Sucrose utilization during ovule and seed development ofGasteria verrucosa (Mill.) H. Duval as monitored by sucrose synthase and invertase localization

Summary The development ofGasteria verrucosa ovules and seeds seems to follow a pattern of growth in which the majority of carbohydrates is first used in the sporophytic tissue (nucellus, integuments, and arillus) around the gametophyte-derived cells. After fertilization the carbohydrates are used for further development of the arillus and seed coat. During the next stage carbohydrates are directed to develop the endosperm, followed by carbohydrate investment in the developing embryo and in storage products. This utilization pattern is deducted from a localization study on sucrose synthase and invertase. These two enzymes break down imported sucrose and are in that perspective used as markers for carbohydrate transport since diffusion is expected to be induced towards cells and tissues with high sucrose-hydrolyzing activities.     

Invertase,sucrose synthase and sucrose phosphate synthase in lyophilized spruce needles; microplate reader assays

Summary Data on changes of apparent activities of enzymes involved in sucrose metabolism of developing spruce needles are presented. Extractable activities of sucrose phosphate synthase (SPS, sucrose synthesis), and sucrose synthase (SS) and acid invertase (both sucrolysis) were determined in small volumes using a novel microplate reader system which combined high rates of activity with good reproducibility and high sample throughput. During a developmental period of up to 18 months after bud break characteristic changes in SPS and SS occurred. During the first 4 months of needle development SS declined while SPS increased which is indicative of a transition from net import to net export of photoassimilates (sink/source transition). After needle maturation both enzymes exhibited parallel annual changes with increasing rates towards autumn, which was mirrored by the pool sizes of sucrose (possibly due to the acquisition of frost hardiness). Acid invertase activity was comparable to that of SS but showed only marginal seasonal changes. Approximately 70% of its total activity was found to be soluble.     

Co-induction of sucrose transport and invertase activities in a thermophilic fungus,Thermomyces lanuginosus

The incorporation of sucrose into the thermophilic fungus,Thermomyces lanuginosus, occurred only in mycelia previously exposed to sucrose or raffinose. Sucrose uptake and invertase were inducible. Both activities appeared in sucrose-induced mycelia at about the same time. Both activities declined almost simultaneously following the exhaustion of sucrose in the medium. The sucrose-induced uptake system was specific for -fructofuranosides as revealed by competition with various sugars. The induction of sucrose uptake system was blocked by cycloheximide, showing that it was dependent on new protein synthesis. Transport of sucrose did not seem to be dependent on ATP. Rather, uptake of this sugar seemed to be driven by a proton gradient across the plasma membrane. The uptake system showed Michaelis-Menten kinetics.Abbreviations FCCP carbonylcyanide p-trifluoromethylphenyl hydrazone - 2,4-DNP 2,4-dinitrophenol     

Tuber-specific expression of a yeast invertase and a bacterial glucokinase in potato leads to an activation of sucrose phosphate synthase and the creation of a sucrose futile cycle

Fluxes were investigated in growing tubers from wild-type potato (Solanum tuberosum L. cv. Desiree) and from transformants expressing a yeast invertase in the cytosol under the control of the tuber-specific patatin promoter either alone (EC 3.2.1.26; U-IN2-30) or in combination with a Zymomonas mobilis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [¹⁴C]sucrose, [¹⁴C]glucose or [¹⁴C]fructose to tuber discs for a 90-min pulse and subsequent chase incubations of 4 and 12 h, and by supplying [¹⁴C]fructose for 2 h and 4 h to intact tubers attached to the mother plant. Contrary to the expectation that this novel route for sucrose degradation would promote starch synthesis, the starch content decreased in the transgenic lines. Labelling kinetics did not reveal whether this was due to changes in the fluxes into or out of starch. However, they demonstrated that glycolysis is enhanced in the transgenic lines in comparison to the wild type. There was also a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated that sucrose phosphate synthase (SPS; EC 2.4.1.14) was responsible for the enhanced recycling of label into sucrose. In agreement, there was a 4-fold and 6-fold increase in the activation status of SPS in U-IN2-30 and GK3-38, respectively, and experiments with protein phosphatase inhibitors indicated that this activation involves enhanced dephosphorylation of SPS. It is proposed that this activation of SPS is promoted by the elevated glucose 6-phosphate levels in the transgenic tubers. These results indicate the pitfalls of metabolic engineering without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation. Received: 21 July 1998 / Accepted: 5 December 1998     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

Tissue- and cell-specific expression of the two sucrose synthase isoenzymes in developing maize kernels

Summary The localization of the two known sucrose synthase isoenzymes of Zea mays L., sucrose synthase 1 and sucrose synthase 2, was studied during kernel development by indirect immunohistochemistry. These enzymes are encoded by the Sh and Sus genes, respectively. Since the antiserum used cross-reacts with both enzymes, tissue sections of Sh and sh kernels were compared. In the latter tissue no sucrose synthase 1 is expressed and thus the signal obtained was ascribed to sucrose synthase 2. We found that the isoenzymes are differentially expressed. While sucrose synthase 1 is expressed only in the endosperm, sucrose synthase 2 is found in almost all tissues of the kernel with cxpression levels specific for cell type and developmental stage. Sucrose synthase 2 is expressed strongly in the aleurone and subaleurone cell layers, where the signal detected is as strong as or even stronger than the sucrose synthase 1 signal in the inner endosperm. The distribution of the enzymes changes characteristically during development.     

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.     

Isoforms of trehalase and invertase of Fusarium oxysporum

Enzymatic assays and native PAGE were used to study trehalase and invertase activities, depending on culture age and different sugar conditions, in cell-free extracts, culture filtrates and ribosomal wash of Fusarium oxysporum. The activity of invertase preceded that of trehalase; in the exponential phase of growth, mainly invertase activity was produced, whereas trehalase activity was high in the stationary phase. In this last phase of growth, the activity of intracellular trehalase was repressed by monosaccharides, whereas disaccharides, especially lactose and starch, enhanced the activity of intracellular and extracellular trehalase. However, invertase activity was not repressed under these conditions and had the maximal activity in the presence of saccharose. Intracellular trehalase appeared in a single, high-molecular weight (120 kDa) form, whereas the extracellular enzyme appeared in a single, low-molecular weight (60 kDa) form. The activity pattern of invertase isoforms indicated the occurrence of three forms of intracellular enzyme with the main activity band at 120 kDa and two isoforms of extracellular enzyme. In the ribosomal wash, high-molecular weight isoforms of both trehalase and invertase were identified. A possible role of trehalase and invertase in carbohydrate metabolism of fungal pathogens is also discussed.     

Sucrose is metabolised by sucrose synthase and glycolysis within the phloem complex of Ricinus communis L. seedlings

Metabolites and enzyme activities were measured in the phloem sap exuding from a cut hypocotyl of germinating castor-bean (Ricinus communis L.) seedlings. The sap contained considerable quantities of adenine nucleotides, uridine nucleotides, uridine diphosphoglucose (UDPGlc), all the major phosphorylated metabolites required for glycolysis, fructose-2,6-bisphosphate and pyrophosphate. Supplying 200 mM glucose instead of sucrose to the cotyledons resulted in high concentrations of glucose in the sap, but did not modify the metabolite levels. In contrast, when 200 mM fructose was supplied we found only a low level of fructose but a raised sucrose concentration in the sap, which was accompanied by a three-to fourfold decrease of UDPGlc, and an increase of pyrophosphate, UDP and UTP. The measured levels of metabolites are used to estimate the molar mass action ratios and in-vivo free-energy change associated with the various reactions of sucrose breakdown and glycolysis in the phloem. It is concluded that the reactions catalysed by ATP-dependent phosphofructokinase and pyruvate kinase are removed from equilibrium in the phloem, whereas the reactions catalysed by sucrose synthase, UDPGlc-pyrophosphorylase, phosphoglucose mutase, phosphoglucose isomerase, aldolase, triose-phosphate isomerase, phosphoglycerate mutase and enolase are close to equilibrium within the conducting elements of the phloem. Since the exuded sap contained negligible or undetectable activities of the enzymes, it is concluded, that the responsible proteins are bound, or sequesterd behind plasmodesmata, possibly in the companion cells. It is argued that sucrose mobilisation via a reversible reaction catalysed by sucrose synthase is particularily well suited to allow the rate of sucrose breakdown in the phloem to respond to changes in the metabolic requirement for ATP, and for UDPGlc during callose production. It is also calculated that the transport of nucleotides in the phloem sap implies that there must be a very considerable uptake or de-novo biosynthesis of these cofactors in the phloem.     

The impact of reduced vacuolar invertase activity on the photosynthetic and carbohydrate metabolism of tomato

The impact of reduced vacuolar invertase activity on photosynthetic and carbohydrate metabolism was examined in tomato (Solanum lycopersicon L.). The introduction of a co-suppression construct (derived from tomato vacuolar invertase cDNA) produced plants containing a range of vacuolar invertase activities. In the leaves of most transgenic plants from line INV-B, vacuolar invertase activity was below the level of detection, whereas leaves from line INV-A and untransformed wild-type plants showed considerable variation. Apoplasmic invertase activity was not affected by the co-suppression construct. It has been suggested that, in leaves, vacuolar invertase activity regulates sucrose content and its availability for export, such that in plants with high vacuolar invertase activity a futile cycle of sucrose synthesis and degradation takes place. In INV-B plants with no detectable leaf vacuolar invertase activity, sucrose accumulated to much higher levels than in wild-type plants, and hexoses were barely detectable. There was a clear threshold relationship between invertase activity and sucrose content, and a linear relationship with hexose content. From these data the following conclusions can be drawn. (i) In INV-B plants sucrose enters the vacuole where it accumulates as hydrolysis cannot take place. (ii) There was not an excess of vacuolar invertase activity in the vacuole; the rate of sucrose hydrolysis depended upon the concentration of the enzyme. (iii) The rate of import of sucrose into the vacuole is also important in determining the rate of sucrose hydrolysis. The starch content of leaves was not significantly different in any of the plants examined. In tomato plants grown at high irradiance there was no impact of vacuolar invertase activity on the rate of photosynthesis or growth. The impact of the cosuppression construct on root vacuolar invertase activity and carbohydrate metabolism was less marked.Abbreviations CaMV Cauliflower Mosaic Virus - WT wild type     

Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase

The aim of this work was to identify which aspects of photosynthetic metabolism respond most sensitively to leaf water deficit. Spinach (Spinacia oleracea L.) leaf discs were floated on sorbitol concentrations of increasing molarity and changes of the protoplast volume were estimated using [¹⁴C]sorbitol and ³H₂O penetration. Detached leaves were also wilted until 10% of their fresh weight was lost. Photosynthesis was studied at very high external CO₂ concentrations, to eliminate the effect of closing stomata. There was no large inhibition of CO₂ fixation after wilting leaves, or until the external water deficit was greater than-1.2 MPa. However, partitioning changed markedly at these moderate water deficits: more sucrose and less starch was made. When an inhibition of CO₂-saturated photosynthesis did appear at a water deficit of-2.0 MPa and above, measurements of chlorophyll-fluorescence quenching and metabolite levels showed the thylakoid reactions were not especially susceptible to short-term water stress. The inhibition was accompanied by a small increase of the triose phosphate: ribulose-1,5-bisphosphate ratio, showing regeneration of ribulose-1,5-bisphosphate was affected. However, there was also a general increase of the estimated concentrations of most metabolites, indicating that there is no specific site for the inhibition of photosynthesis. Increasing water deficit led to a large increase of fructose-2,6-bisphosphate. This is explained in terms of a simultaneous increase of fructose-6-phosphate and inorganic phosphate as the cell shrinks. The high fructose-2,6-bisphosphate led to the accumulation of triose phosphates, and the potential significance of this for protection against photoinhibition is discussed. There was an increase in the extractable activity of sucrose-phosphate synthase. This was only detected when the enzyme was assayed in conditions which distinguish between different kinetic forms which have previously been identified in spinach leaves. It is proposed that activation of sucrose-phosphate synthase is one of the first sites at which spinach leaves respond to a rising water deficit. This could be of importance for osmoregulation.Abbreviations Chl chlorophyll - Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA glycerate-3-phosphate - Pi inorgamic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - triose-P sum of glyceraldehyde-3-phosphate and dehydroxyacetone phosphate - UDPGlc uridine diphosphoglucose     

Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers

Cold storage of potato (Solanum tuberosum L.) tubers is known to cause accumulation of reducing sugars. Hexose accumulation has been shown to be cultivar-dependent and proposed to be the result of sucrose hydrolysis via invertase. To study whether hexose accumulation is indeed related to the amount of invertase activities, two different approaches were used: (i) neutral and acidic invertase activities as well as soluble sugars were measured in cold-stored tubers of 24 potato cultivars differing in the cold-induced accumulation of reducing sugars and (ii) antisense potato plants with reduced soluble acid invertase activities were created and the soluble sugar accumulation in cold-stored tubers was studied. The cold-induced hexose accumulation in tubers from the different potato cultivars varied strongly (up to eightfold). Large differences were also detected with respect to soluble acid (50-fold) and neutral (5-fold) invertase activities among the different cultivars. Although there was almost no correlation between the total amount of invertase activity and the accumulation of reducing sugars there was a striking correlation between the hexose/sucrose ratio and the extractable soluble invertase activitiy. To exclude the possibility that other cultivar-specific features could account for the obtained results, the antisense approach was used to decrease the amount of soluble acid invertase activity in a uniform genetic background. To this end the cDNA of a cold-inducible soluble acid invertase (EMBL nucleicacid database accession no. X70368) was cloned from the cultivar Desirée, and transgenic potato plants were created expressing this cDNA in the antisense orientation under control of the constitutive 35S cauliflower mosaic virus promotor. Analysis of the harvested and cold-stored tubers showed that inhibition of the soluble acid invertase activity leads to a decreased hexose and an increased sucrose content compared with controls. As was already found for the different potato cultivars the hexose/sucrose ratio decreased with decreasing invertase activities but the total amount of soluble sugars did not significantly change. From these data we conclude that invertases do not control the total amount of soluble sugars in coldstored potato tubers but are involved in the regulation of the ratio of hexose to sucrose.The authors are grateful to Heike Deppner and Christiane Prüßner for tuber harvest and technical assistance during the further analysis. We thank Andrea Knospe for taking care of tissue culture, Birgit Schäfer for patient photographic work, Hellmuth Fromme and the greenhouse personnel for attending plant growth and development and Astrid Basner for elucidating the sequence of clone INV-19. The work was supported by the Bundesministerium für Forschung und Technologie (BMFT).     

Relationship between sucrose accumulation and activities of sucrose-phosphatase, sucrose synthase, neutral invertase and soluble acid invertase in micropropagated sugarcane plants

The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) regulates sucrose activity in sugarcane were studied. Micropropagated sugarcane plants were obtained from callus cultures of four Mexican commercially available sugarcane varieties characterized by differences in sugar production, and activities of SPS, SUSY, NI, SAI and concentrations of sucrose were monitored in the sugarcane stem. The results indicated that sucrose accumulation was positively and significantly related to an increase in activity of SPS and SUSY and negatively to a reduction in activity of SAI and NI (P<0.05). SPS explained most of the variations found for sucrose accumulation and least for NI. The relationship between activity of SPS, SUSY, NI and SAI in sugarcane stem was similar in each variety.     

Comparative structure of ovules and seeds inRafflesiaceae

The genera of theRafflesiaceae show a marked diversity in the structure of their ovules and seeds. Evolutionary trends are recognizable in ovule orientation and number of integuments. A change from anatropous ovules inApodantheae andMitrastemoideae towards incomplete anatropy inRafflesieae and orthotropy inCytineae occurs, next to a change from bitegmic ovules inApodantheae towards unitegmy with rudimentary outer integuments inRafflesieae andCytineae and full unitegmy inMitrastemoideae.—The differences in ovule structure are clearly reflected in the seeds. The seeds are essentially exotegmic, have very small embryos and an oily endosperm.—Seed structure strongly confirms the existing subfamilial classification and supports additional arguments for the generic status ofApodanthes. It does not support a separate status of the genusBerlinianche. InRafflesiaceae, seed micromorphology is only of limited use at the species level. As far as known seed dispersal is endo- or exozoochorous in all genera.