Invertase activity, grape berry development and cell compartmentation

The effect of gibberellic acid on grape (Vitis vinifera L., ev. Sultanina) growth, β-fructofuranosidase (EC 3.2.1.26) activity and carbohydrate levels was investigated throughout berry development and ripening. Although the fruits responded to hormone application with the expected increase in size, growth was not correlated with enzymic activity and hexose accumulation. This suggests that there is no direct regulatory relationship between invertase and the rate of assimilate import. However, fructose:glucose ratios changed from 0.1 in green berries to 1.0 in mature samples. The latter situation can be reconciled with the 1:1 stoichiometry of sucrolysis by invertase. It is suggested that this is attributable to a spatial separation of substrate and enzyme in green tissue. Compartmentation studies indicate that mesocarp cell integrity gradually deteriorates during ripening, which allows invertase to leak out of the vacuole into the surrounding tissue. In fact, the protein fraction retrieved from a buffered medium after incubation of ripening berry slices contained a soluble invertase of presumably vacuolar origin with an acid pH-activity profile and a pI of about 4.     

Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.     

Differential sugar uptake by cell suspension cultures of <Emphasis Type="Italic">Rudgea jasminoides</Emphasis>, a tropical woody Rubiaceae

The primary utilization of carbohydrates by cell suspension cultures of Rudgea jasminoides, a native woody Rubiaceae from tropical forests, was investigated. Sucrose, glucose + fructose, glucose, or fructose were supplied as carbon sources. The growth curves of R. jasminoides cultured in glucose + fructose, glucose, or fructose showed similar patterns to that observed when sucrose was supplied to the cells, except that an increase in dry mass was observed at the beginning of the stationary growth phase in the media containing only one monosaccharide. The increase in hexose levels in the media during the early stages of the cultures indicated extracellular hydrolysis of sucrose, which was further supported by the increase in the activity of acid invertase bound to the cell wall. Glucose was preferentially taken up, whereas uptake of fructose was delayed until glucose was nearly depleted from the medium. Measurements of intracellular sucrose content and cytoplasmatic and vacuolar invertases indicate that the enzymatic activity seems to be correlated with a decrease in the hexose flux into the cells of R. jasminoides. Our results indicate that the behavior of cell suspension cultures of R. jasminoides regarding sugar utilization seems to be similar to other dicotyledonous undifferentiated cell suspension cultures.     

Effect of glycine betaine on the sucrose catabolism of an l-lysine producing mutant of Brevibacterium lactofermentum

Summary Effect of exogenous betaine on the growth of an l-lysine-producing mutant of Brevibacterium lactofermentum was examined in a medium containing different carbon sources such as glucose, fructose, or sucrose. The growth rate decreased significantly with a rise in temperature when sucrose was the carbon source. Both the specific sucrose consumption rate and the invertase activity of the mutant decreased with the culture period when the cultivation temperature was 35°C. The addition of betaine restored both growth and invertase activity on medium containing sucrose as the carbon source at 35°C. Betaine protected the invertase activity against the inactivating effects of high temperature in vitro. Furthermore, the addition of exogenous invertase into production medium at 35°C restored the growth rate to that at 32°C. These results indicated that growth decreased on medium containing sucrose at 35°C due to a decrease in invertase activity, and that addition of betaine was an effective way to enhance growth on this medium at a higher temperature. Offprint requests to: Y. Kawahara     

Possible role of soluble invertase in the gibberellic acid berry-sizing effect in Sultana grape

It is well known that post-bloom applications ofgibberellic acid (GA₃) increase seedless grapeberry size by enhancing cell division, or cellenlargement, or both. As a consequence, total waterand sugar per berry are increased. Soluble invertaseis considered to be one of the key enzymes in theaccumulation of sugar in grape berries. To study apossible role of invertase in the GA₃berry-sizing effect, different rates of post-bloomGA₃ were applied to seedless grape cv. Sultanaand hexose concentration and invertase activity weremeasured. GA₃ stimulated both parameters as earlyas 24 and 32 h after applications, respectively.Moreover, the increment in sugar content and enzymeactivity remained throughout the growing of the berries period and, at ripening, increases in hexosescontent (102%) and invertase activity (60%) weredetected when GA₃ was applied at a rate of 45 ppm.At the same GA₃ rate the pericarp cellsdoubled in size. Furthermore, positive correlationswere found between berry-size, invertase activity andhexose content, suggesting that GA₃ stimulationof invertase could be one of the factors involved in theberry sizing-effect of GA₃.     

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).     

Stimulation of acid invertase activity by indol-3yl-acetic acid in tissues undergoing cell expansion

The soluble acid invertase activity of young, excised P. vulgaris internodal segments fell when they were incubated in water, and their elongation ceased within 6–7 h. IAA (10 M) promoted segment elongation and stimulated an increase in the specific activity of acid invertase to a level greater than that originally present. The rate of segment elongation in the presence of IAA was closely and positively correlated with the specific activity of the enzyme. Optimum concentration of IAA for both elongation and stimulation of invertase activity was 10 M. Concurrent protein synthesis was necessary for these responses to IAA. Segments cut from mature, fully-elongated internodes did not responsd to IAA.Inclusion of Ca²⁺, vanadate or mannitol in the incubation medium abolished IAA-induced segment elongation but did not inhibit the stimulation of acid invertase activity by IAA. Auxin-induced elongation and acid invertase activity were both substantially increased in the presence of up to 25 mM D-glucose or up to 50 mM sucrose. Inclusion of either sugar in the medium considerably increased tissue hexose concentrations. Under some circumstances cell growth and invertase synthesis may compete for available hexose substrate.It is concluded that IAA-induced promotion of acid invertase in P. vulgaris internodal segments is not simply an indirect consequence of removal of end-product (hexose) during IAA-induced cell growth and that a more direct action of IAA on enzyme turnover is involved.     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

Regulation of photosynthesis during Arabidopsis leaf development in continuous light

Previous investigations in our laboratory have shown that leaf developmental programming in tobacco is regulated by source strength. One hypothesis to explain how source strength is perceived is that hexokinase acts as a sensor of carbohydrate flux to regulate the expression of photosynthetic genes, possibly as a result of sucrose cycling through acid invertase and hexokinase. We have turned to Arabidopsis as a model system to study leaf development and have examined various photosynthetic parameters during the ontogeny of a single leaf on the Arabidopsis rosette grown in continuous light. We found that photosynthetic rates, photosynthetic gene expression, pigment contents and total protein amounts attain peak levels early in the expansion phase of development, then decline progressively as development proceeds. In contrast, the flux of ¹⁴CO₂ into hexoses increases modestly until full expansion is attained, then falls in the fully expanded leaf. Partitioning of carbon into hexoses versus sucrose increases until full expansion is attained, then falls. The in vitro activities of hexokinase, vacuolar acid invertase, and cell wall acid invertase do not change until the late stages of senescence, when they increase markedly. At this time there are also dramatic increases in hexose pool sizes and in senescence-associated gene (SAG) expression. Taken together, our results suggest that invertase and hexokinase activities do not control the partitioning of label into hexoses during development. We conclude that our data are not readily compatible with a simple model of leaf development, whereby alterations in photosynthetic rates are mediated directly by hexose flux or by hexose pool sizes. Yet, these factors might contribute to the control of gene expression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of carbon sources and auxins on in vitro propagation of banana

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm⁻³. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm⁻³) than at other concentrations of IBA or NAA alone or their combinations.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Effect of controlled carbon dioxide on in vitro shoot multiplication in Feronia limonia (L.) Swingle

The effect of controlled carbon dioxide environment on in vitro shoot growth and multiplication in Feronia limonia (a tropical fruit plant, Family- Rutaceae) was studied. Carbon dioxide available in the ambient air of the growth room was insufficient for in vitro growth of the shoots alone. Also, the presence of sucrose only as the C-source in the medium (without CO₂), was found to be inadequate for sustainable growth and multiplication of shoots. The carbon dioxide enrichment promoted shoot multiplication and overall growth. The promotory effect of CO₂ was independent of the presence of sucrose in the medium. In the presence of both CO₂ and sucrose, an additive effect was observed producing maximum shoot growth. In the absence of sucrose a higher concentration of CO₂ (10.0)g m⁻³ was required to achieve photoautotrophic shoot multiplication comparable to ambient air controls. Highest leaf area per shoot cluster promoting shoot growth and multiplication was recorded under this treatment. Shoots growing on sucrose containing medium under controlled CO₂ environment of 0.6 g m⁻³ concentration evoked better response than ambient air controls (shoots growing on sucrose containing medium) in growth room. This treatment produced the overall best response. The present study highlighted the possibility of photoautotrophic multiplication which might prove useful for successful hardening and acclimatization in tissue culture plants.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

Opposite effects of exogenous sucrose on growth, photosynthesis and carbon metabolism of in vitro plantlets of tomato (L. esculentum Mill.) grown under two levels of irradiances and CO2 concentration

The long-term effects of exogenous sucrose (3 percnt;) on growth, photosynthesis and carbon metabolism ofin vitro tomato plantlets were investigated under two sets of growth conditions that respectively favor source- or sink-limitations of photosynthesis: 1) low photosynthetic photon flux (PPF) (50 μmol m⁻² · s⁻¹) and low CO₂ concentration (400 μmol mol⁻¹) and 2) high PPF (500 μmol m⁻² · s⁻¹ and high CO₂ concentration (4000 μmol mol⁻¹). The supply of sucrose under source-limitation conditions increased the growth, the maximal photosynthetic rate, the chl content, the maximal quantum yield of Photosystem II estimated by the Fv/Fm chl fluorescence ratio as well as the soluble sugars (hexoses, sucrose) and starch contents in roots, young and mature leaves when compared to those of photo-autotrophic plantlets. Also, sucrose feeding under these conditions strongly increased the activity of sucrose synthase (SS) (EC 2.4.1.13) in roots and young leaves whereas the activities of sucrose phosphate synthase (SPS) (EC 2.4.1.14), acid invertase (INV) (EC 3.2.1.26) and ADP-glucose pyrophosphorylase (ADPGppase) (EC 2.7.7.27) were highly stimulated in roots and mature leaves. Contrary to these observations, the supply of sucrose to plantlets developed under high PPF and CO₂ concentration decreased growth and led to a somewhat lower maximal photosynthetic rate relative to photo-autotrophic plantlets. These negative responses to exogenous sucrose were accompanied by stronger accumulations of hexose and starch, larger stimulation of INV in mature leaves developed under conditions of sink limitation than those from source limitation conditions. Moreover, under high PPF and high CO₂ concentration, exogenous sucrose led to a marked repression of the SPS activity and caused much lower stimulations of ADPGppase in mature leaves than those observed at low PPF and low CO₂ concentration. We therefore conclude that under our experimental conditions, the interactive effects of exogenous sucrose and environmental conditions on growth and photosynthesis could be rationalized by the source-sink equilibrium of thein vitro tomato plantlets.     

Light regulation of sink metabolism in tomato fruit : I. Growth and sugar accumulation

Light/dark effects on growth and sugar accumulation in tomato (Lycopersicon esculentum) fruit during early development were studied on intact plants (in vivo) and in tissue culture (in vitro). Through the use of an in vitro culture of tomato fruit, it was possible to investigate the direct effects of light on sink metabolism by eliminating the source tissue. Similar growth patterns were found in vivo and in vitro. Fruit growth in different sugars indicated that sucrose was the best source of carbon for in vitro fruit growth. Fruit growth increased as sucrose concentration increased up to 8%. Darkening the fruit decreased fruit dry weight about 40% in vivo and in vitro. The differences in the CO₂ exchange rate between light and dark grown fruit indicated that light stimulation of fruit growth was due to mechanisms other than photosynthesis. Supporting this conclusion was the fact that light intensities ranging from 40 to 160 micromoles per square meter per second had no significant influence on fruit growth, and light did not increase growth of fruit cultured with glucose or fructose as a carbon source. However, light stimulated fruit growth significantly when sucrose was used as the carbon source. Light-grown fruit took up 30% more sucrose from the same source and accumulated almost twice as much hexose and starch as dark-grown fruit. A possible expansion of an additional sink for carbon by light stimulation of starch synthesis during early development will be discussed.     

Invertase Activity, Carbohydrate Metabolism and Cell Expansion in the Stem of Phaseolus vulgaris L.

In the stem of Phaseolus vulgaris L. the specific activity ofacid invertase was highest in the most rapidly elongating internode.Activity of the enzyme was very low in internodes which hadcompleted their elongation, in young internodes before the onsetof rapid elongation, and in the apical bud. From shortly afterits emergence from the apical bud the elongation of internode3 was attributable mainly to cell expansion. Total and specificactivities of acid invertase in this internode rose to a maximumat the time of most rapid elongation and then declined. Transferof plants to complete darkness, or treatment of plants withgibberellic acid (GA₃), increased the rate of internode elongationand final internode length by stimulating cell expansion. Bothtreatments rapidly increased the total and specific activitiesof acid invertase in the responding internodes; peak activitiesof the enzyme occurred at the time of most rapid cell expansion. In light-grown plants, including those treated with GA₃, rapidcell and internode elongation and high specific activities ofacid invertase were associated with high concentrations of hexosesugar and low concentrations of sucrose. As cell growth ratesand invertase activities declined, the concentration of hexosefell and that of sucrose rose. In plants transferred to darkness,stimulated cell elongation was accompanied by a rapid decreasein hexose concentration and the disappearance of sucrose, indicatingrapid utilization of hexose. No sucrose was detected in theapical tissues of light-grown plants. The results are discussed in relation to the role of acid invertasein the provision of carbon substrates for cell growth. Key words: Cell expansion, Acid invertase, Hexose, Sucrose, Phaseolus