Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development

Levels of activity of the sucrose catabolizing enzymes, acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), were measured during development of new leaves of Citrus sinensis (L.) Osbeck cv. Shamouti. Soluble acid invertase showed a peak activity of 32 nkat (g fresh weight)⁻¹ at ca 60% of full leaf expansion and rapidly declined toward and after full expansion. There was no concomitant increase in an insoluble form of the enzyme. Sucrose synthase activity, measured in the synthesis direction, declined from 33% of full leaf expansion [10 nkat (g fresh weight)⁻¹] 10, and following, full expansion. Highest sucrose synthase activity, measured in the cleavage direction, was 6 nkat (g fresh weight)⁻¹ and showed little change during development. Acid invertase has a K_m of 5 m M for sucrose, while sucrose synthase had a K_m of 118 m M for sucrose. Changes in acid invertase activity correlated with changes in the reducing sugar:sucrose ratio. These results suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding Citrus leaf. Changes in leaf expansion rate and invertase activity did not correlate positively with changes in endogenous free IAA level, as determined by enzyme linked immunoassay.     

Uptake of 13C-glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose-, pentose- and hexose-phosphates

After a lag phase of 2 days, batch-grown cells of carrot ( Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short-term feeding ¹³C-glucose in this period resulted in only minor labelling of sucrose or fructose. CO₂ production from [1-¹³C]- and [6-¹³C]-glucose revealed, that at least 40% of the added glucose passed through the oxidative pentose phosphate pathway (OPPP), up to 40% through glycolysis leaving only minor ¹³C-glucose for incorporation in various cell components in the exponential growth phase. After about 11 days of culture, the medium sugars were exhausted, cells entered the stationary growth phase and consumed stored sugar. Both neutral and acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) increased 50% from day 0 to days 11–13; thereafter their levels decreased again. Labelling with ¹³C-glucose resulted in the accumulation of labelled sucrose and fructose during the stationary growth phase. Sucrose labelling was transient, i.e. after 6 h its level started to decrease again. Labelled fructose, however, evolved slower and increased even after 8 h. In sucrose and fructose up to 20% of the ¹³C-label was exchanged from C-1 to C-6 carbons, indicating intensive cycling of at least 40% of the carbon between hexoses and triose phosphates. In the stationary phase only 10% of the labelled glucose passed through the OPPP and about 30% passed through the respiratory pathway; the remaining 60% was incorporated in cell constituents and sugars. Comparing the various cycles revealed that the regulation of the OPPP operated relatively independently from the cytosolic cycling of hexose phosphates through sucrose and from the cycling between hexose phosphates and triose phosphates.     

Sugar uptake into strawberry fruit is stimulated by abscisic acid and indoleacetic acid

Changes in sugar uptake into strawberry fruits with maturation and the hormonal effect on uptake mechanisms, though important to fruit development, are not known. Therefore, the kinetics of sugar uptake into strawberry ( Fragaria x ananassa Duch cv. Nyoho) fruit tissue and the effects of abscisic acid (ABA) and indoleacetic acid (LAA) on the mechanism of uptake were investigated at 25 and 35 days after pollination (DAP). Uptake of ¹⁴C-sugar was measured over the concentration range of 2 to 30 m M. Uptake kinetics showed a biphasic response to increasing external concentration of ¹⁴C-sugars, and indicated the presence of P -chlorormercuribenzenesulfonic acid (PCMBS)-sensitive and PCMBS-insensitive uptake. The K_m value for each sugar was in the range of 10 to 20 m M. Stage of development had no effect on K_m. but V_max for glucose decreased with maturation. Further, sucrose was not taken up through a PC-MBS-sensitive transport at 35 DAP. ABA, especially 10 μ M , at 25 DAP stimulated uptake of all sugars, mostly through enhanced PCMBS-insensitive uptake but not PC-MBS-sensitive uptake. In contrast to ABA, stimulation of sugar uptake by IAA was most effective at 1 μ M . The PCMBS-insensitive uptake of each sugar was also stimulated by IAA. Further, the PCMBS-sensitive uptake of glucose was enhanced. The developmental change of PCMBS-sensitive sugar uptake and the effect of ABA and IAA on uptake mechanism in this study are considered to be important in influencing the development and enlargement of fruits.     

Accumulation and conversion of sugars by developing wheat grains. 3. Non-diffusional uptake of sucrose, the substrate preferred by endosperm slices

Abstract. Starch synthesis by developing wheat endosperm slices incubated in liquid media was more rapid, at optimum concentration, from sucrose as external substrate than from glucose and/or fructose. Fructose inhibited conversion of sucrose or glucose. The results are consistent with the hypothesis that sucrose is not hydrolysed in the apoplast before uptake.
Besides a diffusional influx and efflux of labelled sucrose there was a non-diffusional influx; it was inhibited by dinitrophenol, potassium arsenate, potassium iodide, and parachloromercuribenzene sulphonate (PCMBS). PCMBS inhibited both uptake and conversion of label from 150 molm⁻³¹⁴C-sucrose by 75%. Uptake and conversion of sucrose were stimulated by lowering pH and by fusicoccin, a promoter of proton extrusion.
Extracellular solutes like raffinosc and polyethylene glycol stimulated net uptake of label from ¹⁴C-sucrose — the larger molecule being more effective — this being due to a non-specific inhibition of diffusional efflux. At too high an osmotic concentration such solutes reduced net uptake; the larger the molecule the lower this transitional concentration.
In conclusion, wheat endosperm is better equipped to convert apoplastic sucrose rather than the hydrolysis products to starch; active loading of sucrose possibly involves proton co-transport; and large molecules in the extracellular solution reduce the diffusional elllux of loaded substrate.     

The development of Pisum sativum explant systems for studies concerning source-sink activities

Using excised fruit/shoot systems of pea ( Pisum sativum L.), the effect of the 10-day-old (post anthesis) fruit (sink) on the translocation of ¹⁴C-sucrose applied to the stipule (source) was investigated. We also examined the influence of various growth regulators on the source-sink relationships using this system. Indole-3-yl-acetic acid (100 or 200 ppm), gibberellic acid (25-100 ppm) increased ¹⁴C-sucrose translocation into the growing fruit by about 13% and 22–29%, respectively. 2-chloroethyl phosphonic acid, 6-benzyladenine and 2-methyl-4-chlorophenoxyacetic acid also increased ¹⁴C-sucrose translocation into the fruit by about 11–26%, 11–23% and 24–37%, respectively. Elevated fruit ¹⁴C-sucrose levels found in other treatments include, methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (300 or 1000 ppm), 2,3,5-triiodobenzoic acid (100 and 300 ppm) and butanedioic acid mono-(2,2-dimethylhydrazide) (30-300 ppm) by about 34%, 39–52% and 7–13%, respectively.     

Evidence for two pathways of phloem loading

The minor veins of small leaf discs, punched out of mature leaves and incubated in ¹⁴C-sucrose solution, appear labeled in macro- and microautoradiographs. Discs with a labeled vein pattern and with labeled sieve tubes in microautoradiographs were found in Beta vulgaris, Vicia faba, Gomphrena globosa and Antirrhinum majus . However, in several other plant species, minor veins appeared unlabeled in macroautoradiographs when the discs were incubated in ¹⁴C-sucrose. Mesophyll cells ( Acer pseudoplatanus, Juglans regia, Fagia, sylvatica, Syringa vulgaris, Laburnum anagyroides ), bundle-sheath cells of major veins ( Salix viminalis, Robinia pseudoacacia, Commelina communis ) or epidermal layers ( Ginkgo biloba, Chlorophytum comosum ) appeared labeled. Lack of radioactivity in sieve tubes of this latter group was confirmed by microauto-radiography. Using ¹⁴C-glucose instead of ¹⁴C-sucrose, leaf discs of Beta vulgaris showed no labeled vein pattern and in microautoradiographs the sieve tubes appeared unlabeled. In view of the by-pass phloem loading, this study provides evidence for two pathways of phloem loading.     

宁夏枸杞果实糖积累和蔗糖代谢相关酶活性的关系

通过对枸杞果实发育过程中果实生长模式、蔗糖、果糖、葡萄糖和淀粉含量及糖代谢相关酶活性的测定,研究了宁夏枸杞果实生长发育过程中糖的代谢积累与相关酶活性的关系.结果表明:(1)宁夏枸杞果实发育呈双S"曲线,果实主要以积累己糖为主.(2)蔗糖磷酸合成酶(SPS)活性在果实发育初期处于下降的趋势,在花后19d开始上升,果实转色后又逐渐下降;蔗糖合成酶(SS)活性总体表现为SS分解方向的活性大于SS合成方向的活性,说明枸杞果实发育过程中,SS的活性主要以分解方向的为主;酸性转化酶(AI)和中性转化酶(NI)的活性随果实发育呈上升趋势,但在果实成熟后期有所下降,且AI和NI活性高于合成酶类的活性,较高的转化酶类活性促进了果实内部己糖的积累.(3)在枸杞果实生长发育中,葡萄糖和果糖含量与AI和NI均呈极显著正相关,而与其它酶不具有相关性.说明AI和NI在宁夏枸杞果实的糖代谢中起着主要的调控作用.     

Activity of sucrose hydrolysing enzymes and sugar accumulation during tomato fruit development

Relationships between the assimilate import rate and the activity of acid invertase and/or sucrose synthase have been investigated in the pericarp, locule and placenta of tomato fruit during development to establish the possible role of sucrose cleavage as the control step for the import of sucrose into these sink tissues. The rate of sucrose cleavage was estimated from the activities of these two enzymes as well as the ratio of hexoses to sucrose (i.e. the sucrose degradation index, SDI) in the tissues of the fruit, based on the assumption that the accumulation of hexoses is the consequence of imported sucrose being degraded by either or both of these two enzymes. The results showed that the change of sucrose synthase activity during fruit development was positively related to both the rate of dry matter accumulation in the fruit tissue and SDI. Although the role of acid invertase in regulating the rate of import during development remains uncertain, the actions of sucrose synthase on sucrose cleavage may regulate the import and compartmentation of sucrose in the early stage of tomato fruit development.     

枇杷果实发育过程中糖积累及相关酶活性变化研究

以'青种'、'霸红'和'鸡蛋白'3个枇杷品种为材料,测定不同果实发育时期果实中蔗糖、葡萄糖和果糖含量以及蔗糖代谢相关酶即酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖积累与酶活性的关系进行了分析.结果表明:在果实膨大期(5月3日)之前,3种枇杷果实的蔗糖、葡萄糖和果糖积累缓慢,之后则迅速积累,存在着明显的转折点;果实成熟(5月23日)之后糖分积累速度趋于平稳.3种枇杷果实在发育过程中转化酶、蔗糖合成酶和蔗糖磷酸合成酶的活性变化与3种糖积累的动态变化趋势相一致.NI和AI活性在果实膨大期之前都较低且没有明显的变化,之后均快速上升;SS和SPS的活性在果实膨大期之前都很低且几乎无变化,随后'鸡蛋白'的活性迅速上升至果实成熟之后便缓慢上升,而'青种'和'霸红'随果实成熟度的增加而升高,但均低于'鸡蛋白'.可见,枇杷果实膨大期是糖分积累代谢活跃期,其糖积累受蔗糖代谢相关酶综合调控.     

‘台农1号’芒果果实发育过程中的糖分积累与相关酶活性研究

以‘台农1号’芒果为材料,测定了果实生长发育过程中淀粉、蔗糖、葡萄糖和果糖含量以及淀粉酶、蔗糖代谢相关酶———酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分与酶活性的关系进行了分析.结果显示,(1)台农1号芒果果实属于单S型生长曲线,发育前期主要积累淀粉、葡萄糖和果糖,果实成熟软化时,淀粉酶活性降至最低,淀粉水解,蔗糖快速积累.(2)酸性转化酶活性在果实整个发育过程中维持最高,完熟时略有降低;蔗糖磷酸合成酶在果实发育前期略有降低,完熟时升至最高;蔗糖合成酶和中性转化酶活性在整个发育期一直很低且较稳定.(3)淀粉含量与淀粉酶活性呈显著正相关,与SPS活性呈极显著负相关,蔗糖、葡萄糖含量均与SPS、SS呈显著、极显著的正相关;果糖含量与SS呈极显著的正相关.研究表明,芒果成熟时淀粉分解、酸性转化酶活性的降低,且蔗糖合成酶和蔗糖磷酸合成酶活性的增加是引起果实蔗糖积累的主要因子.     

Effects of gibberellic acid on patterns of carbohydrate distribution and acid invertase activity in Phaseolus vulgaris

The application of gibberellic acid (GA₃,10 μ M ) as a root drench to 16-day-old plants of Phaseolus vulgaris L. cv. Masterpiece stimulated growth of the stem internodes and reduced root growth. GA₃ treatment did not affect the export of ¹⁴C from a primary leaf to which [¹⁴C]-sucrose was applied, but greatly increased upward translocation to the elongation region of the stem at the expense of transport to the hypocotyl and root system. The observed changes in the patterns of growth and [¹⁴C]-labelled assimilate distribution were correlated with an increase in the specific activity of acid invertase in the elongating stem internodes and a decrease in invertase activity in the hypocotyl and root. Sucrose concentration in the elongating internodes fell substantially after treatment with GA₃ while the concentration of hexose sugars increased. We suggest that by stimulating acid invertase synthesis in the elongating internodes, GA₃ acts to establish a more favourable sucrose gradient between these sinks and source leaves. Under source-limiting conditions this, in turn, will lead to a reduced rate of assimilate translocation to competing sinks in the root system.     

Isolation and sugar uptake characteristics of protoplasts from maize endosperm suspension cultures

The isolation and sugar uptake characteristics of protoplasts from maize ( Zea mays L.) endosperm-derived suspension cultures are described. In contrast with protoplasts from intact developing endosperm, which by virtue of their large size and high starch content are too fragile for sugar uptake experiments, suspension cultures yielded protoplasts capable of withstanding the necessary handling and centrifugations. Intactness of the protoplasts was demonstrated by dye exclusion or accumulation and latency of malate dehydrogenase activity. Uptake of radioactivity from [³H]-inulin did not increase with time, but that from [¹⁴C]-sugars increased over a wide range of external concentrations. Kinetics of fructose, glucose and sucrose uptake were biphasic, and the saturable components of uptake were eliminated by p -chloromercuribenzene sulfonate (PCMBS). Rates of uptake of sucrose and 1'-fluorosucrose were similar, confirming that hydrolysis by cell wall invertase contributes to sucrose uptake by the suspension cultures. The isolation of protoplasts from this tissue source will enable experimental access to plasma membrane sugar carriers which may exist in the intact maize endosperm.     

Growth, sucrose synthase, and invertase activities of developing Phaseolus vulgaris L. fruits

Activities of the sucrose-cleaving enzymes, acid and neutral invertase and sucrose synthase, were measured in pods and seeds of developing snap bean (Phaseolus vulgaris L.) fruits, and compared with ¹⁴C-import, elongation and dry weight accumulation. During the first 10 d post-anthesis, pods elongated rapidly with pod dry weight increase lagging behind by several days. The temporal patterns of acid invertase activity and import coincided closely during the first part of pod development, consonant with a central role for this enzyme in converting imported sucrose during pod elongation and early dry weight accumulation. Later, sucrose synthase became the predominant enzyme of dry weight accumulation and was possibly associated with the development of phloem in pod walls. Sucrose synthase activity in seeds showed two peaks, corresponding to two phases of rapid import and dry weight accumulation; hence, sucrose synthase was associated with seed sink growth. Acid invertase activities in seeds were low and did not show a noticeable relationship with import or growth. All neutral invertase activities, during pod and seed development, were too low for it to have a dominant role in sucrose cleavage. Changes in activities of certain sucrose-cleaving enzymes appear to be correlated with certain sink functions, including import, storage of reserves, and biosynthetic activities. The data supports the association of specific sucrose-cleaving enzymes with the specific processes that occur in the developing pods and seeds of snap bean fruits; for example, acid invertase with pod elongation and sucrose synthase with fruit dry matter accumulation.     

Conversion of sucrose to starch in the developing Pennisetum typhoides grain

Developing grains of pearl millet ( Pennisetum typhoides Burm. S & H cv. PIB 155) were sampled and analyzed for starch and its free-sugar precursors. The activities of invertase, sucrose-ADP (UDP) glucosyl transferase and of α-amylase and β-amylase in relation to the rate of starch accumulation in the developing grain were assayed. By culturing detached ears, the incorporation of ¹⁴C from free sugar precursors to starch was studied. The starch content gradually increased until grain maturity. The rate of starch accumulation was maximum around 12 days after anthesis. Around this period, the activities of sucrose-ADP(UDP) glucosyl transferase and α-amylase, β-amylase were also at a peak. Invertase activity was high during the early period of grain development but gradually declined as the grains matured. In the most actively metabolising milky grains, incorporation of ¹⁴C from [¹⁴C]-sugars to starch was maximum in the mid mid-milky grains. Addition of 20 m M K⁺ to the culture solution did not affect the incorporation of ¹⁴C from supplied sucrose to the free sugar pool and to the starch of the grain, but Mg²⁺ supply at 20 m M concentration lowered ¹⁴C incorporation from exogenous sucrose to grain free sugars, although the utilization of the latter for starch synthesis was enhanced.     

Distribution of 14C-sucrose applied to leaves at different nodes of okra (Abelmoschus esculentum) during flowering and early pod growth

The distribution pattern of ¹⁴C-sucrose from ¹⁴C-sucrose applied to vegetative okra plants and leaves 1–9 on separate plants during the green pod development stage were investigated in relation to duration and leaf position. Results indicated bi-directional transport of assimilates to both apical and basal portions of the stem. Within 48 h ¹⁴C moved to all plant parts; stem and leaves appeared to be strong sinks. In plants fed at the vegetative stage, 48 h after feeding, 66% of the fed activity was exported from the fed leaf. At the pod development stage, about 35% of the activity exported from the fed leaf was present in green pods and 65% in vegetative parts. In plants where leaf 1–9 was fed, irrespective of the position of the fed leaf, the subtending fruit was the strongest sink among the reproductive parts. Leaves and stems were the principal sinks.     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

Translocation of 14C, carbohydrate content and activity of the enzymes of sucrose metabolism in rose petals temperatures

Both export of ¹⁴C from the source leaves of roses (Rosa × hybrida cv. Golden Times) and import of ¹⁴C to the petals were reduced by plant exposure to low night temperature. However, the import was affected to a greater extent than the export. During all stages of flower bud development the concentration of reducing sugars in petals of roses grown at reduced night temperature was lower than in petals of plants grown at higher night temperature. There was no significant difference in starch content in response to the night temperature, and the content of starch decreased toward complete flower bud opening. The concentration of sucrose in flowers at the low night temperature remained low during all stages of flower bud development, while at the high night temperature the concentration of sucrose increased during flower bud development, reaching a peak at the stage when petals start to unfold. At both temperatures the concentration of sucrose declined at complete flower opening. The activity of sucrose synthase (EC 2.4.1.14) was inhibited by low temperature in young rose shoots more than in the petals, while the activity of acid invertase (EC 3.2.1.26) was affected similarly in both tissues by the temperature treatments.     

The directional control of sucrose and asparagine transport in lupin by abscisic acid

Injections of exogenous abscisic acid into the primary flowerhead of Lupinus luteus cv. Weiko III reduced the movement of ¹⁴C-sucrose into the flowerhead from the uppermost leaves. Sucrose transported from below the lateral branches subtending the flowerhead, was diverted into the lateral branches by injection of the exogenous abscisic acid into the flowerhead. ¹⁴C-sucrose was also diverted from a lateral branch injected with exogenous abscisic acid to all other parts of the plant, particularly the main stem and leaves, and the roots. Transport of ¹⁴C-asparagine administered at the cotyledonary node was directed from the flowerhead into the subtending lateral branches by injection of abscisic acid into the flowerhead. Transport of both ¹⁴C-sucrose and ¹⁴C-asparagine into the flowerhead was reduced at least three fold at physiological levels of abscisic acid. No significant correlation was found between the amount of ¹⁴C-asparagine entering a sink and the dry weight of the tissues of that sink. It is concluded that distribution of ¹⁴C-sucrose and ¹⁴C-asparagine between the flowerheads and lateral branches of L. luteus is actively and dynamically controlled and that abscisic acid levels play a significant part in that control. It is suggested that the relative levels of endogenous abscisic acid in plant organs could serve as an important factor in the directional control of assimilate transport in plants.     

网纹甜瓜发育果实糖分积累与蔗糖代谢参与酶的关系

随着网纹甜瓜果实的发育,果实中葡萄糖和果糖的含量增加,蔗糖的快速积累发生在果实发育的中后期,高蔗糖积累型果实中蔗糖积累速率明显快于低蔗糖积累型.蔗糖磷酸合成酶活性在果实发育的前期短暂下降, 而后稳步上升,在果实发育的中后期高蔗糖积累型果实中该酶的活性显著高于低蔗糖积累型果实;随着果实发育,蔗糖合成酶的分解活性降低而合成活性升高.酸性和中性转化酶在未成熟果实中活性较高,而在成熟果实中很低; 高蔗糖积累型果实中酸性转化酶活性显著低于同期低蔗糖积累型果实.合成蔗糖的酶活性小于分解蔗糖的酶活性时蔗糖几乎没有积累.根据这些结果推测,转化酶活性的下降、蔗糖磷酸合成酶活性的增加以及蔗糖合成酶分解活性的下降和合成活性的增加,是引起果实蔗糖积累的主要内在因子.     

Does light-promoted export from Commelina benghalensis leaves result from differential light-sensitivity of the cells in the mesophyll-to-sieve tube path?

In contrast to the light-promoted uptake by mesophyll cells, light slightly inhibited sucrose uptake by stripped leaf disks of Commelina benghalensis L. This phenomenon appeared to result from a light-promoted vein-associated release, as light stimulated photosynthate release from stripped disks and inhibited that from mesophyll cells. In the -presence of the resorption-blocker p -chloromercuriphenylsulfonic acid, (PCMBS) the release of preloaded [¹⁴C]-sugars (sucrose, glucose) and [¹⁴C]-amino acids (alanine, asparagine, proline, valine, α-aminoisobutyric acid) from stripped disks was doubled in the light. Illumination enhanced by 20 to 60% the release of endogenous leaf cell compounds (sucrose, H₂PO-₄, K⁺, Mg²⁺, Ca²⁺) from stripped disks in the presence of PCMBS. Light also increased the export of [¹⁴C]-assimilates from intact leaves by 20% after pulse-labelling with ¹⁴CO₂. A model for loading is proposed, based on the differential light sensitivities of the plasma membranes in the mesophyll-to-sieve tube path.