Progress 09/15/03 to 09/14/06
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
|
Progress 10/01/04 to 09/30/05
Outputs
A complete developmental analysis of citric acid content in 6 different Citrus fruit cultivars was completed, and the second year analysis was initiated. The study aimed at determining factors regulating citrate content in citrus fruits also investigated activities of enzymes involved in citric acid metabolism such as citrate synthase and aconitase. The selected cultivars are characterized for their differences in citric acid content due to climatic influence or to genetic variation. Except for sweet lime, all remaining cultivars started at about 3% citric acid at 100 days after flowering (DAF). Citric acid sharply increased in acid limes throughout development to a final content of to 5.5% at maturity (300 DAF). In all Valencia fruits, citric acid declined with samples from Puerto Rico declining first, followed by Florida and California. At 300 DAF, Valencia fruit from Puerto Rico and Florida had the same citric acid content (0.5%), whereas California fruit retain
1.2% acid. Acid content in an isogenic Valencia line (OR) developed at UF remained relatively constant at 2.2%. As expected, citric acid content in Sweet lime was the lowest and virtually non-existent throughout development. Citrate synthase activity did not follow a particular relationship with citric acid content. Whereas activity remained fairly constant in sweet lime, and Valencia cultivars from CREC and OR, it declined drastically between 100 and 150 DAF in Valencia from Puerto Rico and California. The most unexpected result was the sharp decline in activity in samples from acid lime being the lowest activity after 150 DAF at 10 m-units/gm fresh fruit. Aconitase activity was measurable only at early stages of fruit development between 90 and 120 DAF. Differences were minor between these dates and activity disappeared after 120 DAF in all cultivars.
Impacts
Understanding the mechanisms of acid synthesis, accumulation and degradation in Citrus fruits is fundamental for the future design of experimental approaches aimed at controlling its concentration. Although relatively low in concentration compared to sugars, citric acid is key to flavor and quality. The information gathered in this project will describe several metabolic processes that lead to the synthesis, accumulation and degradation of citric acid in Citrus fruits. Once these precesses have been described, desired genetic modifications can proceed altering maturity standards to satisfy consumer demands. In Florida, this may translate to the development of varieties which mature earlier the season, and varieties that can remain fresh on the tree for extended periods expanding the fresh fruit season to a year round industry.
Publications
- No publications reported this period
|
Progress 10/01/03 to 09/30/04
Outputs
Investigation of factors involved in the regulation of citric acid content in Citrus fruit commenced with the 2004-2005 fruit crop season. Although the granting period indicates research starting on 10/01/2003, for developmental studies as the one here proposed, is was necessary to delay until the flowering period of 2004. This delay was done in order to sample fruit from one continuous growing season. Fragmented developmental studies conducted between two growing seasons are meaningless, given the enormous variations due to environmental factors and internal biological rhythms. Several fruit cultivars were selected for their unique physiological characteristics in relation to acid metabolism, and for the differences in acidity resulting from variations in growing locations. These included acid lime (Citrus aurantifolia), sweet lime (Citrus limettioides), Valencia oranges (Citrus sinensis) grown under three different climatic conditions (California, Florida and Puerto
Rico), and a unique Valencia clone with altered acid metabolism. Fruit were sampled on a monthly basis starting in June 2004, taking into account the different flowering dates from different locations. Juice cells were extracted and samples processed according to appropriate methodology for enzyme analysis. Enzymes of citrate synthesis and degradation were assayed for activity in triplicates for each fruit samples. This part of the research was conducted in Florida. Concurrent fruit samples were extracted in Puerto Rico for acid analysis. On a per gram fresh weight basis, citrate synthase activity was highest early in development for all cultivars except for sweet limes, whose activity was approximately 20% of other cultivars. In all cases, activity rapidly declined until approximately 150 days after anthesis. From this date on, activity remained very low. Sweet lime citrate synthase activity, which was the lowest of all cultivars, remained almost constant throughout development.
Aconitase, enzyme of citrate degradation, activity was also elevated early in development, but dropped to unmeasurable levels at around 180 days after anthesis. By 230 days after anthesis, aconitase activity was detected again but at approximately 10% of that measured in younger fruit. Interestingly, no second activity peak was measured for acid or sweet limes. At the time of this report, several sapling points remain for the completion of the first year study, and any conclusions or suggestions will de delayed until then. Also, acid analysis and western blots for citrate synthase will be performed once all samples have been collected to eliminate experimental errors.
Impacts
Understanding the mechanisms of acid synthesis, accumulation and degradation in Citrus fruits is fundamental for the future design of experimental approaches aimed at controlling its concentration. Although relatively low in concentration compared to sugars, citric acid is key to flavor and quality. The information gathered in this project will describe several metabolic processes that lead to the synthesis, accumulation and degradation of citric acid in Citrus fruits. Once these precesses have been described, desired genetic modifications can proceed altering maturity standards to satisfy consumer demands. In Florida, this may translate to the development of varieties which mature earlier the season, and varieties that can remain fresh on the tree for extended periods expanding the fresh fruit season to a year round industry.
Publications
|
|