Invertase-mediated Interconversion of Sucrose and Hexoses During Their Translocation in Growing Pearl Millet Plant

Metabolism of free sugars, particularly sucrose, in various plant tissues enroute from leaf sheaths to grains in growing pearl millet was studied. With the enhancement in growth, the levels of both reducing and non-reducing sugars declined in middle and basal leaf sheaths but increased in flag leaf sheath towards plant maturity. In sheath, wall-bound invertase was more active than soluble invertases and the activities of all these enzymes rose towards maturity. Besides hexoses and sucrose, some fructose polymers were also detected in the internodes. In contrast with internodes, where the levels of total free sugars declined till around anthesis, in penultimate node their levels continuously increased, but attained peak values at 65 CAS in middle- and basal nodes. In both these tissues, arriving sucrose encounters invertases but in nodes wall-bound invertase appears to be the pivotal one. On feeding (U-¹⁴C)-sucrose to the detached ear-heads a large proportion of ¹⁴C was incorporated into hexoses alone in peduncle and rachis. PCMBS and HgCl₂ inhibited the metabolism of sucrose supplied to peduncle and rachis pointing to the involvement of invertases in sucrose cleavage in these organs. Through the regulated operation of invertase(s), the nodes seem to maintain a controlled flow of free sugars from source to sink tissue.     

Bioregulation of carbohydrate metabolism in relation to source-sink operation during grain-filling phase of growth in wheat

Mobilization of free sugars from vegetative tissues to grain and their transformation to starch in relation to activities of some relevant enzymes during growth and development were investigated in wheat (Triticum aestivum L.). Vegetative tissues, viz. flag-leaf, flag-leaf sheath, nodes and internodes contained high concentration of free sugars from 70 DAS to 18 DPA and that was in the order of accumulation--flag-leaf sheath> flag-leaf and internodes > nodes. In these tissues, major portion of 14C appeared in endogenous sucrose, irrespective of the nature of (U-14C]-sugars supplied. In photosynthetic structures above flag-leaf node, namely peduncle, rachis and bracts, the free sugar make-up was maximum at anthesis (90 DAS). Activity of soluble acid invertase (EC 3.2.1.26) was high in these tissues during early stages of grain growth but reverse was true for soluble neutral invertase (EC 3.2.1.27) activity. In apical and basal portions of grain, free sugars were more or less similarly distributed in concentration. Linear and rapid accumulation of starch in endosperm paralleled with a decline in accumulation of this polymer in pericarp-aleurone. In the latter tissue, the activities of starch hydrolyzing enzymes, i.e alpha- and beta-amylases (3.2.1.1 and 3.2.1.2) were high during initial stages of grain growth. During active grain-filling, alkaline inorganic pyrophosphatase (EC 3.6.1.1) seemed to play a vital role during starch accumulation in endosperm, whereas the involvement of 3-PGA phosphatase (EC 3.1.3.38) was almost confined to pericarp-aleurone. Impairement of ear head photosynthesis by shading depressed starch synthesis (approximately 50%) indicating, thereby, the significant role of current photosynthates during grain-filling. The results suggested that grain growth in wheat was influenced by an efficient operation of source as well as regulatory factors, including enzymes, constituting intrinsic potential of grain sink.     

Carbohydrate metabolism during fruit development in sweet pepper (Capsicum annuum) plants

Growth, accumulation of sugars and starch, and the activity of enzymes involved in sucrose mobilization were determined throughout the development of sweet pepper fruits. Fruit development was roughly divided into three phases: (1) an initial phase with high relative growth rate and hexose accumulation, (2) a phase with declining growth rate and accumulation of sucrose and starch, and (3) a ripening phase with no further fresh weight increase and with accumulation of hexoses, while sucrose and starch were degraded. Acid and neutral invertase (EC 3.2.1.26) were closely correlated to relative growth rate until ripening and inversly correlated to the accumulation of sucrose. Acid invertase specifically increased during ripening, concurrently with the accumulation of hexoses. Sucrose synthase (EC 2.4.1.13) showed little correlation to fruit development, and in periods of rapid growth the activity of sucrose synthase was low compared to the invertases. However, during late fruit growth sucose synthase was more active than the invertases. We conclude that invertase activities determine the accumulation of assimilates in the very young fruits, and a reactivation of acid invertase is responsible for the accumulation of hexoses during ripening. During late fruit growth, before ripening, sucrose synthase is transiently responsible for the sucrose breakdown in the fruit tissue. Results also indicate that pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) and its activator fructose-2,6-bisphosphate (Fru2,6bisP) are involved in the regulation of the sink metabolism of the fruit tissue.     

Transport and Metabolism of Sucrose versus Hexoses in Relation to Growth in Etiolated Pea Stem

Sucrose, supplied to detached pea (Pisum sativum L. var Alaska) epicotyls through cut bases, supported better growth of apical tissue than supplied glucose and/or fructose. The hexoses were converted mainly to sucrose in basal regions of the epicotyl but some moved as such through the epicotyl and accumulated at the apex (plumule) at a rate faster than sucrose. A greater proportion of the carbon derived from supplied hexoses than from sucrose was used for synthesis of ethanol-insoluble products throughout the epicotyl. By use of asymmetrically labeled sucrose, it was shown that neither hexose moiety was used preferentially for the synthesis of metabolites. Supplied sucrose moved as such only up to the region of cell elongation where it was hydrolyzed and completely equilibrated before moving into more apical regions. The results indicate that better growth with supplied sucrose than hexose could not have resulted from differential effects on cell division, more rapid uptake or transport of sucrose, enhanced wall synthesis, or cleavage by sucrose synthase. It is concluded that transported sucrose versus hexoses must undergo or evoke different reactions which affect growth in the region of cell elongation.     

肉苁蓉和寄主梭梭体内可溶性糖分积累与蔗糖代谢相关酶活性研究

通过测定不同发育时期肉苁蓉和寄主梭梭体内主要糖类物质含量和蔗糖代谢相关酶活性,以研究寄生植物与寄主植物的糖代谢及其关系。结果表明:未寄生肉苁蓉的梭梭以积累葡萄糖为主,而寄生肉苁蓉的梭梭在夏季休眠期以积累葡萄糖为主,进入秋季旺盛生长期时以积累蔗糖为主。肉苁蓉的糖分积累与梭梭不同,己糖含量约占可溶性总糖的62.45%,而蔗糖仅为可溶性总糖的4.98%,故肉苁蓉为己糖积累型。寄主梭梭同化枝内蔗糖磷酸合成酶活性较转化酶活性和蔗糖合成酶活性低,其中寄生肉苁蓉的梭梭的分解酶类活性高于未寄生肉苁蓉的梭梭。肉苁蓉体内转化酶活性较低,而蔗糖合成酶和蔗糖磷酸合成酶活性较高,且蔗糖合成酶活性高于蔗糖磷酸合成酶活性,表现为肉苁蓉中的分解酶类活性高于合成酶类活性,较高的分解酶类活性促进了蔗糖的分解,从而促进了糖分由寄主梭梭向肉苁蓉的不断转移。总体来看,肉苁蓉和寄主梭梭体内糖分的代谢主要以蔗糖合成酶为主,其它2种酶为辅协同参与调控。     

Roles of cell-wall invertases and monosaccharide transporters in the growth and development of Arabidopsis

The hydrolysis of sucrose by cell-wall invertases (cwINV) and the subsequent import of hexoses into target cells appears to be crucial for appropriate metabolism, growth and differentiation in plants. Hexose uptake from the apoplast is catalysed by monosaccharide/H+ symporters (Sugar Transport Proteins or STPs), which have the potential to sense sugars. Import of extracellular hexoses may generate signals to orchestrate cellular activities, or simply feed metabolic pathways distinct from those fed by sucrose. It is predicted that Arabidopsis has six cwINV genes and at least 14 STP genes. These genes show different spatial and temporal patterns of expression, and several knock-out mutants have been isolated for analysis. AtSTP1 transports glucose, galactose, xylose, and mannose, but not fructose. It accounts for the majority of the AtSTP activity in vegetative tissues and its activity is markedly repressed by treatment with exogenous sugars. These observations are consistent with a role in the retrieval of cell-wall-derived sugars, for example, during carbohydrate limitation or cell expansion. The AtSTP1 gene is also expressed in developing seeds, where it might be responsible for the uptake of glucose derived from imported sucrose. The large number of AtcwINV and AtSTP genes, together with complex patterns of expression for each, and the possibility that each protein may have more than one physiological function, provides the plant with the potential for a multiplicity of patterns of monosaccharide utilization to direct growth and differentiation or to respond flexibly to changing environmental conditions.     

Synthesis and Storage of Sucrose in Relation to Activities of Its Metabolizing Enzymes in Sugarcane Cultivars Differing in Maturity

Metabolic changes in the contents of sucrose and hexoses in relation to the activities of invertase, sucrose synthase and sucrose-phosphate synthase in early (CoJ 64) and late (Co 1148) maturing cultivars of sugarcane have been studied. During early stages of cane growth, lower activities of sucrose synthase and sucrose-phosphate synthase in leaf blade In CoJ 64 over Co 1148 were observed. However, sucrose content in sheath/blade was higher in CoJ 64 than in Co 1148. With the advancing age, the activity of soluble acid invertase (pH 5.4) in stem declined more rapidly in CoJ 64. This resulted in building up of high ratio of sucroselinvert sugars in stem tissue of this cultivar. Feeding uniformly-labelled sucrose and glucose to the cut discs of leaf sheath resulted in higher uptake of ¹⁴C in CoJ 64 than in Co 1148. Uptake by stem tissue discs of ¹⁴C from sucrose was less than that from hexoses. Based on these results, it is suggested that (i) the rapid fall in the activity of soluble acid invertase in stem concomitant with fast accumulation of sucrose in this tissue is an index of early maturity of the cane, and (ii) high content of sucrose in sheath is a reflection of an efficient translocation of this sugar in early maturing cultivars.     

Phytohormone-mediated transformation of sugars to starch in relation to the activities of amylases, sucrose-metabolising enzymes in sorghum grain

Indole-acetic acid (IAA) and abscisic acid (ABA) were fed throughcomplete liquid medium (containing 2, 4, 8% sucrose) to detached earheads of sorghum. The effect of these phytohormones on interconvertion ofsugarsand their transformation to starch in relation to the activities of-, -amylases, sucrose-synthase (synthesis), sucrose-phosphatesynthase and soluble invertases was studied in the grain. This effect on theuptake of (U-¹⁴C) sucrose by detached ear heads and incorporation of¹⁴C into free sugars and starch of grain and into free sugars ofinflorescence parts was also studied. At concentrations of up to 4%sucrose in the culture medium, IAA increased the content of total free sugarsinthe grain. However, accumulation of starch and activities of - and-amylases increased when lAA was present even beyond the 4%sucroseconcentration in the culture medium. At all sucrose concentrations, the effectsof ABA and IAA were opposite. With 4% sucrose, both phytohormones causedmaximum accumulation of starch in the grain. ABA enhanced the relativeproportion of sucrose in the sugar pool with a concomitant reduction in theactivities of soluble acid (pH 4.8) and neutral (pH 7.5) invertases. Incontrast, IAA decreased the sucrose proportion of grain sugars with asimultaneous elevation and reduction in the activities of invertases andsucrose-phosphate synthase, respectively. Irrespective of sucrose concentrationin the culture medium, the activity of sucrose synthase (synthesis) wasenhancedwith IAA as well as ABA at their 10 M concentration. IAA alsoenhanced incorporation of ¹⁴C from (U-¹⁴C) sucrose intothe EtOH extract (principally constituted by free sugars) and starch of thegrain, but ABA caused the reverse effect. Based on the results, it is suggestedthat IAA and ABA have contrasting effects on the transformation of sucrose tostarch in sorghum grain where its capacity to synthesise starch is modulatedpositively by IAA and negatively by ABA.     

甜高粱茎秆不同节间糖分累积与相关酶活性的变化

为了进一步了解甜高粱茎秆糖分代谢的规律,利用高效液相色谱等方法测定了考利、拉马达和MN-2747等3个甜高粱品种成熟期6个节间果糖、葡萄糖和蔗糖含量以及中性转化酶(NI)、可溶性酸性转化酶(SAI)、蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)的酶活性,并对其变化规律和相关性进行了分析。结果表明:不同品种间,果糖、葡萄糖和蔗糖含量变化范围较大,分别为2.32～4.34mg/g、2.30～4.14mg/g和35.92～95.92mg/g。随着节间的变化,3个品种果糖和葡萄糖均呈现"U"型变化趋势,而蔗糖无明显的变化规律,只是略有增高的趋势。3个品种成熟期茎秆中NI、SAI、SPS和SS酶活性普遍较低,随着节间的提高均呈现降低的趋势。节间蔗糖含量与SAI酶活性呈显著负相关(R=-0.71,P0.01),与NI、SPS和SS酶活性无明显相关性。SAI可能为甜高粱茎秆糖分代谢的关键调控酶。     

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [¹⁴C]stachyose or [¹⁴C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [¹⁴C]stachyose, but some degree of vein labeling with [¹⁴C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and α-galactosidase and extensively metabolized exogenously supplied ¹⁴C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.     

Sucrose uptake and metabolism in a double layer system for micropropagation ofRosa multiflora

Uptake and metabolism of sucrose in micropropagatedRosa multiflora using the double layer technique was investigated. In the multiplication as well as the root induction stage, hydrolysis of sucrose in the culture medium was observed. A mathematical model was developed to quantify sucrose hydrolysis and the uptake of sucrose, glucose and fructose, based on the time series for the different sugars in the culture medium. These data were linked to a study of the sugar metabolism in the microshoots. After 48 h of incubation on¹⁴C-[U]-glucose containing medium, the incorporated label was mainly detected in the ethanol soluble fraction; within this fraction sucrose was the most important compound. This indicates a significant re-synthesis of sucrose in the plant material after the uptake of hexose. To assess the extent that different enzymes of sucrose metabolism (invertases, sucrose synthase and sucrose-P-synthase) were involved, their activity in different plant parts (of final stage III microshoots) were assayed. A decreasing gradient for sucrose metabolising enzymes from the roots toward the leaves gave a good indication of how the different tissues depend on sucrose absorbed from the medium.     

Hexoses as phloem transport sugars: the end of a dogma?

According to most textbooks, only non-reducing carbohydrate species such as sucrose, sugar alcohols, and raffinose-family sugars function as phloem translocates. Occasional abundance of reducing sugar species (such as hexoses) in sieve-tube sap has been discarded as an experimental artefact. This study, however, discloses a widespread occurrence of hexoses in the sieve-tube sap. Phloem exudation facilitated by EDTA provided evidence that many of the members of two plant families (Ranunculaceae and Papaveraceae) investigated translocate >80% of carbohydrates in the form of hexoses. Representatives of other families also appear to translocate appreciable amounts of hexoses in the sieve tubes. Promoting effects of EDTA, activities of sucrose-degrading enzymes, and sugar uptake by micro-organisms on hexose contents of phloem exudates were checked. The rate of sucrose degradation is far too low to explain the large proportions of hexoses measured in phloem exudates; nor did other factors tested seem to stimulate the occurrence of hexoses. The validity of the approach is further supported by the virtual absence of hexoses in exudates from species that were known as exclusive sucrose transporters. This study urges a rethink of the existing views on carbohydrate transport species in the phloem stream. Hexose translocation is to be regarded as a normal mode of carbohydrate transfer by the phloem equivalent to that of sucrose, raffinose-family sugars, or sugar alcohols.     

Characteristics of sugar uptake in hypocotyls of cotton

Uptake of sucrose and hexoses by cotton (Gossypium hirsutum L.) hypocotyl segments from free space was shown to be an active, carrier-mediated process. Separate carriers existed for hexoses and sucrose. Accumulated sugars appeared in both soluble and insoluble fractions of the tissue. At optimum temperature and pH, sucrose uptake rate versus concentration was fit by a rectangular hyperbola with V(max) of 14 micromoles per gram fresh weight per hour and K(m) of 8 mm. Sucrose was the principal sugar found in the free space in vivo, and invertase activity was essentially absent from that space except after aging.     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

Invertase activity in sinks undergoing cell expansion

During the development of roots, internodes and leaves, closely correlated changes occur in the rates of cell expansion, specific activities of acid invertase and concentrations of hexose sugars and sucrose. Rates of cell growth and acid invertase activities frequently exhibit closely coupled responses to environmental changes and to growth regulator treatments. The possibility is considered that, by controlling the availability of hexose substrates for cellular metabolism, acid invertase may regulate cell growth. Potential mechanisms regulating the in vivo activity of acid invertases are reviewed and attention is drawn to the need for more information on the sub-cellular localization of the enzyme.     

Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development.

Carbohydrate metabolism was investigated during spruce somatic embryogenesis. During the period of maintenance corresponding to the active phase of embryogenic tissue growth, activities of soluble acid invertase and alkaline invertase increased together with cellular glucose and fructose levels. During the same time, sucrose phosphate synthase (SPS) activity increased while sucrose synthase (SuSy) activity stayed constant together with the cellular sucrose level. Therefore, during maintenance, invertases were thought to generate the hexoses necessary for embryogenic tissue growth while SuSy and SPS would allow cellular sucrose to be kept at a constant level. During maturation on sucrose-containing medium, SuSy and SPS activities stayed constant whereas invertase activities were high during the early stage of maturation before declining markedly from the second to the fifth week. This decrease of invertase activities resulted in a decreased hexose:sucrose ratio accompanied by starch and protein deposition. Additionally, carbohydrate metabolism was strongly modified when sucrose in the maturation medium was replaced by equimolar concentrations of glucose and fructose. Essentially, during the first 2 weeks, invertase activities were low in tissues growing on hexose-containing medium while cellular glucose and fructose levels increased. During the same period, SuSy activity increased while the SPS activity stayed constant together with the cellular sucrose level. This metabolism reorganization on hexose-containing medium affected cellular protein and starch levels resulting in a decrease of embryo number and quality. These results provide new knowledge on carbohydrate metabolism during spruce somatic embryogenesis and suggest a regulatory role of exogenous sucrose in embryo development.