Progress 09/15/05 to 09/15/06
Outputs
Tonoplast isolation and characterization of all 6 cultivars, and continuation of acidity measurements constituted the major advances during the second year. All tonoplast samples (40 in total) were assayed individually. For each variety, between 5 and 8 samples were collected depending on variety and developmental requirements. ATPase activity and formation of a pH gradient resulting from the V-ATPase H+ activity and accumulation within the vesicles was measured simultaneously. Testing for tonoplast integrity and H+ leakage involves an ATP trap, established by adding mannose and hexokinase. Depletion of ATP disrupts further H+ pumping into the interior of the vesicle. In the absence of H+ pumping, leakage becomes the overriding factor in the quenching of acridine orange. Leakage of protons reverses the quenching of acridine orange and observed as an increase in OD. In all fruit samples, maximum ΔpH measured by changes in OD of acridine orange was approximately
0.8 units. Interestingly, steady state ΔpH was achieved faster in sweet limes vesicles than in acid limes. When plotted against time of development, the capacity of vesicles acidification per unit ATPase activity in all Valencia's declined at similar rates. Acidification in both lime samples remained fairly constant. Although the rate of decline was similar in all varieties, the absolute values were distinct. Acid limes had the highest ΔpH/unit V-ATPase, followed by sweet lime. All 4 Valencia cultivars had the lowest ΔpH/unit V-ATPase. Highest pH gradients are evidently formed by acid lime tonoplast vesicles followed by sweet lime and Valencia cultivars. A major difference amongst cultivars was the degree of membrane H+ leakage. In sweet limes, a large recovery in absorbance indicates movement of H+ out of the vesicle until the establishment of equilibrium. The degree of proton leakage was much lower in the Valencia sample shown. In the highly acid limes, however, there
was virtually no had H+ leakage. Therefore, at an early stage of development, drastic differences in H+ leakage are noted which are directly proportional to fruit acidity at maturity. The tonoplast of the highly acid lime was practically impermeable at all stages of development. In sharp contrast, sweet lime tonoplast was highly permeable at early stages, degree of leakage declining throughout development. In general, Valencia tonoplast also became less permeable throughout development except for Ory Lee cultivar. This exception amongst Valencia cultivars is noteworthy given that this particular cultivar differs from the others in its high acid content. In fact, the permeability of Ory Lee is much more comparable to acid limes than to any other Valencia cultivar. This crucial difference is more evident when data for all varieties is plotted in the same figure. Acid lime and Ory Lee Valencia (the two most acidic fruits) are the only two varieties with virtually no tonoplast H+ leakage.
Sweet lime however had substantial leakage during initial stages of development. Other Valencia cultivars were intermediate and no significant differences were noted.
Impacts
Differences between the capacities of vacuole acidification, H+ permeability, citrate synthesis, and utilization impact different Citrus varieties within their distinctive acid characteristics. In our studies, determinations of various physiological properties are discovering differences that can be used to devise future experiments to control acidity (and hence quality) in Citrus fruits. The results of this project, together with recent data from other laboratories, will allow us to determine the individual steps to modify in order to manipulate acid concentration in citrus fruits. Furthermore, this data may be applicable to other fruits such as tomato, pineapple, etc. The impact of this research is also due to its timelines. As all the molecular aspects of all elements controlling fruit acidity are unraveled, we now describe which factor and at which time changes need to be made.
Publications
- No publications reported this period
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Progress 09/15/05 to 09/14/06
Outputs
The final of experimentation year was devoted to completion of experiments dealing with citrate uptake into isolated tonoplast vesicles. Previously isolated vesicles from all varieties being investigated were tested for total V-ATPase activity and their capacity to create a pH gradient. In addition, proton leakage through tonoplast (a prime characteristic of highly acid varieties) was determined after addition of an ATPase trap. Tonoplast vesicles from all varieties, independent from final vacuolar pH, were able to generate similar pH gradient under the experimental conditions used. The data on proton leakage showed that although vesicles in all varieties were virtually impermeable to protons at later stages of development, leakage was inversely proportional to final acidity in early stages. Citrate acid accumulation was tested under 2 separate conditions. In one condition, ATP/citrate translocator was isolated, whereas in the other condition total citrate accumulation
was tested. Total acid accumulation is the sum of ATP/citrate translocator and citrate entering electrochemically. All these measurements were made at 6 different stages of development. The data does not allow to distinguish developmental differences other than both limes had a higher ATP citrate transporter capacity during the final developmental stages. Western blot analysis of cytosolic aconitase was completed and results are in accordance to acid utilization during later stages of development.
Impacts
The data generated in this year extends the overall understanding of citrate accumulation in Citrus fruits. These data were instrumental in completing the overall series of measurements needed to generate a complete assessment of citrate uptake in citrus fruits. This data will add to the volume of data published aimed at understanding the fundamental mechanism of citrate synthesis and uptake in Citrus fruits. The data will impact not only physiologists but molecular biologists as well.
Publications
- No publications reported this period
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