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Kurt D. Nolte, Andrew D. Hanson and Douglas A. Gage

Proline and various betaines can function as osmoprotectants and cryoprotectants when accumulated in the cytoplasm of cells. Genetic engineering can raise levels of these compounds and thereby improve stress resistance; Citrus species are potential candidates for this. Before attempting such engineering, it is necessary to characterize the natural osmoprotectants of Citrus and related genera. We therefore surveyed 55 cultivated and wild species of the Aurantioideae, analyzing proline and betaines in leaves of mature trees. Some citrus relatives accumulated proline alone; others accumulated proline and proline betaine, as did all Citrus species studied. The levels of these two compounds ranged from about 20 to 100 μmol·g-1 dry mass, and were significantly inversely correlated. Proline betaine is known to be synthesized from proline and to be a better osmoprotectant. Because Citrus species all have more proline than proline betaine, there is scope for engineering more of the latter. Many species had small amounts of hydroxyproline betaine; other betaines were essentially absent. The lack of other betaines means that it would also be rational to engineer the accumulation of glycine betaine or similar compounds.

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Kurt D. Nolte, Gregory W. Erdos and Karen E. Koch

Localization of sucrose synthasa (SS), an enzyme Previously shown to be highly active in transport tissues of citrus fruit, was further defined via immunohistochemical analysis of stage II calamondin fruit. Using the indirect immunogold technique, 8 μm sections were first reacted with rabbit anti-SS polyclonal serum followed by incubation with 5 nm gold conjugated goat-anti-rabbit IgG. Little immunolabel was observed in the majority of peel tissues, however an abundant immunoreaction was evident in parenchyma cells directly adjacent to the segment epidermis surrounding juice sacs. Antibody was not associated with this epidermnl layer. Similarly, in juice vesicle stalks (JVS) the internal parenchyma cells showed significant SS localization compared to minimal immunoreaction in the epidermal layers of the JVS. Although the antigen did not appear to be specifically localized within the vascular bundles, an extensive distribution of the enzyme was associated with the parenchymatous cells immediately adjacent to vascular strands.

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Kurt D. Nolte, Eugene A. Nothnagel and Charles W. Coggins Jr.

Studies were conducted to determine whether certain physiological effects of gibberellic acid (GA3) on the peel of citrus fruits may be attributed to GA3 interaction with cellular membranes. Excised mesocarp tissue from pummelo [Citrus maxima (Burm.) Merrill] fruits was analyzed for electrolyte and K+ release over time in varying concentrations of GA3. Electrolyte leakage and K+ efflux was significantly reduced (up to 30%) when tissue was incubated in the presence of GA3. GA3 improved the viability of mechanically isolated protoplasts during 72 hr of storage at 7C, as shown by the use of fluorescein diacetate. These results suggest that some of the GA3-elicited responses in citrus fruits may be membrane related.