Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques

Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.     

The transport of sugars to developing embryos is not via the bulk endosperm in oilseed rape seeds

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [(14)C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [(14)C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [(14)C]Suc converted to hexose falls dramatically. Instead, most of the [(14)C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.     

Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize

Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP-dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP-linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi-linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed.     

Invertase Activity in Normal and Mutant Maize Endosperms during Development

A bound invertase and two soluble invertases are found in the developing endosperm of maize (Zea mays L.). The two soluble invertases can be separated on diethylaminoethyl-cellulose and Sephadex columns and distinguished by their kinetic constants. One soluble invertase, invertase I, is present from the 10- to 28-day stages of endosperm development with maximal activity per normal endosperm at the 12-day stage. In two endosperm mutant lines, shrunken-1 and shrunken-2, there is a second increase in invertase I activity later in development which could be a secondary effect caused by the abnormal metabolism in these lines. Another soluble invertase, invertase II, is present in the embryo upon germination and is also found in the very young developing endosperm (6-day stage). The third form of invertase, bound invertase, is present in the endosperm by the 6-day stage, and its activity remains approximately constant during development.     

Genetic evidence that invertase-mediated release of hexoses is critical for appropriate carbon partitioning and normal seed development in maize

 Cell wall-bound invertase (CWI) is spatially and temporally the first enzyme which metabolizes the incoming sucrose in developing seed of maize (Zea mays). Our previous studies have shown that the cell wall-bound invertase-2 (INCW2) isozyme encoded by the wild-type gene of the Miniature1 (Mn1) seed locus plays a critical role in seed development. Null mutations of the gene, such as the mn1 seed mutant which lacks invertase activity, are associated with a loss of ∼70–80% of the normal seed weight. We show here that under in vitro kernel culture conditions the hexose-based medium was similar to the sucrose-based medium in promoting the normal development of kernels of the Mn1, but not of the mutant mn1, genotype. Anatomical, biochemical, and immunohistological data showed that the mn1 kernels retain their mutant phenotype regardless of the presence of sucrose or hexoses in the culture media. The most drastic changes in the mn1 seed mutant were associated with a significant reduction in the size of the endosperm, but not in the pattern or the level of starch localization. Because Mn1 expression was temporally coincident with the endosperm cell divisions, INCW2 must play a critical role in providing hexose sugars for mitotic division, and only a minor role in generating carbon skeletal substrates for starch biosynthesis in the early stages of endosperm development. Furthermore, a lack of the wild-type seed phenotype of the mn1 mutant in hexose media suggests that a metabolic release of hexoses catalyzed by INCW2, rather than an exogenous source, is critical for both generating appropriate sugar-sensing signals for gene expression and for normal endosperm development. Received: 8 April 1998 / Accepted: 14 August 1998     

Import of sucrose and its transformation to starch in the developing sorghum caryopsis

Levels of soluble and bound invertases and amylases were studied in relation to the changes in the free sugars and the accumulation of starch in the developing sorghum [Sorghum bicolor (L.) Moench, cv. spv. 351] caryopsis and its associated bractspedicel. Besides sucrose, glucose and fructose as the principal sugars, small amounts of sugars of the raffinose series were detected in the developing caryopsis. Through out the period of caryopsis development, the amount of reducing sugars was higher than that of sucrose. With the advancement in the development of the caryopsis, the contents and levels of sucrose rose with a concomitant fall in the activity of soluble acid (pH 4.8) invertase (EC 3.2.1.26) in the endosperm. In the pericarp-aleurone layer, the activity of soluble acid invertase predominated over soluble neutral (pH 7.5) invertase (EC 3.2.1.27). The activity of bound acid invertase declined with the ageing of the caryopsis. In bracts-pedicel, the activity of bound invertase and the levels of reducing sugars peaked around 18 days post anthesis. In these organs, the level of starch gradually decreased concomitantly with an increase in its level in the developing caryopsis. Amylases (EC 3.2.1.1 and 3.2.1.2) are distributed in the endosperm as well as in the pericarp-aleurone layer. On culturing detached ears in [U-¹⁴C]-sucrose solution for 6 h in the dark at 25°C, 80–90% of the ¹⁴C of extracted major sugars (i.e. sucrose + glucose + fructose) of the caryopsis appeared in sucrose alone. In comparison with the effects of glucose or fructose, transport into the caryopsis of ¹⁴C from [U-¹⁴C]-sucrose supplied to detached ears was promoted by the addition to the radiolabelled sucrose solution of 1% unlabelled sucrose. Addition to the [U-¹⁴C]-sucrose solution fed to the detached ears of 20 mM NaN₃ or HgCl₂ or galactose, lowered the amount of ¹⁴C in the free sugars and starch of the earyopsis.     

Metabolism of carbohydrates during the development of seeds of the brazilian rubber tree [Hevea brasiliensis (Willd. Ex Adr. de Juss) Muell.-Arg.]

This work aimed at the assessment of the metabolism of carbohydrate during the development of the seeds of Brazilian rubber trees. The enzymatic activity of the acid invertase, neutral invertase and sucrose synthase (SuSy) and the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose were evaluated separately in each part of the fruit and seed—pericarp, seed coat, embryo and endosperm—on different days after the pollination (DAP). Based on the results obtained in this study, it is possible to conclude that in the beginning of the development of the rubber tree seeds, until 95 DAP, the endosperm presents high concentration of RS and low concentration of sucrose. After this period, the endosperm of the seed initiates starch accumulation and the concentration of RS decreases followed by the increase in the concentration of sucrose, presenting, after 120 DAP, an inversion of concentration of these two sugars. In the embryo, the levels of TSS, RS and sucrose show significant increase with the progress of the seed development. In the endosperm, the transition of the division phase and cell expansion for the storage of reserve material seem to occur around 120 DAP and is to be controlled mainly by the enzymes acid invertase and SuSy, while in the embryo, such transition seems to occur around 135 DAP and is to be controlled mainly by the enzymes acid and neutral invertases.     

Effect of carbon source and sucrose concentration on growth and hexose accumulation of grape berries cultured in vitro

Using in vitro culture of isolated small berries of Vitis vinifera L. cv. Sultana, it was possible to study the effect of different carbon sources and sucrose concentration on fruit growth, hexose accumulation and soluble invertase activity during the first stage of berry development by eliminating the source tissue. Berries cultured in vitro lack stage III of berry development which is characterised by massive accumulation of water and sugars, and thereby berries reached only 30% of the weight of those grown in the plant. Sucrose and glucose were both good carbon sources for berry growth, while fructose was not as good. Berry growth, hexose accumulation and invertase activity increased as sucrose concentration increased up to 15% in the medium. Furthermore, the onset of hexose accumulation in cultured berries depended on the concentration of sucrose in the medium, starting earlier at higher concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sucrose metabolism during endosperm development in wheat (Triticum aestivum)

This work reports changes in sucrose synthase and invertase activities throughout endosperm development in wheat, together with the associated substrates and metabolites, sucrose, UDP, glucose, fructose and UDP-glucose. Throughout endosperm development, sucrose synthase had consistently higher activity than invertase and indeed invertase activity did not change appreciably. The observed variation in pattern and amounts of glucose and fructose present during the mid- and late stages of endosperm development confirmed the suggestion that invertase was not the preferred pathway of sucrose catabolism. Kinetic parameters for sucrose synthase were determined in crude extracts. Estimates of UDP and sucrose concentrations suggest that sucrose synthase is unlikely to achieve its potential maximum velocity. This limitation may however be overcome in part by the apparent excess catalytic activity measured during endosperm 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.     

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.     

Soluble invertase expression is an early target of drought stress during the critical,abortion-sensitive phase of young ovary development in maize

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.     

An invertase inactivator in maize endosperm and factors affecting inactivation

A protein present in the developing endosperm of maize (Zea mays L.) causes a loss of invertase activity under certain conditions of incubation. This protein, designated an inactivator, inactivates invertase I of maize even in the presence of other proteins. No inactivation of invertase II of maize or yeast invertase has been observed. The inactivator and invertase I are found only in the endosperm. The quantity of inactivator increases in the normal endosperm during development while invertase I activity decreases. However, the altered levels of invertase I activity in several endosperm mutant lines do not result from different quantities of inactivator. The inactivator can decrease invertase I activity during a preincubation period before addition of sucrose; inactivation is noncompetitive. Invertase I activity decreases curvilinearly with an increase in inactivator concentration. At high buffer concentrations or low inactivator concentrations in the reaction mixture, a latent period is observed when invertase I is not inactivated. Inactivation increases with an increase in temperature and a decrease in pH.     

Characterization and distribution of invertase activity in developing maize (Zea mays) kernels

Invertase ( β -fructofuranoside fructohydrolase, EC 3.2.1.26) activity in developing maize ( Zea mays L. inbred W64A) was separated into soluble and particulate forms. The particulate form was solubilized by treatment with 1 M NaCl or with other salts. However, CaCl₂ inhibited invertase activity, and neither detergents nor 0.5 M methyl mannoside were effective in solubilizing the invertase activity. The soluble and particulate invertases were both glycoproteins, both had pH optima of 5.0 and K_m values for sucrose of 2.83 and 1.84 m M , respectively. The apparent molecular weight of salt-solubilized invertase was 40 kDa. Gel filtration of the soluble invertase showed multiple peaks with apparent molecular weights ranging from 750 kDa to over 9 000 kDa. Histochemical staining of cell wall preparations for invertase activity suggested that the particulate invertase is associated with the cell wall. Also, nearly all the invertase activity was localized in the basal endosperm and pedicel tissues, which are sites of sugar transport. No invertase activity was found in the upper endosperm, the embryo or in the placento-chalazal tissue. In contrast, sucrose synthase (EC 2.4.1.13) activity was found primarily in the embryo and the upper endosperm, which are areas of active biosynthesis of storage compounds.     

Growth regulator effects on storage root development in carrot

The major carbohydrates stored in carrots are sucrose, glucose and fructose. The ratio of sucrose to reducing sugars varies between cultivars, with early forcing types generally having a higher level of reducing sugars while storage types have a greater proportion of sucrose.In an early forcing cultivar, Super Sprite, high acid invertase activity was correlated with low levels of stored sucrose. As acid invertase activity decreased, the levels of reducing and non-reducing sugars appeared to be related to a balance between alkaline invertase and sucrose synthetase activities.Foliar applications of gibberellic acid at 35 and 42 days after sowing reduced the root/shoot ratio while similar applications of chlormequat chloride marginally increased the ratio. Both growth regulators temporarily increased sucrose stograge, but only gibberellic acid consistently reduced hexose accumulation.Gibberellic acid reduced acid invertase activity following both applications while only reducing the activities of sucrose synthetase after the first application and alkaline invertase after the second application, respectively. Chloremequat chloride increased acid invertase activity after the first application but otherwise has no effect on the activities of the enzymes studied. The significance of changes in assimilate partitioning are discussed in relation to published schemes on carbohydrate storage in root vegetables.     

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).     

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.