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Larry Parsons and Ed Etxeberria

Moderate water stress applied to citrus trees can increase fruit °Brix. Florida citrus growers have been required to use less irrigation as competition for water increases. The goal of this study was to see if irrigation could be eliminated in the fall and winter with few negative consequences. Water stress was imposed over a 3-year period on `Hamlin' and `Valencia' oranges (an early and late variety, respectively) by stopping irrigation in late October and restarting it in March. `Hamlin' fruit was harvested after 2 months of stress and `Valencia' fruit was harvested after 4 to 5 months of stress. In most years, °Brix was increased by water stress. Over a 3-year period, `Hamlin' yield was not affected by the water stress, but `Valencia' yield decreased slightly in the 3rd year. The increase in °Brix is beneficial economically, and, over 3 cm of irrigation water per year can be saved.

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Ed Etxeberria and Pedro Gonzalez

The mechanisms of sucrose uptake into the vacuole and sucrose efflux from the vacuole were studied using tonoplast vesicles from red beet at two distinct developmental stages. Vesicles from both developing and mobilizing hypocotyls (sucrose uptake and efflux, respectively) accumulated sucrose against a concentration gradient. However, higher rates and maximal levels of sucrose accumulation were obtained with tonoplast from developing hypocotyls. ATP-dependent sucrose efflux was more pronounced in vesicles from mobilizing hypocotyls. Despite the apparent overlapping, the data indicate that the physiological mechanisms for sucrose uptake and sucrose efflux are separated in time and governed by the developmental state of the cell. Chemical name used: adenosine 5′-triphosphate (ATP).

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Ed Etxeberria and Pedro Gonzalez

By combining the principles of density gradient separation and two phase partitioning, we devised a system to obtain highly pure plasmalemma and tonoplast vesicles from citrus (Citrus limettioides Tan.) juice cells. Both tonoplast and plasmalemma fractions were virtually free from golgi, endoplasmic reticulum and mitochondria contamination. Plasmalemma and tonoplast samples were also clean from each other cross-contaminants. Immediately after isolation, 72% of the plasmalemma and 82% of the tonoplast vesicles were oriented rightside-out according to enzyme marker activities. After freezing and thawing, however, plasmalemma vesicles re-oriented evenly but orientation of tonoplast vesicles remained unchanged. Differential changes in marker activities before and after freezing and thawing indicated that the low levels of plasmalemma contamination within the tonoplast fractions were due to the presence of a separate population of plasmalemma vesicles and not to the existence of hybrid vesicles. The method described in this communication allows for future studies on photoassimilate accumulation in cells of important horticultural storage organs.

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Larry R. Parsons* and Ed Etxeberria

Earlier work has shown that moderate water deficits imposed on citrus trees can increase fruit Brix without adversely affecting yield. Increased water restrictions have been imposed on citrus growers as Florida's population continues to increase. The objectives of this study were to determine: 1) the effects of no irrigation in the fall and winter on orange fruit quality parameters in Florida; and 2) amount of potential irrigation water savings. Trees were irrigated identically in the spring and summer. A non-irrigation treatment was started on Hamlin and Valencia oranges in September and October, respectively, while controls continued to be irrigated following established irrigation practices. An additional non-irrigated treatment was started at the same time on the Valencias that consisted of a soil covering with a water barrier (Tyvek) to exclude rainfall. Stem water potential was monitored during the fall and winter to estimate differences in water stress among the treatments. Brix and organic acids increased in fruit from non-irrigated treatments when compared to fruit from irrigated trees. Results also demonstrate that reduced irrigation did not affect yield greatly. Amount of irrigation savings was determined for both cultivars that differ in maturity dates.

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Brandon R. Hockema and Ed Etxeberria

The nature of sink strength in orange fruit and changes occurring during drought stress were investigated. Potted trees of `Hamlin' orange [Citrus sinensis (L.) Osbeck] grafted on Troyer citrange [Citrus sinensis × Poncirus trifoliata (L.) Raf.] were irrigated using a microsprinkler system creating either well-watered or water-stressed conditions, as determined by stem water potentials. Fruit were harvested every other week from trees of both well-watered and drought-stressed treatments during the final stage of fruit development when sugars accumulate rapidly. Fruit quality indices and activities of sucrose synthase (SuSy), invertase, sucrose-P synthase, sucrose-P phosphatase, V-ATPase, and V-PPase were measured. Acids and soluble sugar concentrations were elevated in drought-stressed fruit, whereas juice pH decreased in those same fruit. Results indicate that increased sink strength in fruit from stressed trees was accompanied by an increase in SuSy activity and lowered juice pH. The remaining enzymes examined in this experiment showed no changes in activity between control and treated fruit, as was the case for plasmalemma and tonoplast sucrose carriers. Based on the present data, we conclude that SuSy and vacuolar pH are the predominant factors controlling photoassimilate accumulation in orange fruit under enhanced sink conditions brought about by imposition of a mild drought stress.

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Ed Etxeberria, William M. Miller and Diann Achor

Fruit etching is an alterative means to label produce. Laser beam-generated pinhole depressions form dot-matrix alphanumerical characters that etch in the required price-look-up information. Pinhole depressions can disrupt the cuticular and epidermal barriers, potentially weakening the natural protection against pathogens. In the present study we describe the anatomical and morphological characteristics of the pinhole depressions in the cuticle/epidermis, and the changes taking place during storage of two fruits: avocado (Persea americana) and tomato (Lycopersicon esculentum). These fruits represent the extremes from a thick, non-edible peel to a thin edible peel. On both tomato and avocado, etching depressions were fairly similar in diameter and depth, averaging 200 μm and 25 μm, respectively, for energy impact durations of 30 μs for tomato and 45 μs for avocado. Immediately after etching, the two- to five-cell-deep depressions contained cuticle/wax deposits. Additional cuticle/wax material was deposited in and around the depressions during storage as demonstrated by confocal, fl uorescent, and light microscopy. In addition, the cells underlining the etch depression increased phenolic and lignin deposits in their walls, creating a potential barrier against pathogenic organisms.

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Naveen Kumar, Fnu Kiran and Ed Etxeberria

Citrus fibrous roots are vital for absorption and transport of water, nutrients, and other endogenous plant growth regulators. Efficient functioning of these roots in Huanglongbing (HLB)-affected citrus trees is important for their survival. One-year-old ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck) trees on Swingle citrumelo were budded with HLB-infected budwood to determine the HLB-induced pathological responses at the ultrastructural level of different fibrous root orders. The fibrous root mass was dissected into four root orders: fourth-order (attached to a thick rudimentary taproot), third-order (attached to the fourth-order root), and second-order roots (attached to the third-order root). We were not able to study the ultrastructure of the first-order (attached to the second-order root) roots in this study. Severe loss in fibrous root mass was observed within 1 year following HLB infection. All root orders displayed various degrees of HLB symptoms. The fourth-order roots comprised normal phloem and disintegrated phloem. Some vascular bundles had completely disintegrated phloem tissue, whereas others showed normal ultrastructure. The fourth-order roots were also deficient in starch granules compared with controls. The pattern of phloem disintegration was similar in the third- and second-order roots. A thick layer of necrotic phloem developed near cortical cells, while the rest of the phloem structure remained normal in the third- and second-order roots. Cortical cells of both third and second orders were enriched with starch granules; therefore, soluble carbohydrates are most likely not the limiting factor for root decline in these root orders. The xylem anatomy displayed heptarch to pentarch morphology in the various root orders. These observations confirmed that various root orders in the fibrous root system are distinct and exhibit varied pathological responses during HLB pathogenesis. We propose that photosynthates deprived fourth-order roots in conjunction with necrotic phloem promoted decline in all root orders and impaired the translocation process to aboveground plant parts.

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Pedro Gonzalez, James P. Syvertsen and Ed Etxeberria

Although citrus trees are considered relatively salt-sensitive, there are consistent differences in Na+ and Cl tolerance among different citrus rootstocks. We grew uniform seedlings of rough lemon (RL) and the more Na+-tolerant Swingle citrumelo (SC) with and without 50 mm NaCl for 42 days. Salinity reduced leaf chlorophyll and plant transpiration rate (Ep) more in RL than SC. Confocal laser scanning analyses using the Na+-specific cell-permeant fluorescent probe CoroNa-Red revealed a higher capacity for Na+ sequestration in root tissue vacuoles of SC than in RL roots and that cell walls within the stele acted as Na+ traps. In leaves, however, RL had significantly higher Na+-dependent fluorescence than SC. Thus, the sequestration of Na+ in root tissue vacuoles and its immobilization by cell walls were key contributing mechanisms enabling SC leaves to maintain lower levels of Na+ than RL leaves. Examination of intracellular distribution of CoroNa-Green fluorescence in SC root protoplasts verified a vacuolar localization for Na+ in addition to the presence of a 2- to 6-μm unidentified endosomal compartment containing significantly higher Na+ concentrations.

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Ed Etxeberria, Pedro Gonzalez and Javier Pozueta-Romero

To investigate the mechanisms of sucrose transport and its accumulation into `Murcott' mandarin (Citrus reticulata Blanco) fruit, developmental changes in determinants of sink strength such as sucrose metabolizing enzymes, and sucrose transport across both plasmalemma and tonoplast were analyzed. Concurrently with sucrose levels, sucrose synthase, sucrose phosphate synthase and sucrose phosphate phosphatase increased throughout fruit development. Plasmalemma and tonoplast vesicles isolated from fruit collected at different developmental stages were analyzed for their transport capabilities. Sucrose uptake into energized plasmalemma vesicles was abolished by gramicidin, which is in accordance with the presence of an active symport mechanism of sucrose transport from the apoplast into the cytosol. Unexpectedly, tonoplast vesicles were shown to lack active transport mechanism of sucrose into the vacuole. More importantly, however, and in conformity with recent findings showing the occurrence of an endocytic mechanism of ion uptake in maize (Zea mays L.) root cells, citrus (Citrus L.) juice cells were shown to incorporate membrane impermeable dyes into their vacuoles in the presence of sucrose. High definition confocal microscopy revealed the co-localization of membrane impermeable markers in cytoplasmic vesicles and the formation of vesicles at the plasmalemma. The data provide evidence for an endocytic system of transport that allows direct incorporation of sucrose from the apoplast to the vacuole bypassing both the plasmalemma and tonoplast.

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Ed Etxeberria, Pedro Gonzalez and Javier Pozueta

To determine whether the mechanisms of sucrose accumulation into the low acid `Sweet Lime' (Citrus limmetioides Blanco) juice cells are consistent with those previously reported for the more acidic cultivars, we followed similar developmental changes in determinants of sink strength. In addition, we followed the incorporation and distribution of quantum dots and fluorescent endocytic probes into the cell with time of incubation. As in other citrus fruits, sucrose levels, sucrose synthase, sucrose phosphate synthase, and sucrose phosphate phosphatase increased throughout fruit development. The pH however, was much higher than in the more acidic cultivars. Sucrose uptake into energized plasmalemma vesicles was inhibited by gramicidin, in accordance with the presence of an active symport mechanism of sucrose from the apoplast into the cytosol. On the contrary, tonoplast vesicles were shown to lack active transport mechanism of sucrose into the vacuole. In conformity with recent findings showing the occurrence of an endocytic mechanism in `Murcott' mandarin, `Sweet Lime' juice cells were shown to incorporate membrane-impermeable dyes into their vacuoles in the presence of sucrose. High-definition confocal microscopy revealed the co-localization of membrane-impermeable markers in cytoplasmic vesicles, in membrane-bound intermediate structures such as the endosome and multi-vesicular body, and the eventual distribution of such fluorescent particles. The data provide strong evidence for an endocytic system of transport that allows direct incorporation of sucrose from the apoplast to the vacuole and for the visualization of intermediate distribution and cargo centers in the cell.