Sucrose phosphate synthase and other sucrose metabolizing enzymes in fruits of various species

Recent reports have suggested that sucrose phosphate synthase (EC 2.4.1.14), a key enzyme in sucrose biosynthesis in photosynthetic “source” tissues, may also be important in some sucrose accumulating “sink” tissues. These experiments were conducted to determine if sucrose phosphate synthase is involved in sucrose accumulation in fruits of several species. Peach (Prunus persica NCT 516) and strawberry (Fragaria x ananassa cv. Chandler) fruits were harvested directly from the plant at various stages of fruit development. Kiwi (Actinidia chinensis), papaya (Carica papaya), pineapple (Ananas comosus) and mango (Mangifera indica) were sampled in postharvest storage over a period of several days. Carbohydrate concentrations and activities of sucrose phosphate synthase, sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. All fruits contained significant activities of sucrose phosphate synthase. Moreover, in fruits from all species except pineapple and papaya, there was an increase in sucrose phosphate synthase activity associated with the accumulation of sucrose in situ. The increase in sucrose concentration in peaches was also associated with an increase in sucrose synthase activity and, in strawberries, with increased activity of both sucrose synthase and neutral invertase. The hexose pools in all fruits were comprised of equimolar concentrations of fructose and glucose, except in the mango. In mango, the fructose to glucose ratio increased from 2 to 41 during ripening as sucrose concentration more than doubled. The results of this study indicate that activities of the sucrose metabolizing enzymes, including sucrose phosphate synthase, within the fruit itself, are important in determining the soluble sugar content of fruits of many species. This appears to be true for fruits which sweeten from a starch reserve and in fruits from sorbitol translocating species, raffinose saccharide translocating species, and sucrose translocating species.     

Comparison of Isoprene Emission, Intercellular Isoprene Concentration and Photosynthetic Performance in Water-Limited Oak (Quercus pubescens Willd. and Quercus robur L.) Saplings

Abstract: The influence of prolonged water limitation on leaf gas exchange, isoprene emission, isoprene synthase activities and intercellular isoprene concentrations was investigated under standard conditions (30 °C leaf temperature and 1000 μmol photons m^-2 s^-1 PPFD) in greenhouse experiments with five-year-old pubescent oak ( Quercus pubescens Willd.) and four-year-old pedunculate oak ( Quercus robur L.) saplings. Net assimilation rates proved to be highly sensitive to moderate drought in both oak species, and were virtually zero at water potentials (Ψ_pd) below - 1.3 MPa in Q. robur and below - 2.5 MPa in Q. pubescens . The response of stomatal conductance to water stress was slightly less distinct. Isoprene emission was much more resistant to drought and declined significantly only at Ψ_pd below - 2 MPa in Q. robur and below - 3.5 MPa in Q. pubescens . Even during the most severe water stress, isoprene emission of drought-stressed saplings was still approximately one-third of the control in Q. robur and one-fifth in Q. pubescens . Isoprene synthase activities were virtually unaffected by drought stress. Re-watering led to partial recovery of leaf gas exchange and isoprene emission. Intercellular isoprene concentrations were remarkably enhanced in water-limited saplings of both oak species during the first half of the respective drought periods with maximum mean values up to ca. 16 μl l^-1 isoprene for Q. pubescens and ca. 11 μl l^-1 isoprene for pedunculate oak, supporting the hypothesis that isoprene serves as a short-term thermoprotective agent in isoprene-emitting plant species.     

Species distinction in Irish populations of Quercus petraea and Q. robur: morphological versus molecular analyses

BACKGROUND AND AIMS: Populations of oak (Quercus petraea and Q. robur) were investigated using morphological and molecular (AFLP) analyses to assess species distinction. The study aimed to describe species distinction in Irish oak populations and to situate this in a European context. METHODS: Populations were sampled from across the range of the island of Ireland. Leaf morphological characters were analysed through clustering and ordination methods. Putative neutral molecular markers (AFLPs) were used to analyse the molecular variation. Cluster and ordination analyses were also performed on the AFLP markers in addition to calculations of genetic diversity and F-statisitcs. KEY RESULTS: A notable divergence was uncovered between the morphological and molecular analyses. The morphological analysis clearly differentiated individuals into their respective species, whereas the molecular analysis did not. Twenty species-specific AFLP markers were observed from 123 plants in 24 populations but none of these was species-diagnostic. Principal Coordinate Analysis of the AFLP data revealed a clustering, across the first two axes, of individuals according to population rather than according to species. High F(ST) values calculated from AFLP markers also indicated population differentiation (F(ST) = 0.271). Species differentiation accounted for only 13 % of the variation in diversity compared with population differentiation, which accounted for 27 %. CONCLUSIONS: The results show that neutral molecular variation is partitioned more strongly between populations than between species. Although this could indicate that the populations of Q. petraea and Q. robur studied may not be distinct species at a molecular level, it is proposed that the difficulty in distinguishing the species in Irish oak populations using AFLP markers is due to population differentiation masking species differences. This could result from non-random mating in small, fragmented woodland populations. Hybridization and introgression between the species could also have a significant role.     

Occurrence of sucrose and sucrose metabolizing enzymes in achlorophyllous algae

The presence of sucrose and the enzymes related to sucrose metabolism, i.e. sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13), sucrose phosphate synthase (SPS) (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was demonstrated in Prototheca zopfii, a colorless alga. The levels of enzyme activities were lower than those obtained in Chlorella vulgaris, which is generally considered the photosynthetic counterpart of P. zopfii. Whem enzyme activities were measured in bleached cells of C. vulgaris, the levels were of the same order than those found in P. zopfii. These results would indicate that the sucrose metabolizing enzymes are not related to the algae ability to carry on photosynthesis.     

Isoprene synthase activity and its relation to isoprene emission in Quercus robur L. leaves

Pedunculate oak ( Quercus robur L.) is known as a strong isoprene (2-methyl-1,3-butadiene) emitter. Diurnal changes in isoprene emission were determined by branch enclosure measurements. In contrast to the diurnal cycle in emission rates, specific isoprene synthase activity in the leaves remained unchanged. Based on in vitro enzyme activity and its temperature dependency, an isoprene synthesis capacity at specific leaf temperatures was calculated. The comparison of these 'leaf temperature-dependent enzyme capacities' and the measured emission rates revealed that the enzyme activity of isoprene synthase is comparable to the observed isoprene emission rates. In addition, variation in the isoprene synthase activity of the leaves due to changes in light intensity during leaf development was investigated. A 50% reduction of light intensity by shading of single branches reduced isoprene synthase activity by ≈ 60% compared with full sunlight. The calculation of isoprene synthesis capacities based on enzymatic data obtained under optimum reaction conditions, corrected for actual leaf temperature and related to leaf surface area, provides a sound basis for predicting the isoprene emission potential of plants.     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Sucrose metabolism and fruit growth in parthenocarpic vs seeded blueberry (Vaccinium ashei) fruits

Although fruit set and development are induced by applications of gibberellins, final fruit weight of gibberellin-induced parthenocarpic fruit is often less than that of pollinated fruit. We examined changes in the activities of sucrose-metabolizing enzymes and sugar accumulation in developing fruits of cultivated blueberry (Vaccinium ashei Reade) and their correlation with fruit growth upon pollination or exogenous applications of gibberellic acid (GA₃). The objective was to determine if differences in fruit growth could be attributed to differences in enzyme activities and subsequent sugar accumulation in fruits. The fruit development period of GA₃-treated fruits was 15 days longer than that of pollinated fruits. At maturity, GA₃-treated fruit accumulated an average of 180 mg dry weight while pollinated fruit accumulated 390 mg dry weight. Dry weight accumulation in nonpollinated fruits was negligible and these fruits abscised by 45 days after bloom (DAB). The total carbon (C) cost (dry weight C + respiratory C) for fruit development was 109 and 244 mg C fruit^-1 for GA₃-treated and pollinated fruits, respectively. Hexose concentration increased to 100 mg (g fresh weight)^-1 at ripening in both GA₃-treated and pollinated fruits. Nonpollinated fruits reached a maximum hexose concentration at 45 DAB. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities reached a maximum of ≤5.0 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits. Soluble acid invertase (EC 3.2.1.26) activity increased to about 60 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits at ripening, while in nonpollinated fruits, a maximum soluble acid invertase activity of 0.12 μmol (g fresh weight)^-1 h^-1 was measured at 24 DAB. Insoluble acid invertase activity declined during the early stages of fruit growth and remained relatively low throughout fruit development. Neutral invertase activity was low throughout development, increasing to 5 μmol (g fresh weight)^-1 h^-1 at ripening in GA₃-treated and pollinated fruits. Our studies demonstrate that blueberry fruit development does not appear to be limited by sucrose metabolizing enzyme activity and/or the ability to accumulate sugars in either GA₃-treated or pollinated fruits.     

The influence of chilling on photosynthesis and activities of some enzymes of sucrose metabolism in Lycopersicon esculentum Mill

The effects of chilling stress on leaf photosynthesis and sucrose metabolism were investigated in tomato plants (Lycopersicon esculentum Mill. cultivar Marmande). Twenty-one-day-old seedlings were grown in a growth chamber at 25/23 °C (day/night) (control) and at 10/8 °C (day/night) (chilled) for 7 days. The most evident effect of chilling was the marked reduction of plant growth and of CO₂ assimilation as measured after 7 days, the latter being associated with a decrease in stomatal closure and an increase in Ci. The inhibition in photosynthetic rate was also related to an impairment of photochemistry of photosystem II (PSII), as seen from the slight, but significant change in the ratio of F_v/F_m. The capacity of chilled leaves to maintain higher q_P values with respect to the controls suggests that some protection mechanism prevented excess reduction of PSII acceptors. The results of the determination of starch and soluble sugar content could show that chilling impaired sucrose translocation. The activity of leaf invertase increased significantly in chilled plants, while that of other sucrose-metabolizing enzymes was not affected by growing temperature. Furthermore, the increase in invertase (neutral and acid) activity, which is typical of senescent tissue characterized by reduced growth, seems to confirm that tomato is a plant which is not a plant genetically adapted to low temperatures.     

Activities of Sucrose Synthase and Acid Invertase in Pea Seedling Organs

The organ topography of sucrose synthase and soluble acid invertase in pea seedlings at heterotrophic stage (3–14 days) was studied. Sucrose synthase was most active in the roots, with the highest activity on the 6–8th days. In the leaves, its activity decreased from day 3 to day 14. In the stems, sucrose synthase activity was at an invariantly low level. The patterns of sucrose synthase activity in etiolated and green plants were similar. As distinct from sucrose synthase, invertase activity was the highest in the stem, especially in etiolated plants. The peak of its activity was observed on the 6-8th days. In the leaves, invertase activity was lower but its pattern was the same. In the roots, acid invertase activity decreased from the 3rd day and did not depend on illumination. The conclusion is that differences in sucrose synthase and acid invertase activities in roots, leaves, and stem are determined by differences in the import of hydrolytic products of stored compound from the cotyledons as well as by different demands of these organs for these products for the processes of organ expansion and for the maintenance of organ metabolism.     

Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants

We assessed the effects of doubling atmospheric CO₂ concentration, [CO₂], on C and N allocation within pedunculate oak plants (Quercus robur L.) grown in containers under optimal water supply. A short-term dual ¹³CO₂ and ¹⁵NO₃^? labelling experiment was carried out when the plants had formed their third growing flush. The 22-week exposure to 700 μl l^?1 [CO₂] stimulated plant growth and biomass accumulation (+53% as compared with the 350 μl l^?1 [CO₂] treatment) but decreased the root/shoot biomass ratio (-23%) and specific leaf area (-18%). Moreover, there was an increase in net CO₂ assimilation rate (+37% on a leaf dry weight basis; +71% on a leaf area basis), and a decrease in both above- and below-ground CO₂ respiration rates (-32 and -26%, respectively, on a dry mass basis) under elevated [CO₂]. ¹³C acquisition, expressed on a plant mass basis or on a plant leaf area basis, was also markedly stimulated under elevated [CO₂] both after the 12-h ¹³CO₂ pulse phase and after the 60-h chase phase. Plant N content was increased under elevated CO₂ (+36%), but not enough to compensate for the increase in plant C content (+53%). Thus, the plant C/N ratio was increased (+13%) and plant N concentration was decreased (-11%). There was no effect of elevated [CO₂] on fine root-specific ¹⁵N uptake (amount of recently assimilated ¹⁵N per unit fine root dry mass), suggesting that modifications of plant N pools were merely linked to root size and not to root function. N concentration was decreased in the leaves of the first and second growing flushes and in the coarse roots, whereas it was unaffected by [CO₂] in the stem and in the actively growing organs (fine roots and leaves of the third growth flush). Furthermore, leaf N content per unit area was unaffected by [CO₂]. These results are consistent with the short-term optimization of N distribution within the plants with respect to growth and photosynthesis. Such an optimization might be achieved at the expense of the N pools in storage compartments (coarse roots, leaves of the first and second growth flushes). After the 60-h ¹³C chase phase, leaves of the first and second growth flushes were almost completely depleted in recent ¹³C under ambient [CO₂], whereas these leaves retained important amounts of recently assimilated ¹³C (carbohydrate reserves?) under elevated [CO₂].     

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.     

Interactive effects of mycorrhization and elevated carbon dioxide on growth of young pedunculate oak (Quercus robur L.) trees

Pedunculate oak (Quercus robur L.) was germinated and grown at ambient CO₂ level and 650 ppmv CO₂ in the presence and absence of the ectomycorrhizal fungus Laccaria laccata for a total of 6 month under nutrient non-limiting conditions. Mycorrhization and elevated atmospheric CO₂ each supported the growth of the trees. Stem height, stem diameter, and dry matter accumulation of pedunculate oak were increased by mycorrhization. Elevated atmospheric CO₂ enhanced stem height, stem diameter, fresh weight and dry weight, as well as lateral root formation of the trees. In combination, mycorrhization and elevated atmospheric CO₂ had a more than additive, positive effect on tree height and biomass accumulation, and further improved lateral root formation of the trees. From these findings it is suggested that the efficiency of the roots in supporting the growth of the shoot is increased in mycorrhized oak trees at elevated atmospheric CO₂.Abbreviations DW dry weight - FW fresh weight - RWC relative water content     

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.     

Expression analysis of genes associated with sucrose accumulation in sugarcane (Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage

Sucrose synthesis/accumulation in sugarcane is a complex process involving many genes and regulatory sequences that control biochemical events in source–sink tissues. Among these, sucrose synthase (SuSy), sucrose phosphate synthase (SPS), soluble acid (SAI) and cell wall (CWI) invertases are important. Expression of these enzymes was compared in an early (CoJ64) and late (BO91) maturing sugarcane variety using end‐point and qRT‐PCR. Quantitative RT‐PCR at four crop stages revealed high CWI expression in upper internodes of CoJ64, which declined significantly in both top and bottom internodes with maturity. In BO91, CWI expression was high in top and bottom internodes and declined significantly only in top internodes as the crop matured. Overall, CWI expression was higher in CoJ64 than in BO91. During crop growth, there was no significant change in SPS expression in bottom internodes in CoJ64, whereas in BO91 it decreased significantly. Apart from a significant decrease in expression of SuSy in mature bottom internodes of BO91, there was no significant change. Similar SAI expression was observed with both end‐point and RT‐PCR, except for significantly increased expression in top internodes of CoJ64 with maturity. SAI, being a major sucrose hydrolysing enzyme, was also monitored with end‐point PCR expression in internode tissues of CoJ64 and BO91, with higher expression of SAI in BO91 at early crop stages. Enzyme inhibitors, e.g. manganese chloride (Mn⁺⁺), significantly suppressed expression of SAI in both early‐ and late‐maturing varieties. Present findings enhance understanding of critical sucrose metabolic gene expression in sugarcane varieties differing in content and time of peak sucrose storage. Thus, through employing these genes, improvement of sugarcane sucrose content is possible.     

The effects of seed size, cotyledon reserves, and herbivory on seedling survival and growth in Quercus rugosa and Q. laurina (Fagaceae)

In a greenhouse experiment, seedling survival of two oak species (Quercus rugosa and Q. laurina) was greatly affected by the excision of cotyledons 1 mo after germination, with a greater impact on Q. laurina. The effect of seed size was also significant for both species, with a positive correlation between seed mass and survival and growth. The effect of cotyledon excision on seedling growth persisted throughout the first growing season in Q. rugosa and was not analyzed for Q. laurina due to the low number of seedlings that survived cotyledon excision. Seed size significantly affected seedling height, diameter, leaf area, and biomass at 6 mo. Seed size and cotyledon retention affected the ability of Q. rugosa to recover from herbivory, as both factors had a significant effect on relative growth rates after aerial biomass removal. The results show that seedlings originating from large seeds can better endure loss of cotyledons and aerial biomass and thus are better equipped to confront stress early in their lives.