A Similar Dichotomy of Sugar Modulation and Developmental Expression Affects Both Paths of Sucrose Metabolism: Evidence from a Maize Invertase Gene Family

Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for invertases. Toward this end, a family of maize invertases was cloned and characterized. Here, we show that invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.     

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.     

Interconversion of free sugars in relation to activities of enzymes catalyzing synthesis and cleavage of sucrose in growing stem tissues of sorghum

Free sugar interconversion and activities of soluble acidic (pH 4.8) and neutral (pH 7.5) invertases, sucrose synthase (synthesis) and sucrose phosphate synthase were investigated in the growing nodes and internodes of sorghum (Sorghum vulgare). The results were substantiated with incorporation of 14C from supplied sucrose and hexoses into endogenous sugars of these stem tissues. With the advancement in plant growth, the content of total free sugars in apical nodes and internodes increased till 70 DAS (flowering stage) followed by a decline. In the corresponding basal tissues, the sugar build-up continued even beyond this stage of plant growth. Compared with basal stem tissues, the apical ones contained high activities of soluble invertases and a low proportion amongst free sugars of sucrose. The activities of sucrose-hydrolyzing enzymes were higher as compared with those of sucrose-synthesizing ones in both nodes and internodes and with the growth of plant, the activity of neutral invertase increased in these tissues. More 14C from supplied sucrose and hexoses appeared in extracted sugars from cut discs of apical nodes and internodes in comparison with their basal counterparts. 14C from supplied sucrose appeared in glucose, fructose and from supplied hexoses appeared in sucrose. The results suggest that in apical nodes and internodes, where a rapid cell division and cell expansion occur, sucrose is obligatorily inverted to meet the increased requirement of hexoses and there is a compartmentalized synthesis and cleavage of sucrose in the nodes and internodes of growing sorghum plant.     

The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in the tap roots

Sucrose utilisation in sink tissues depend on its cleavage and is mediated by two different classes of enzymes, invertase and sucrose synthase, which determine the mechanism of phloem unloading. Cloning of two extracellular (BIN35 and BIN46) and one vacuolar invertase (BIN44) provided the basis for a detailed molecular analysis of the relative contribution of the sucrose cleaving enzymes to the sink metabolism of sugar beets (Beta vulgaris) during development. The determination of the steady state levels of mRNAs has been complemented by the analysis of the corresponding enzyme activities. The present study demonstrates an inverse regulation of extracellular invertase and sucrose synthase during tap root development indicating a transition between functional unloading pathways. Extracellular cleavage by invertase is the dominating mechanism to supply hexoses via an apoplasmic pathway at early stages of storage root development. Only at later stages sucrose synthase takes over the function of the key sink enzyme to contribute to the sink strength of the tap root via symplasmic phloem unloading. Whereas mRNAs for both extracellular invertase BIN35 and sucrose synthase were shown to be induced by mechanical wounding of mature leaves of adult plants, only sucrose synthase mRNA was metabolically induced by glucose in this source organ supporting the metabolic flexibility of this species.     

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.     

A role for 'futile cycles' involving invertase and sucrose synthase in sucrose metabolism of tomato fruit.

Current concepts of the factors determining sink strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon an understanding of the relative roles of sucrose synthase, sucrose phosphate synthase and invertase, derived from studies in mutants and transformed plants. These enzymes participate in at least four futile cycles that involve sugar transport between the cytosol, vacuole and apoplast. Key reactions are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy), (2) sucrose re-synthesis via either SuSy or sucrose phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent transport of hexoses to the cytosol where they are once more converted into sucrose, and (5) rapid synthesis and breakdown of starch in the amyloplast. In this way futile cycles of sucrose/hexose interchange govern fruit sugar content and composition. The major function of the high and constant invertase activity in red tomato fruit is, therefore, to maintain high cellular hexose concentrations, the hydrolysis of sucrose in the vacuole and in the intercellular space allowing more efficient storage of sugar in these compartments. Vacuolar sugar storage may be important in sustaining fruit cell growth at times when less sucrose is available for the sink organs because of exhaustion of the carbohydrate pools in source leaves.     

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.     

Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism.

The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ.     

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.     

Changes in the activities of carbon metabolizing enzymes with pod development in lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> L.)

Activities of some key enzymes of carbon metabolism sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were investigated in relation to the carbohydrate status in lentil pods. Sucrose remained the dominant soluble sugar in the pod wall and seed, with hexoses (glucose and fructose) present at significantly lower levels. Sucrose synthase is the predominant sucrolytic enzyme in the developing seeds of lentil (Lens culinaris L.). Acid invertase was associated with pod elongation and showed little activity in seeds. Sucrose breakdown was dominated by alkaline invertase during the development of podwall, while both the sucrose synthase and alkaline invertase were active in the branch of inflorescence. A substantial increase of sucrolytic enzymes was observed at the time of maximum seed filling stage (10–20 DAF) in lentil seed. The pattern of activity of sucrose synthase highly paralleled the phase of rapid seed filling and therefore, can be correlated with seed sink strength. It seems likely that the fruiting structures of lentil utilize phosphoenol pyruvate carboxylase for recapturing respired carbon dioxide. Higher activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could be effectively linked to the deposition of protein reserves.     

Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits

Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 ± 0.3 micromole per hour per gram fresh weight and 2.2 ± 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality.     

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。     

Extracellular invertase: key metabolic enzyme and PR protein

Extracellular invertase is the key enzyme of an apoplasmic phloem unloading pathway and catalyses the hydrolytic cleavage of the transport sugar sucrose released into the apoplast. This mechanism contributes to long-distance assimilate transport, provides the substrate to sustain heterotrophic growth and generates metabolic signals known to effect various processes of primary metabolism and defence responses. The essential function of extracellular invertase for supplying carbohydrates to sink organs was demonstrated by the finding that antisense repression of an anther-specific isoenzyme provides an efficient method for metabolic engineering of male sterility. The regulation of extracellular invertase by all classes of phytohormones indicates an essential link between the molecular mechanism of phytohormone action and primary metabolism. The up-regulation of extracellular invertase appears to be a common response to various biotic and abiotic stress-related stimuli such as pathogen infection and salt stress, in addition to specific stress-related reactions. Based on the observed co-ordinated regulation of source/sink relations and defence responses by sugars and stress-related stimuli, the identified activation of distinct subsets of MAP kinases provides a mechanism for signal integration and distribution within such complex networks. Sucrose derivatives not synthesized by higher plants, such as turanose, were shown to elicit responses distinctly different from metabolizable sugars and are rather perceived as stress-related stimuli.     

Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves : estimation by alternate substrate utilization

The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1′-deoxy-1′-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [¹⁴C]Sucrose and [¹⁴C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [¹⁴C]sucrose was supplied than when [¹⁴C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.     

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.     

Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.