In order to determine whether the changes in the demand for the transported carbohydrates in apple source leaves are associated with specific carbohydrate enzyme changes, we made source–sink manipulation by girdling or defoliation. The girdle was applied to side branches with several fully expanded leaves, and the defoliation was conducted by removing about 90% of source leaves in apple seedlings. 3-year-old apple (Malus domestica Borhk. cv. Gala) seedlings were grown in a 15/9-h light (≈700 μmol photons/m2 per s, 22 °C)/dark (18 °C) in the growth chamber. When the demand for transported carbohydrates from a particular source leaf is limited by girdling, carbohydrates including starch, sorbitol and sucrose accumulated in the source leaves, meanwhile girdling reduced net photosynthetic rates (Pn) dramatically from 12.8 initially to 4.6 μmol CO2/m2 per s over a 7-day period. When the demand for transported carbohydrate in the remaining source leaves was increased by defoliation, all carbohydrate levels decreased while Pn of individual leaves increased from 13.6 initially to a maximum of 19.8 μmol CO2/m2 per s after 7 days. These Pn changes in the carbohydrate depleted and accumulated leaves were due mainly to changes in the photosynthetic capacity as indicated by Pn-Ci curve measurements. The carbohydrate enzyme activities were also dramatically changed during the 7-day experimental period. The activity of aldose-6-phosphate reductase (E.C. 184.108.40.206), an important enzyme in sorbitol biosynthesis, increased significantly from 27.5 to 39.2 μmol/h per g FW in the carbohydrate depleted leaves while it remained unchanged in the girdled leaves, the activity of sucrose-6-phosphate synthase (SPS, E.C. 220.127.116.11), a key enzyme for sucrose biosynthesis, increased from 15.4 to 23.0 μmol/h per g FW in the depleted leaves and declined from 17.4 to 8.2 μmol/h per g FW in the girdled leaves, the activity of fructose 1,6 bisphosphatase (E.C. 18.104.22.168), another key enzyme for sucrose biosynthesis in non-Rosaceae species showed a similar pattern as SPS, ADPglucose-pyrophosphorylase (E.C. 22.214.171.124), a key enzyme for starch biosynthesis, decreased a small amount in the girdled leaves but increased markedly from 42.9 to 56.0 μmol/h per g FW in the depleted leaves. These results indicated the specific roles of the enzymes in the partitioning of carbon between sorbitol, sucrose and starch in apple source leaves.
Cytosolic fructose-1,6-bisphosphatase (cytoFBPase) (EC 126.96.36.199) occupies a strategic site in sucrose synthesis and has been demonstrated to play a key role in carbon partitioning between sucrose and starch in non-sorbitol forming plants. In addition to sucrose and starch, Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family. To understand the biochemical regulation of photosynthetic carbon partitioning between sorbitol, sucrose and starch in sorbitol synthesizing species, we purified cytoFB-Pase to apparent homogeneity from apple leaves. The enzyme was a homotetramer with a subunit mass of 37 kDa. It was highly specific for fructose-1,6-bisphosphate with a Km of 3.1 μm and a Vmax of 48 units/mg protein. Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mm and 62 μM, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+-activated enzyme activity. Fructose-6-phosphate was found to be a mixed type inhibitor with a Ki of 0.47 mm. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. Adenosine monophosphate (AMP) was a non-competitive inhibitor for the enzyme. F2,6BP interacted with AMP to inhibit the enzyme in a synergistic way. Dihydroxyacetone phosphate did not have inhibitory effect on apple leaf cytosolic FBPase activity. Sorbitol increased the susceptibility of the enzyme to the inhibition by F1,6BP. The presence of sorbitol in the reaction mixture led to a reduction in the enzyme activity.
Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.
Photosynthesis and carbohydrate metabolism in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] source leaves were monitored during a 7-day period after source-sink manipulations by girdling or partial defoliation treatments. In the girdling treatment, sorbitol, sucrose, glucose, and starch accumulated in leaves, and net photosynthetic rates (Pn) at 350 μL·L-1 CO2 decreased during a 7-day period. Pn measured at 1000 μL·L-1 [CO2] was also decreased but the changes were less. Stomatal conductance and intracellular CO2 concentration decreased markedly in leaves of girdled shoots. When shoots were partially defoliated, starch and glucose concentrations in remaining source leaves declined steadily during the 7-day study period. Sorbitol and sucrose concentrations decreased during the first 2 days after defoliation, then increased the following 5 days. Pn of the remaining leaves measured at ambient and elevated CO2 levels were enhanced markedly. Aldose-6-phosphate reductase activity in source leaves increased markedly from 27.5 to 39.2 μmol·h-1·g-1 fresh weight (FW) after partial defoliation but remained unchanged in leaves after girdling. Selective and maximum sucrose phosphate synthase (SPS) activities increased following partial defoliation and decreased following girdling. ADP-glucose pyrophosphorylase activity remained relatively unchanged in the partial defoliation treatments but increased markedly in the girdled-shoot leaves. These results suggested that girdling-induced photosynthetic inhibition is mainly due to stomatal limitation, however, the photosynthesis enhancement by partial defoliation may be due primarily to acceleration of photosynthetic capacity per se. These studies showed that the metabolism of sorbitol, sucrose and starch, three photosynthetic end products in mature apple leaves, was coordinately regulated in source leaves in response to source-sink manipulations.
The reaction catalyzed by ADP-glucose pyrophosphorylase (AGPase) to form ADP-glucose is a regulatory and rate-limiting step in starch synthesis in plants. In response to decreased sorbitol synthesis, starch synthesis was up-regulated in the transgenic apple plants. In this study, we examined both redox and metabolite regulation of AGPase to understand the mechanism responsible for the up-regulation of starch synthesis. No difference in the monomerization/dimerization of apple leaf AGPase small subunits was observed between the transgenic plants and the untransformed control. NADP-dependent malate dehydrogenase, indicative of chloroplastic redox status, did not show significant change in the transgenic plants either. Determination of key metabolites with nonaqueous fractionation indicated that concentrations of hexose phosphates (mainly glucose-6-phosphate and fructose-6-phosphate) were higher in both the cytosol and chloroplasts of the transgenic plants than in the control, whereas 3-phosphoglycerate (PGA) concentration in the chloroplast was not higher in the transgenic plants. We conclude that accumulation of hexose-phosphates results in a decrease in inorganic phosphate (Pi) concentration and an increase in PGA/Pi ratio in the chloroplast, leading to up-regulation of starch synthesis via activating AGPase.
Considering starch synthesis was enhanced in leaves of transgenic apple trees with decreased sorbitol synthesis, we hypothesized that starch degradation must be up-regulated correspondingly to maintain carbon supply to sink tissues. Compared with the untransformed control, mature leaves of the transgenic plants had a larger drop in starch concentration between dusk and pre-dawn, higher maltose concentration, and higher activities of two key enzymes in starch degradation: -amylase and cytosolic glucosyltransferase during the day and night. 14C-maltose and 14C-glucose were fed to the apple leaves to study the fate of starch breakdown products in the synthesis of sorbitol and sucrose. Under light, a larger proportion of both 14C-maltose and 14C-glucose were converted to sorbitol than to sucrose in the untransformed control, whereas conversion of 14C-maltose and 14C-glucose to sucrose predominated over that to sorbitol in the transgenic apple leaves. The leaf samples fed with 14C-maltose and 14C-glucose in the dark are still being analyzed, but it appears that sucrose is the main product in both the untransformed control and the transgenic plants. These results support the hypothesis that starch degradation is up-regulated in the transgenic plants.
Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family, but its physiological role remains unclear. In this study, we determined the effect of decreased sorbitol synthesis on the antioxidant system that scavenges reactive oxygen species (ROS) in apple leaves. Sorbitol synthesis was decreased in apple leaves by antisense inhibition of aldose-6-phosphate reductase activity. Dehydroascorbate reductase (DHAR), glutathione reductase, and catalase (CAT) activities increased in the leaves of the transgenic plants with decreased sorbitol synthesis, whereas superoxide dismutase, ascorbate peroxidase, NADH dependent and NADPH dependent monodehydroascorbate reductase activity did not show significant changes. Ascorbate and glutathione concentrations were higher in leaves of the transgenic plants compared with the control. The effect of decreased sorbitol synthesis on the antioxidant enzyme activity was dependent on leaf developmental stages. Larger changes in the enzyme activities of CAT, DHAR, and GR were observed in the old leaves than in the young leaves. These results suggest that sorbitol may play a role in ROS scavenging in apple leaves.
Big fruit size and nice red pigmentation combined with good flavor should be the major target for red-fleshed kiwifruit (Actinidia spp.) breeding programs. Genetic diversity and plant characteristics were evaluated on a set of kiwifruit accessions with predominantly red flesh to identify the superior individuals for further breeding or study of commercial application. The leading phenotypic characters varied widely among the accessions. Accession R reached average fruit weight ≈100 g, whereas it ranged from 43.15 to 84.71 g for the other accessions. Fruits of L and Q were flatter in shape than the others. The core volume accounted for fruit proportions ranging from 2.33% to 11.42%. ‘Chuhong’, ‘Honghua’, and K exhibited a round fruit apex, whereas most others showed a depressed apex. R, L, and Q had the highest a* values in the inner pericarp and also the most appealing visual coloration. Results revealed significantly higher soluble solid content (SSC), total sugar, and sugar/acid ratio in Q, R, and L. The 12 pairs of simple sequence repeat (SSR) markers were successfully used to characterize the genetic variability and confirm true-to-type identity for four accessions. However, the limited number of markers had no ability to discriminate among the other 11 accessions. Based on additional 28 SSRs, six of the indistinguishable accessions were confirmed to be genetically different, and three seemed to belong to the same clone vine. The results demonstrated that application of SSR data could improve the efficiency of identifying red-fleshed kiwifruit germplasm.