Progress 02/12/98 to 01/31/04
Outputs
Compared with the abundant literature on sucrose and starch metabolism, few studies have been preformed on the regulation of sorbitol synthesis. The properties of purified aldose-6-phosphate reductase (A6PR)were examined. In an effort to better understand the regulatory mechanisms of sorbitol synthesis we investigated the effects of metabolites and cations on A6PR activity in apple leaves. We found that Pi inhibited A6PR by decreasing the vmax and increasing the km for Glc6P. The divalent metal ions, Ca2+ and Mg2+ increased A6PR activity by lowering the km for Glc6P. Conversely, Zn2+ and Cu2+ inhibited A6PR activity. There results are indicative of the crucial role that A6PR performs in regulating photosynthetic carbon metabolism in apple leaves and in related sorbitol-forming species.
Impacts
The effects of drought stress is poorly understood in tree fruits such as apple and peach. Contributions of this project will determine how changes in photosynthesis, carbon partitioning and yield stability are altered under water limiting conditions. This development will significantly improve fruit production in Maryland and nationwide.
Publications
- Zhou, R., R.C. Sicher and B. Quebedeaux. 2003, Regulation of apple leaf aldose-6-phospate reductase activity by inorganic phosphate and divalent cations. Functional Plant Biology. 29:569-574.
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Progress 01/01/02 to 12/31/02
Outputs
Aldose-6-phosphate reductase (A6PR) has been purified to apparent homogeneity from apple (Malus domestica Borhk. cv. Gala) fully developed leaves. Divalent ions including Ca2+, Mg2+, Mn2+, Zn2+ and Cu2+ were shown to affect A6PR activity. The effect of Ca2+, Mg2+, or Mn2+ was metal ion concentration dependent and glucose-6-phosphate concentration dependent. In the range of 0.5 to 5 millimolar concentration Ca2+, Mg2+ and Mn2+ stimulated A6PR activity at lower glucose-6-phosphate concentration (below 10 mM) but decreased A6PR activity at higher Glc-6P level. Higher concentration of metal ions (above 5 mM) resulted in a decrease in A6PR activity. A6PR displayed Michaelis- Menten kinetics in the presence of 2.5 mM MgCl2 or without divalent ions. The apparent Km for Glc6-P decreased from 12.2 mM for the control to 2.8 mM in the presence of 2.5 mM MgCl2 in the assay mixture. Also, 2.5 mM MgCl2 lowered the Km for sorbitol 6-phosphate from 3.1 mM for the control to 1.3 mM
when A6PR activity was measured in the oxidative direction. In contrast to the above, Zn2+ and Cu2+ dramatically inhibited A6PR activity. The enzyme activity was decreased about 50 and 70%, respectively, when enzyme preparations were preincubated with 2 mM Zn2+ and Cu2+ for 60 min at room temperature. The inactivation of A6PR activity by 2 mM Zn2+ could be partially reversed by dialysis or by chelating with 20 mM EDTA. NADPH and NADP, which are substrates for A6PR in the oxidative and reductive directions, respectively, partially protected the enzyme from inactivation by Zn2+. The above findings suggested that Mg2+ and Ca2+ were mixed-type nonessential activators of A6PR that decreased the Km for sugar-phosphates and lowered the overall Vmax.
Impacts
The effects of drought stress is poorly understood in tree fruits such as apple and peach. Contributions of this project will determine how changes in photosynthesis, carbon partitioning and yield stability are altered under water limiting conditions. This development will significantly improve fruit production in Maryland and nationwide.
Publications
- Zhou, R., R.C. Sicher and B. Quebedeaux. 2002, apple Leaf Sucrose - Phosphate synthase Is Inhibited by Sorbitol-6-Phosphate. Functional Plant Biology.29:569-574.
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Progress 01/01/01 to 12/31/01
Outputs
In Rosaceae species including many important tree fruits such as apple, peach, pear, and cherry, etc., the principal photosynthetic end products are sorbitol and sucrose. Recent studies strongly suggest that sorbitol and sucrose play different roles in plant growth and development. However, biochemical factors controlling carbon partitioning between sorbitol and sucrose are unknown. Compared to other crop plants, there are few studies on the functions and regulation of sucrose biosynthesis in the Roseceae species of tree fruits. It is very difficult to get a clear view of carbohydrate metabolism in Rosaceae plants without some basic knowledge on the regulation of sucrose metabolism. During this period we report the first characterization of the kinetic and regulatory properties of apple leaf sucrose phosphate synthase (SPS), the key enzyme for sucrose biosynthesis. The long-term objectives of our studies are to investigate the relationship between sucrose synthesis
and sorbitol metabolism. SPS was shown to be present in apple leaves with an activity of 15.1 micromol per(g fresh weight)per hour. Apple SPS from mature leaves was purified 34-fold to a final specific activity of 15.3 micromol per(mg protein)per hour. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (Km=0.36 mM) and uridine 5'-diphosphoglucose (Km=6.49 mM). In apple, glucose-6-phosphate was found to be an activator of SPS and the activation was dependent upon the fructose-6-phosphate concentration. At a concentration of 2 mM, glucose-6-phosphate significantly decreased the Km for fructose-6-phosphate and increased SPS activity. However, higher concentrations of glucose-6-phosphate did not further stimulate SPS activity. In contrast to SPS from other species, inorganic phosphate had little or no inhibitory effects on apple SPS. The apple leaf enzyme was inhibited 7 to 10% by 10 mM of inorganic phosphate when fructose-6-phosphate concentrations were in the
range of 2 to 10 mM. We observed that sorbitol-6-phosphate, an intermediate metabolite in sorbitol biosynthesis, was a competitive inhibitor of SPS with a Ki of 1.83 mM. Sorbitol-6-phosphate also inhibited glucose-6-activation of SPS. Our results suggest that sucrose biosynthesis may be altered by the products of sorbitol biosynthesis in apple leaves.
Impacts
The effects of drought stress is poorly understood in tree fruits such as apple and peach. Contributions of this project will determine how changes in photosynthesis, carbon partitioning and yield stability are altered under water limiting conditions. This development will significantly improve fruit production in Maryland and nationwide.
Publications
- Quebedeaux, B.,Z. Wang, R. Zhou, and V.R. Reddy. 2000. A Model Database for Apple Growth, Phenology and Photosynthesis. Proceedings of the 30th Annual Crop Simulation Workshop. Biological Systems Simulation Group. 30:30.
- Zhou,R., R.C. Sicher and B. Quebedeaux. 2001. Diurnal changes in Carbohydrate Metabolism in Apple Mature Leaves. Australian Journal of Plant Physicology. 28(11):000.
- Lamp, W.O., G.R. Nielsen and B. Quebedeaux. 2001. Potato Leafhopper Injury Disrupts Basal Transport of C14-labeled photoassimilates in Alfalfa. Journal of Economic Entomology. 94(1):93-97.
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Progress 01/01/00 to 12/31/00
Outputs
Net photosynthesis, individual carbohydrate concentrations, and the activities of some key enzymes involved in carbohydrate biosynthesis were monitored in mature apple leaves during a natural day night cycle at regular intervals throughout the diurnal cycle. Sorbitol was the major carbohydrate in apple mature leaves. Its concentration increased gradually after dawn and reached its highest level of 18.0 mg.g-1 fresh weight and then declined to its lowest level of 9.6 mg.g-1 at the end of the dark period. Starch also showed a similar diurnal pattern except for the low absolute concentration with a maximum of 4.6 mg.g-1 fresh weight and a minimum of 1.8 mg.g-1 fresh weight. The pronounced changes in sorbitol and starch concentrations indicate both sorbitol and starch are temporarily stored in apple leaves. Aldose-6-phosphate reductase (A6PR) showed significant diurnal fluctuations in activity whereas the activity of sucrose phosphate synthase (SPS) under both saturating
and limiting substrate conditions showed no variations during the day and night. The highest A6PR activity under natural light conditions appeared at 1000 h with 37.2 umol.h-1.g-1 fresh weight, which was about 30% higher than that in the night. The diurnal alteration of A6PR was also found under controlled environmental conditions in growth chambers. Treatments of 14/10 hour light/dark photoperiod, continuous light and continuous darkness did not change the pattern of diurnal variation in A6PR activity. Significant correlation between A6PR activity, sorbitol concentration and photosynthetic rate suggest that the processes of photosynthesis and carbohydrate biosynthesis are coordinated.
Impacts
The effects of drought stress is poorly understood in tree fruits such as apple and peach. Contributions of this project will determine how changes in photosynthesis, carbon partitioning and yield stability are altered under water limiting conditions. This development will significantly improve fruit production in Maryland and nationwide.
Publications
- Quebedeaux, B., Z. Wang, R. Zhou and V. R. Reddy, 2000. A Model Database for Apple Growth, Phenology and Photosynthesis. Proceedings of the 30th Annual Crop Simulation Workshop. Biological Systems Simulation Group. 30:30.
- Zhou, R., R. C. Sicher and B. Quebedeaux. 2001. Diurnal Changes in Carbohydrate Metabolism in Apple Mature Leaves. Australian Journal of Plant Physiology. (In Press)
- Lamp, W.O., G.R. Nielsen and B. Quebedeaux. 2001. Potato Leafhopper Injury Distrups Basal Transport of 14C-labeled photoassimilates in Alfalfa. Journal of Economic Entomology. 94(1):93-97.
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Progress 01/01/99 to 12/31/99
Outputs
In order to determine whether the changes in the demand for 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 percent of source leaves in apple seedlings. 3 year old apple (Malus domestica Borhk. cv. Gala) seedlings were grown in a 15h/9h light (ca 700 umol photons.m-2.s-1,22C)/dark (18C) 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 umol CO2.m-2.s-1 over a 7 day period. When the demand for transported carbohydrates 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 umol CO2.m-2.s-1 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. 1.1.1.200), an important enzyme in sorbitol biosynthesis, increased significantly from 27.5 to 39.2 umol .h-1.g-1FW in the carbohydrate depleted leaves while it remained unchanged in the girdled leaves; the activity of sucrose-6-phosphate synthase (SPS, E.C. .4.1.14), a key enzyme for sucrose biosynthesis, increased from 15.4 to 23.0 umol.h-1.g-1FW in the depleted leaves and declined from 17.4 to 8.2 umol.h-1.g-1FW in the girdled leaves; the activity of fructose 1,6 bisphosphatase (E.C.3.1.3.11), another key
enzyme for sucrose biosynthesis in non-Rosaceae species showed a similar pattern as SPS; ADP glucose-pyrophosphorylase (E.C.2.7.7.27), a key enzyme for starch biosynthesis, decreased a small amount in the girdled leaves but increased markedly from 42.9 to 56.0 umol.h-1.g-1FW 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.
Impacts
The effects of drought stress is poorly understood in tree fruits such as apple and peach. Contributions of this project will determine how changes in photosynthesis, carbon partitioning and yield stability are altered under water limiting conditions. This development will significantly improve fruit production in Maryland and nationwide.
Publications
- Pan,Q.,Z.Wang, and B.Quebedeaux. 1998. Responses of the apple plant to CO2 enrichment: changes in photosynthesis sorbitol, soluble sugars, and starch. Australian Journal of Plant Physiology. 25:293-297.
- Wang, Z.,Z.Yuan and B.Quebedeaux. 1998. Photoperiod alters partitioning of newly-fixed 14C and reserve carbon into sorbitol, sucrose and starch in apple leaves, stems, and roots. Australian Journal of Plant Physiology. 25:503:506.
- Wiesman,Z., S. Lavee, N. Avidan and B. Quebedeaux. 1998. Molecular characterization of common olive cultivars in the Middle East. Journal of the ASHS. 123(5):837-841.
- Wang,Z., Q.Pan, and B. Quebedeaux. 1999. Carbon partitioning into sorbitol, sucrose, and starch in source and sink apple leaves as affected by elevated CO2. Environmental and Experimental Botany. 41:39-46.
- Nielsen,G.R., C.Fuentes, B.Quebedeaux, Z.Wang and W.O.Lamp. 1999. Alfalfa physiological response to potato leafhopper injury depends on leafhopper and alfalfa developmental state. Entomologia Experimentalis et Applicata. 90-247-255.
- Quebedeaux,B.,Z.Wang,R.Zhou and V.R.Reddy. 1999. A Database for Developing Simulation Models for Apple Growth and Photosynthate Partitioning. Proceedings of The 29th Annual Crop Simulation Workshop. Biological Systems Simulation Group. 29:20.
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