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.
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).
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.
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.
Kenneth Marsh, Pedro Gonzalez, and Ed Echeverría
Tonoplast vesicles isolated from juice cells of mature `Valencia' oranges [Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (V-PPiase) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ΔpH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PPiase was not able to significantly amplify the existing ΔpH. Although not able to function as a H+ pump, V-PPiase effectively synthesized PPi in the presence of inorganic phosphate (Pi). Formation of PPi by V-PPiase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PPiase from mung bean [Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PPiase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PPiase of mature orange juice cells acts as a source of inorganic pyrophosphate (PPi) but not as a H+ pump. We propose that synthesis of PPi provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage.
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.
Diego Pozueta-Romero, Pedro Gonzalez, Ed Etxeberria, and Javier Pozueta-Romero
Sucrose accumulation into sink cells consists of multiple components collectively characterized by a biphasic kinetics curve. Whereas the hyperbolic phase at low external sucrose concentration denotes a membrane-bound, carrier-mediated component, the linear nonsaturable phase at higher concentrations has been suggested to represent facilitated diffusion. We recently demonstrated the occurrence of fluid phase endocytosis (FPE) uptake of sucrose in heterotrophic cells. To investigate the involvement of this process within both phases of the sucrose accumulation curve, we analyzed the effect of phloridzin and latrunculin-B (sucrose/H+ symport and endocytosis inhibitors, respectively) in the accumulation of sucrose and the endocytic marker Alexa-488 in turnip (Brassica campestris L.) storage parenchyma cells. At low external sucrose concentration, phloridzin, but not latrunculin-B, greatly reduced sucrose accumulation. By contrast, at high external sucrose concentration, phloridzin and latrunculin-B significantly inhibited sucrose accumulation. In addition, latrunculin-B exerted a pronounced inhibitory effect on Alexa-488 uptake at any external sucrose concentration. Our results indicate that carrier-mediated and endocytic uptake (at different magnitudes) of sucrose take place at any external sucrose concentration; that within the hyperbolic phase of the sucrose accumulation curve, most of the sucrose accumulating in the cell enters via plasmalemma-bound carrier(s); and that within the linear phase, plasmalemma-bound carriers and FPE are determinants of sucrose accumulation, with the involvement of FPE increasing parallel to external sucrose. Confocal laser scanning microscopy observations confirmed the increased involvement of FPE at higher external sucrose concentrations.
Ed Etxeberria, Pedro Gonzalez, Ariel Singerman, and Timothy Ebert
Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candidatus Liberibacter asiaticus (CLas) titer has an inherent element of imprecision because CLas titer varies considerably within the tree canopy and with calendar seasons. In addition, the destructive sampling method used to determine CLas titer entails a different set of leaves per sampling period adding to the inconsistency and inexactitude of the results. To overcome these ambiguities and to reduce the numerical variability between samples, we developed an experimental method that analyzes portions of the same treated leaves for up to four sampling periods. By assaying subsamples of adjacent locations of the same leaf, random variability was significantly reduced, and comparative analysis can be carried out with greater precision.
Andreas Brune, Mathias Müller, Lincoln Taiz, Pedro Gonzalez, and Ed Etxeberria
Vacuolar acidification was investigated in `Palestine' sweet (Citrus limmetioides Tanaka) and `Persian' acid lime [(Citrus aurantifolia (Christm.) Swingle] (vacuolar pHs of 5.0 and 2.1, respectively) using tonoplast vesicles isolated from juice cells. The ATPase activity of tonoplast-enriched vesicles from sweet limes was strongly inhibited by bafilomycin A1 and NO3 -, but was unaffected by vanadate. In contrast, the ATPase activity in acid lime membranes was only slightly inhibited by bafilomycin A1 and NO3 - and was strongly inhibited by high concentrations of vanadate. The vacuolar origin of the acid lime vesicles was confirmed by immunoblotting. After solubilization and partial purification of the two enzymes by gel filtration, their inhibitor profiles were largely unchanged. Based on equal ATPase activities, vesicles from sweet and acid limes were able to generate similar pH gradients. However, in tonoplast vesicles from sweet limes, the maximum ΔpH was reached four times faster than in those from acid limes. Addition of ethylenediamine tetraacetic acid (EDTA) to chelate Mg+2 after the maximal ΔpH was attained resulted in collapse of the pH gradient in vesicles from sweet limes, whereas no change in ΔpH was observed in vesicles from acid limes, indicating a less H+ permeable membrane. Vacuolar ATPases from both cultivars exhibited identical pH optima and showed similar Mg+2 dependence, but only the acid lime ATPase activity was inhibited by Ca+2. These data confirm that the vanadate-sensitive form of the V-ATPase found in lemon and acid limes is specific to hyperacidifying tissues rather than to citrus juice cells. Sweet lime vacuoles bear the classical V-ATPase also found in vegetative plant tissues.
Giuseppe Cimò, Riccardo Lo Bianco, Pedro Gonzalez, Wije Bandaranayake, Edgardo Etxeberria, and James P. Syvertsen
The most important worldwide problem in citrus production is the bacterial disease Huanglongbing (HLB; citrus greening) caused by a phloem-limited bacterium Candidatus Liberibacter asiaticus. The earliest visible symptoms of HLB on leaves are vein yellowing and an asymmetrical chlorosis referred to as “blotchy mottle,” thought to be the result of starch accumulation. We tested the hypothesis that such visible symptoms are not unique to HLB by stem girdling 2-year-old seedlings of two citrus rootstocks with and without drought stress in the greenhouse. After 31 days, girdling had little effect on shoot growth but girdling increased the relative growth rate of shoots in drought-stressed trees. Starch content in woody roots of non-girdled trees was three to 19 times higher than in girdled trees. In non-girdled trees, drought stress induced some starch accumulation in roots, but there were no effects of drought stress on root starch or sucrose in girdled trees. Girdling induced a 4-fold greater starch content in leaves on well-watered trees but leaf sucrose content was unaffected. Girdling reduced leaf transpiration in well-watered trees but net assimilation of CO2 was unaffected by girdling or leaf starch accumulation. Leaves on girdled trees clearly had visible blotchy mottle symptoms but no symptoms developed on non-girdled trees. The increase in leaf starch, up to 50% dry weight (DW), resulted in an increase in leaf DW per leaf area (LA) and an artificial reduction of many leaf nutrients on a DW basis. Most of these differences disappeared when expressed on a LA basis. Leaf boron (B), however, was inversely related to leaf starch when both were expressed on a LA basis. In the absence of HLB, girdling increased leaf starch, decreased root starch, and duplicated the asymmetric blotchy mottled visual leaf symptoms that have been associated with HLB-infected trees. This supports our contention that such symptoms generally attributed to HLB are not uniquely related to HLB infection, but rather are directly related to starch accumulation and secondarily to nutrient deficiencies in leaves.