transport into the phloem of ricinus communis l. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K+ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K+ concentration in the exudate. High concentrations of K+, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na+ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.