Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestris pv vesicatoria

Cell wall-bound invertase (cw-Inv) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. There is increasing evidence that pathogens interfere with sink-source interaction, and induction of cw-Inv activity has frequently been shown in response to pathogen infection. To investigate the role of cw-Inv, transgenic tomato (Solanum lycopersicum) plants silenced for the major leaf cw-Inv isoforms were generated and analyzed during normal growth and during the compatible interaction with Xanthomonas campestris pv vesicatoria. Under normal growth conditions, activities of sucrolytic enzymes as well as photosynthesis and respiration were unaltered in the transgenic plants compared with wild-type plants. However, starch levels of source leaves were strongly reduced, which was most likely caused by an enhanced sucrose exudation rate. Following X. campestris pv vesicatoria infection, cw-Inv-silenced plants showed an increased sucrose to hexose ratio in the apoplast of leaves. Symptom development, inhibition of photosynthesis, and expression of photosynthetic genes were clearly delayed in transgenic plants compared with wild-type plants. In addition, induction of senescence-associated and pathogenesis-related genes observed in infected wild-type plants was abolished in cw-Inv-silenced tomato lines. These changes were not associated with decreased bacterial growth. In conclusion, cw-Inv restricts carbon export from source leaves and regulates the sucrose to hexose ratio in the apoplast. Furthermore, an increased apoplastic hexose to sucrose ratio can be linked to inhibition of photosynthesis and induction of pathogenesis-related gene expression but does not significantly influence bacterial growth. Indirectly, bacteria may benefit from low invertase activity, since the longevity of host cells is raised and basal defense might be dampened.     

Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds: analysis of tobacco seed development and effects of overexpressing apoplastic invertase

Wild-type tobacco (Nicotiana tabacum L.) seed development was characterized with respect to architecture and carbohydrate metabolism. Tobacco seeds accumulate oil and protein in the embryo, cellular endosperm and inner layer of the seed coat. They have high cell wall invertase (INV) and hexoses in early development which is typical of seeds. INV and the ratio of hexose to sucrose decline during development, switching from high hex to high suc, but not until most oil and all protein accumulation has occurred. The oil synthesis which coincides with the switch is mostly within the embryo. INV activity is greater than sucrose synthase activity throughout development, and both activities exceed the demand for carbohydrate for dry matter accumulation. To investigate the role of INV-mediated suc metabolism in oilseeds, genes for yeast INV and/or hexokinase (HK) were expressed under a seed-specific napin promoter, targeting activity to the apoplast and cytosol, respectively. Manipulating the INV pathway in an oilseed could either increase oil accumulation and sink strength, or disrupt carbohydrate metabolism, possibly through sugar-sensing, and decrease the storage function. Neither effect was found: transgenics with INV and/or HK increased 30-fold and 10-fold above wild-type levels had normal seed size and composition. This contrasted with dramatic effects on sugar contents in the INV lines.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Quantitative trait loci for sucrose, starch, and hexose accumulation before heading in rice.

We studied the storage of sucrose, starch, and hexose before heading in rice (Oryza sativa L.) plants by quantitative trait locus (QTL) analysis with a population of backcross inbred lines (BILs) of japonica cv. Nipponbare x indica cv. Kasalath. Carbohydrates are accumulated in the rice plant before heading and are translated to the panicle after heading. A higher capacity for accumulation is thus a main target for improvement in yield. The form of carbohydrate (sucrose, starch, or hexose) differs depending on the organ in which it is stored. There was no correlation between starch and sucrose or hexose contents in BILs, and the positions of QTLs controlling starch differed from those for sucrose and hexose accumulation. These results suggest that the genetic control of accumulation differs between starch and sugars. QTLs that control the ratio of sucrose to starch content were detected, suggesting the existence of a mechanism(s) that determines this ratio. On chromosome 1, sucrose-phosphate synthase 1, the key enzyme in sucrose synthesis was close to the peaks of the likelihood odds ratios in QTLs for sucrose or hexose content. These results suggest that SPS1 is related to conversion of carbohydrate to sucrose as accumulated form in a plant before heading.     

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.     

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.     

Acid invertase confers heat tolerance in rice plants by maintaining energy homoeostasis of spikelets

Heat stress impairs both pollen germination and pollen tube elongation, resulting in pollination failure caused by energy imbalance. Invertase plays a critical role in the maintenance of energy homoeostasis; however, few studies investigated this during heat stress. Two rice cultivars with different heat tolerance, namely, TLY83 (heat tolerant) and LLY722 (heat susceptible), were subjected to heat stress. At anthesis, heat stress significantly decreased spikelet fertility, accompanied by notable reductions in pollen germination on stigma and pollen tube elongation in ovule, especially in LLY722. Acid invertase (INV), rather than sucrose synthase, contributed to sucrose metabolism, which explains the different tolerances of both cultivars. Under heat stress, larger enhancements in NAD(H), ATP, and antioxidant capacity were found in TLY83 compared with LLY722, whereas a sharp reduction in poly(ADP-ribose) polymerase (PARP) activity was found in the former compared with the latter. Importantly, exogenous INV, 3-aminobenzamide (a PARP inhibitor), sucrose, glucose, and fructose significantly increased spikelet fertility under heat stress, where INV activity was enhanced and PARP activity was inhibited. Therefore, INV can balance the energy production and consumption to provide sufficient energy for pollen germination and pollen tube growth under heat stress.     

Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.     

Soluble acid invertase activity in leaves is independent of species differences in leaf carbohydrates, diurnal sugar profiles and paths of phloem loading

Leaf sucrose, starch, hexose and maximum extractable soluble acid invertase activity were compared throughout the day in source leaves of 13 plant species chosen for their putative phloem-loading type (apoplastic or symplastic). Four species which represent the different phloem-loading types (tomato, barley, maize and Fuchsia ) were studied in detail. Using this information we wished to determine whether a positive correlation between foliar carbohydrates and acid invertase activity exists in leaves from different species and, furthermore, whether this relationship is determined by phloem-loading type. Acid invertase activity was relatively constant throughout the day in all species. The extent of sucrose, hexose and starch accumulation and the sucrose: starch ratio measured at a given time were species-dependent. No correlations were found between foliar soluble acid invertase activity and the hexose, sucrose or starch content of the leaves in any of the species, regardless of phloem-loading type. The species examined could be divided into three distinct groups: (1) high sucrose, low invertase; (2) low sucrose, low invertase; and (3) low sucrose, high invertase. The absence of an inverse relationship between leaf sucrose, hexose or starch contents and endogenous soluble acid invertase suggests that this enzyme is not directly involved in carbon partitioning in leaves but serves an auxiliary function.     

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.     

Diurnal Changes in Maize Leaf Photosynthesis : III. Leaf Elongation Rate in Relation to Carbohydrates and Activities of Sucrose Metabolizing Enzymes in Elongating Leaf Tissue

Maize (Zea mays L. cv. Pioneer 3184) leaf elongation rate was measured diurnally and was related to diurnal changes in the activities of sucrose metabolizing enzymes and carbohydrate content in the elongating portion of the leaf. The rate of leaf elongation was greatest at midday (1300 hours) and was coincident with the maximum assimilate export rate from the distal portion of the leaf. Leaf elongation during the light period accounted for 70% of the total observed increase in leaf length per 24 hour period. Pronounced diurnal fluctuations were observed in the activities of acid and neutral invertase and sucrose phosphate synthase. Maximum activities of sucrose phosphate synthase and acid invertase were observed at 0900 hours, after which activity declined rapidly. The activity of sucrose phosphate synthase was substantially lower than that observed in maize leaf source tissue. Neutral invertase activity was greatest at midday (1200 hours) and was correlated positively with diurnal changes in leaf elongation rate. There was no significant change in the activity of sucrose synthase over the light/dark cycle. Sucrose accumulation rate increased during a period when leaf elongation rate was maximal and beginning to decline. Maximum sucrose concentration was observed at 1500 hours, when the activities of sucrose metabolizing enzymes were low. At no time was there a significant accumulation of hexose sugars. The rate of starch accumulation increased after the maximum sucrose concentration was observed, continuing until the end of the light period. There was no delay in the onset of starch mobilization at the beginning of the dark period, and essentially all of the starch was depleted by the end of the night. Mobilization of starch in the elongating tissue at night could account for a significant proportion of the calculated increase in the tissue dry weight due to growth. Collectively, the results suggested that leaf growth may be controlled by the activities of certain sucrose metabolizing enzymes and may be coordinated with assimilate export from the distal, source portion of the leaf. Results are discussed with reference to diurnal photoassimilation and export in the distal, source portion of the leaf.     

Effects of nitrogen and phosphorus deficiencies on levels of carbohydrates, respiratory enzymes and metabolites in seedlings of tobacco and their response to exogenous sucrose

A simple method of growing plants in agar was exploited to investigate the effect of long-term nitrogen (N) and phosphorus (P) deficiencies on respiratory metabolism and growth in shoots and roots of Nicotiana tabacum seedlings, and their interaction with exogenously supplied sucrose. Levels of hexose phosphates and 3-phosphoglyceric acid (3-PGA) were low in P-deficient shoots and roots and high in N-deficient shoots and roots. The ratio of hexose phosphates to 3-PGA and levels of fructose-2,6-bisphosphate were high in P-deficient plants and low in N-deficient plants. These data reflect differences in the way metabolism was perturbed, yet both deficiencies were associated with increased root growth relative to shoot growth, starch accumulation in the shoots, and soluble carbohydrate accumulation, especially hexoses, in the roots. Enzymes for sucrose degradation (sucrose synthase, acid and alkaline invertase) and glycolysis (phosphofructokinase, pyrophosphate-dependent phospho-fructokinase and pyruvate kinase) remained unaltered or declined in the shoots and roots. The accumulation of hexoses in roots of N- and P-deficient plants may result from maintenance of high invertase activities relative to sucrose synthase and glycolytic enzymes in the roots. The possibility that hexose accumulation may drive preferential root growth osmotically in N and P deficiencies is discussed. The addition of sucrose to roots to further investigate the interaction of carbohydrates with growth and allocation in low N and low P produced clear effects even though endogenous levels of soluble carbohydrate were already high in the nutrient-deficient plants. In complete nutrition, growth was stimulated, protein content particularly of the roots was increased and there was a preferential increase in activity of sucrose synthase in roots. At low P, enzyme activities in roots were increased, including sucrose synthase, and protein content increased, particularly in the roots, but there was no increase in growth. In N-deficient plants, exogenous sucrose led to decreased protein, Rubisco and chlorophyll content in shoots, in contrast to the other conditions, and a higher protein content and a general increase of catabolic enzyme activities and growth in the roots.