Development of Sink Capacity of the "Storage Root" in a Radish Variety with a Low Ratio of "Storage Root" to Shoot

Controling mechanisms of sink capacity are poorly understood.Previously we suggested that sucrose synthase (SuSy), but notinvertase, plays an important role for sink capacity of theradish "storage root" in a variety, Raphanus sativus L. (cv.White Cherish) [plant Cell Physiol. (1999) 40: 369]. With thisvariety about 50% of the total dry weight (DW) was in the "storageroot" at 21 d after sowing (DAS). We investigated the sink capacityof another radish variety, R. sativus L. (cv. Kosena) with alow ratio of "storage root" to shoot. With the latter varietyonly 3% of the total DW was in the "storage root" at 21 DAS.Sink activity (increase in DW of the "storage root" per unitof DW present per unit of time) of the "storage root" in Kosenaas well as White Cherish was strongly related to the level andactivity of SuSy but not to the activity of invertase. Theseresults confirmed that SuSy rather than invertase may be criticalfor the development of the sink activity of the radish "storageroot" and that the reaction products of UDP-glucose and fructoseare utilized for sink growth including biosynthesis of the cellwall. In Kosena photosynthates seemed to be partitioned mainlyinto developing leaves and fibrous roots. Differences in partitioningof photosynthates among various sinks with these two varietiesare discussed including anatomical considerations. (Received July 19, 1999; Accepted September 30, 1999)     

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.     

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.     

Spatial and temporal distribution of sucrose synthase in the radish hypocotyl in relation to thickening growth.

Sucrose synthase (SuSy) is a key enzyme in the development of storage root of radish. Clarification of its spatial and temporal expression during the thickening growth of radish hypocotyl, which later develops into storage root, was carried out immunologically using light microscopy. Sequential harvests at 3, 7, 11 and 13 d after sowing (DAS) were performed on two radish cultivars having different sink capacity. A very low level of SuSy was observed 3 DAS for both cultivars. White Cherrish (WC; strong storage root) showed the maximum level of SuSy between 7 and 11 DAS with increased cell development (thickening), while in Kosena (K; low storage root) the level remained high after 13 d of growth. A high level of SuSy was found in companion cells, which was consistent with previous observations, but SuSy was also found in the xylem parenchyma and in some cortical cells. The level of SuSy differed according to the localization and depended highly on cell development. Both cell division and cell enlargement were stimulated in WC compared with K. The role of SuSy during thickening growth of radish hypocotyl is discussed in terms of utilizing photosynthates.     

Sucrose-metabolizing enzymes in transport tissues and adjacent sink structures in developing citrus fruit

Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import remains to be determined.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Sucrose‐cleaving enzymes and carbohydrate pools in Lilium longiflorum floral organs

The activities of soluble invertase (EC 3.2.1.26), cell wall invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) were determined in Easter lily ( Lilium longiflorum Thunb. cv. Nellie White) floral organs during flower development. These enzyme activities were correlated with dry weight gains and carbohydrate pools to investigate the importance of their expression in maintaining sink strength of floral organs. In the early stages of flower bud development, anthers exhibited the highest rates of dry weight gain and activity of sucrolytic enzymes. Once anther growth was completed, the dry weight gain of tepal, filament, stigma and style increased with a concomitant increase in hexose concentrations and invertase activity. Although all three enzymes capable of catalyzing sucrose cleavage were present in every flower organ of L. longiflorum , soluble invertase was the predominant enzyme in all flower organs except stigma where cell wall invertase dominated. Soluble invertase activity was highly correlated with dry weight gain in most of the flower organs.     

Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

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.     

Rhythmic growth and carbon allocation in Quercus robur. Sucrose metabolizing enzymes in leaves

The high sucrose phosphate synthase (SPS) capacity and the low soluble acid invertase activity of mature leaves of the first flush of leaves remained stable during second flush development. Conversely, fluctuations of sucrose synthase (SS) activity were in parallel with the sucrose requirement of the second flush. Sucrose synthase activity (synthesis direction) in first flush leaves could increase in 'response' to sink demand constituted by the second flush growth. Only the ptotosynthates provided by flush mature leaves were translocated for a current flush, while the starch content of these leaves remained stable. After their emergence, second flush leaves showed an increase in SPS and SS (Synthetic direction) activities. The high sucrose synthesis in second flush leaves was used for leaf expansion. When young leaves were 30% fully expanded (stage II20), SPS activity showed little change whereas SS activity declined rapidly toward and after full leaf expansion. The starch accumulation in the young leaves occured simultaneously with their expansion. Developing leaves showed a high level of acid invertase activity until maximum leaf expansion (stage II1). In first and second flush leaves, changes in acid invertase activity correlated positively with changes in reducing sugar concentrations. Alkaline invertase and sucrose synthase (cleavage direction) activities showed similar changes with low values when compared with those of acid invertase activity, especially in second flush leaves. The present results suggest that soluble acid invertase was the primary enzyme responsible for sucrose catabolism in the expanding common oak leaf.     

Sink development, sucrose metabolising enzymes and carbohydrate status in turnip (Brassica rapa L.)

Accumulation of 60–70 % of biomass in turnip root takes place between 49–56 days after sowing. To understand the phenomenon of rapid sink filling, the activities of sucrose metabolising enzymes and carbohydrate composition in leaf blades, petiole and root of turnip from 42–66 days of growth were determined. An increase (2–3 folds) in glucose and fructose contents of roots accompanied by an increase in activities of acid and alkaline invertases was observed during rapid biomass accumulating phase of roots. The observed decrease in the activities of acid and alkaline invertases along with sucrose synthase (cleavage) in petiole during this period could facilitate unrestricted transport of sucrose from leaves to the roots. During active root filling period, a decrease in sucrose synthase (cleavage) and alkaline invertase activities was also observed in leaf blades. A rapid decline in the starch content of leaf blades was observed during the phase of rapid sink filling. These metabolic changes in the turnip plant led to increase in hexose content (35–37 %) of total dry biomass of roots at maturity. High hexose content of the roots appears to be due to high acid invertase activity of the root.     

Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants

A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study β-1,3-glucan synthase activity changes. All tissues contained an active β-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active β-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in β-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that β-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development.     

Expression analysis of genes associated with sucrose accumulation in sugarcane under normal and GA<Subscript>3</Subscript>-induced source–sink perturbed conditions

Sugarcane accumulates high amount of sucrose, thus making it one of the important cash crops worldwide. The final destination of sucrose accumulation in sugarcane is sink tissue, i.e., stalk, supplied by the source, i.e., leaf, to fulfill the need of plant growth, respiration, storage, and other metabolic activities. Signals between sink and source tissues regulate sucrose accumulation in sink and possibly the negative feedback from the sink restrains further accumulation in the stalk. However, perturbation of this negative feedback may help to improve sugar yield. This can be achieved by the application of GA₃ (Gibberellic acid), a plant growth regulator, known to excite physiological responses and modify the source–sink metabolism through their effect on photosynthesis, which in turn improves sink strength by redistribution of the photoassimilates. In the present study, GA₃ applied canes showed prominent increase in invertase activity, at early stage of the application, to provide hexoses. This in turn helped increase the internodal length and cane capacity for additional accumulation of sucrose, thereby increasing sink strength. At maturity, sucrose% and brix% were found higher in middle and top portions of the GA₃-applied canes. Expression analysis of various sucrose metabolising genes viz., sucrose phosphate synthase (SPS), sucrose synthase (SuSy), soluble acid invertase, neutral invertase, and cell wall invertase (CWI) was carried out at different growth stages, using quantitative RT-PCR. CWI, which plays key role in phloem unloading in sink tissues, exhibited higher expression in GA₃ samples at the elongation stage which decreased with maturity, whereas both SuSy and SPS, involved in regulation of sucrose accumulation, showed a variable level of expression. Thus, GA₃ application on cane may improve the sucrose content in stalk and thus assuage maneuvering source–sink dynamics in sugarcane.     

Exaggerated root respiration accounts for growth retardation in a starchless mutant of Arabidopsis thaliana

The knock‐out mutation of plastidial phosphoglucomutase (pgm) causes a starchless phenotype in Arabidopsis thaliana, and results in a severe growth reduction of plants cultivated under diurnal conditions. It has been speculated that high soluble sugar levels accumulating during the light phase in leaf mesophyll might cause a reduction of photosynthetic activity or that shortage of reduced carbon during the night is the reason for the slow biomass gain of pgm. Separate simultaneous measurements of leaf net photosynthesis and root respiration demonstrate that photosynthetic activity per unit fresh weight is not reduced in pgm, whereas root respiration is strongly elevated. Comparison with a mutant defective in the dominating vacuolar invertase (AtβFruct4) revealed that high sucrose concentration in the cytosol, but not in the vacuole, of leaf cells is responsible for elevated assimilate transport to the root. Increased sugar supply to the root, as observed in pgm mutants, forces substantial respiratory losses. Because root respiration accounts for 80% of total plant respiration under long‐day conditions, this gives rise to retarded biomass formation. In contrast, reduced vacuolar invertase activity leads to reduced net photosynthesis in the shoot and lowered root respiration, and affords an increased root/shoot ratio. The results demonstrate that roots have very limited capacity for carbon storage but exert rigid control of supply for their maintenance metabolism.     

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.     

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.     

Photosynthate Partitioning and Enzymes of Sucrose Metabolism in Sugarbeet Roots

Populations of sugarbeet (Beta vulgaris L.) plants that differed in taproot/leaf weight ratio and in photosynthate partitioning between taproots and fibrous roots did not differ in root/shoot ratio as indicated by relative dry weight distribution. Based on the hypothesis that dry weight distribution is influenced by the metabolism of imported sucrose, we examined the relationships between the activity of the enzymes of sucrose metabolism and dry weight distribution as a function of genotype and ontogeny. A decreased specific activity of acid invertase in taproots was associated with increased taproot/fibrous root weight ratio at 21 and at 28 days post-emergence. Alkaline invertase activity was negatively correlated with taproot/fibrous root weight ratio at 28 days. Sucrose synthetase specific activities of taproots were not correlated with dry matter distribution. Acid invertase may influence photosynthate partitioning between the taproot and fibrous roots via regulation of sucrose levels in the region of fibrous root initiation.     

Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.     

Invertase,sucrose synthase and sucrose phosphate synthase in lyophilized spruce needles; microplate reader assays

Summary Data on changes of apparent activities of enzymes involved in sucrose metabolism of developing spruce needles are presented. Extractable activities of sucrose phosphate synthase (SPS, sucrose synthesis), and sucrose synthase (SS) and acid invertase (both sucrolysis) were determined in small volumes using a novel microplate reader system which combined high rates of activity with good reproducibility and high sample throughput. During a developmental period of up to 18 months after bud break characteristic changes in SPS and SS occurred. During the first 4 months of needle development SS declined while SPS increased which is indicative of a transition from net import to net export of photoassimilates (sink/source transition). After needle maturation both enzymes exhibited parallel annual changes with increasing rates towards autumn, which was mirrored by the pool sizes of sucrose (possibly due to the acquisition of frost hardiness). Acid invertase activity was comparable to that of SS but showed only marginal seasonal changes. Approximately 70% of its total activity was found to be soluble.