Vacuoles Of Leaf Cells Research Articles

Divergent roles of IREG/Ferroportin transporters from the nickel hyperaccumulator Leucocroton havanensis.

In response to our ever-increasing demand for metals, phytotechnologies are being developed to limit the environmental impact of conventional metal mining. However, the development of these technologies, which rely on plant species able to tolerate and accumulate metals, is partly limited by our lack of knowledge of the underlying molecular mechanisms. In this work, we aimed to better understand the role of metal transporters of the IRON REGULATED 1/FERROPORTIN (IREG/FPN) family from the nickel hyperaccumulator Leucocroton havanensis from the Euphorbiaceae family. Using transcriptomic data, we identified two homologous genes, LhavIREG1 and LhavIREG2, encoding divalent metal transporters of the IREG/FPN family. Both genes are expressed at similar levels in shoots, but LhavIREG1 shows higher expression in roots. The heterologous expression of these transporters in A. thaliana revealed that LhavIREG1 is localized to the plasma membrane, whereas LhavIREG2 is located on the vacuole. In addition, the expression of each gene induced a significant increase in nickel tolerance. Taken together, our data suggest that LhavIREG2 is involved in nickel sequestration in vacuoles of leaf cells, whereas LhavIREG1 is mainly involved in nickel translocation from roots to shoots, but could also be involved in metal sequestration in cell walls. Our results suggest that paralogous IREG/FPN transporters may play complementary roles in nickel hyperaccumulation in plants.

Enhanced Na+ and Cl− sequestration and secretion selectivity contribute to high salt tolerance in the tetraploid recretohalophyte Plumbago auriculata Lam.

Enhanced secretion of Na+ and Cl- in leaf glands and leaf vacuolar sequestration of Na+ or root retention of Cl-, combined with K+ retention, contribute to the improved salt tolerance of tetraploid recretohalophyte P. auriculata. Salt stress is one of the major abiotic factors threatening plant growth and development, and polyploids generally exhibit higher salt stress resistance than diploids. In recretohalophytes, which secrete ions from the salt gland in leaf epidermal cells, the effects of polyploidization on ion homeostasis and secretion remain unknown. In this study, we compared the morphology, physiology, and ion homeostasis regulation of diploid and autotetraploid accessions of the recretohalophyte Plumbago auriculata Lam. after treatment with 300mM NaCl for 0, 2, 4, 6, and 8 days. The results showed that salt stress altered the morphology, photosynthetic efficiency, and chloroplast structure of diploid P. auriculata to a greater extent than those of its tetraploid counterpart. Moreover, the contents of organic osmoregulatory substances (proline and soluble sugars) were significantly higher in the tetraploid than in the diploid, while those of H2O2 and malondialdehyde (MDA) were significantly lower. Analysis of ion homeostasis revealed that the tetraploid cytotype accumulated more Na+ in stems and leaves and more Cl- in roots but less K+ loss in roots compared with diploid P. auriculata. Additionally, the rate of Na+ and Cl- secretion from the leaf surface was higher, while that of K+, Mg2+, and Ca2+ secretion was lower in tetraploid plants. X-ray microanalysis of mesophyll cells revealed that Na+ mainly accumulated in different cellular compartments in the tetraploid (vacuole) and diploid (cytoplasm) plants. Our results suggest that polyploid recretohalophytes require the ability to sequester Na+ and Cl-(via accumulation in leaf cell vacuoles or unloading by roots) and selectively secrete these ions (through salt glands) together with the ability to prevent K+ loss (by roots). This mechanism required to maintain K+/Na+ homeostasis in polyploid recretohalophytes under high salinity provides new insights in the improved maintenance of ion homeostasis in polyploids under salt stress.

Insight into the subcellular mechanism of maximizing Cd accumulation in hyperaccumulator Solanum nigrum L. under the action of biodegradable chelating agent

Under the promotion of biodegradable chelating agents, hyperaccumulators can accumulate large amounts of heavy metals. However, in practical application, excessive accumulation of heavy metals will lead to the reduction of biomass, resulting in the reduction of heavy metal accumulation efficiency. Therefore, here we seek to investigate the dynamic process of vacuolar compartmentalization regulated by biological chelators, to provide guidance for improving the accumulation efficiency of heavy metals and alleviating the reduction of biomass. In this study, subcellular isolation was used to study the changes of enzyme activities, total cadmium (Cd), Cd2+, glutathione and reactive oxygen species (ROS) contents in protoplasts and vacuoles of leaf cells of Solanum nigrum L. The results indicate that vacuolar compartmentalization is a dynamic regulation process of continuous glutathione consumption. Under the promotion of biodegradable chelating agent N, N-bis glutamic acid (GLDA), more glutathione was induced and preferentially entered the vacuole to chelate and store Cd. Moreover, for large amounts of Cd accumulation, the insufficient supply of glutathione (GSH) preferentially maintains vacuolar homeostasis and causes redox damage outside the vacuole. These results showed that insufficient glutathione production led to the reduction of biomass. Therefore, in the practical application of GLDA, the consumption inside and outside the vacuole can be met by controlling the production of glutathione, so as to increase the efficiency of heavy metal accumulation.

Insight into the Vacuolar Compartmentalization Process and the Effect Glutathione Regulation to This Process in the Hyperaccumulator Plant Solanum nigrum L.

Vacuole compartmentalization plays an important role in the storage of heavy metals in hyperaccumulators. Is the vacuolar compartmentation a simple shielding process or a dynamic process that continuously consumes cell sap resources? How does glutathione affect the process of vacuolar compartmentalization? These unknown questions are very important to understand the mechanism of vacuole compartmentalization and can provide a guide for the design of hyperaccumulator plants by genetic engineering. Therefore, this study explored the enzyme activities, total cadmium, Cd2+, glutathione, oxidized glutathione, and reactive oxygen species contents in protoplasts and vacuoles of leaf cells in Solanum nigrum L. through subcellular separation. The results showed that vacuolar compartmentalization was a dynamic process that actively induced the related substances produced by cell sap to enter the vacuole for detoxification. When regulating the decreased glutathione content with buthionine sulfoximine, the total cadmium and combined cadmium in protoplasm decreased significantly, but the vacuole still maintained a high proportion of cadmium content and stable ROS content, which indicated that various external resources were preferentially used to maintain cadmium storage and homeostasis in vacuole rather than outside vacuole. These findings could guide the use of genetic engineering to design hyperaccumulator plants.

Salt tolerance in relation to elemental concentrations in leaf cell vacuoles and chloroplasts of a C4 monocotyledonous halophyte.

Halophytes accumulate and sequester high concentrations of salt in vacuoles while maintaining lower levels of salt in the cytoplasm. The current data on cellular and subcellular partitioning of salt in halophytes are, however, limited to only a few dicotyledonous C3 species. Using cryo‐scanning electron microscopy X‐ray microanalysis, we assessed the concentrations of Na, Cl, K, Ca, Mg, P and S in various cell types within the leaf‐blades of a monocotyledonous C4 halophyte, Rhodes grass (Chloris gayana). We also linked, for the first time, elemental concentrations in chloroplasts of mesophyll and bundle sheath cells to their ultrastructure and photosynthetic performance of plants grown in nonsaline and saline (200 mM NaCl) conditions. Na and Cl accumulated to the highest levels in xylem parenchyma and epidermal cells, but were maintained at lower concentrations in photosynthetically active mesophyll and bundle sheath cells. Concentrations of Na and Cl in chloroplasts of mesophyll and bundle sheath cells were lower than in their respective vacuoles. No ultrastructural changes were observed in either mesophyll or bundle sheath chloroplasts, and photosynthetic activity was maintained in saline conditions. Salinity tolerance in Rhodes grass is related to specific cellular Na and Cl distributions in leaf tissues, and the ability to regulate Na and Cl concentrations in chloroplasts.

Visualization and quantification of cadmium accumulation, chelation and antioxidation during the process of vacuolar compartmentalization in the hyperaccumulator plant Solanum nigrum L.

Hyperaccumulators store metals in the vacuoles of leaf cells. To investigate the role of vacuolar compartmentalization in Cd accumulation, chelation and induced antioxidation, we quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH) and reactive oxygen species (ROS) in leaf cells of Solanum nigrum L. The results confirmed that vacuoles were, indeed, the main storage compartments for Cd. We then found that with increased Cd treatment concentration, the proportion of vacuolar Cd in protoplasts showed its ultimate storage capacity (82.24 %–83.40 %), and the Cd concentration stored in the protoplast maintained at a certain level (73.81–77.46 mg L−1). Besides, studies on different forms of Cd showed that the chelation state was dominant in the protoplast. The large level appearance of Cd2+ outside the vacuole revealed the limitations of vacuolar Cd2+ sequestration. The relationships between the combined forms of Cd and GSH outside the vacuole (R2 = 0.9906) showed GSH was mainly distributed to important compartments for chelation, not to vacuoles. We also demonstrated the presence of ROS-induced oxidative stress and detoxification mediated by the antioxidant GSH in vacuoles, suggesting that sequestration into vacuoles is an active process accompanied by chelation and antioxidant-mediated detoxification.

Biochemical and Physiological Changes in Response to Salinity in Leaves and Roots of Two Durum Wheat (Triticum durum Desf.) Genotypes

Abstract Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response, two wheat (Triticum durum Desf.) cultivars, Karim and Azizi, which are of agronomic significance in Tunisia, were grown under non-saline and saline conditions (100 mM). Leaves and roots of control and salt-stressed plants were harvested after 11 days of salt treatment. Karim cultivar may behave as a typical Na+ include, which compartmentalizes Na+ within the leaf cell vacuoles where it could be used as an osmoticum to lower the osmotic potential necessary for the maintenance of the plant hydric status. While, accumulation of K+ was greater in Karim cultivar compared to Azizi, in both organs, presenting an important manifestation of salinity tolerance. Significant changes in metabolism of antioxidative system were observed, with an increase in protein tyrosine nitration, which indicates that salinity stress induces a nitro-oxidative stress.

Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants.

Plant cells orchestrate an array of molecular mechanisms for maintaining plasmatic concentrations of essential heavy metal (HM) ions, for example, iron, zinc and copper, within the optimal functional range. In parallel, concentrations of non-essential HMs and metalloids, for example, cadmium, mercury and arsenic, should be kept below their toxicity threshold levels. Vacuolar compartmentalization is central to HM homeostasis. It depends on two vacuolar pumps (V-ATPase and V-PPase) and a set of tonoplast transporters, which are directly driven by proton motive force, and primary ATP-dependent pumps. While HM non-hyperaccumulator plants largely sequester toxic HMs in root vacuoles, HM hyperaccumulators usually sequester them in leaf cell vacuoles following efficient long-distance translocation. The distinct strategies evolved as a consequence of organ-specific differences particularly in vacuolar transporters and in addition to distinct features in long-distance transport. Recent molecular and functional characterization of tonoplast HM transporters has advanced our understanding of their contribution to HM homeostasis, tolerance and hyperaccumulation. Another important part of the dynamic vacuolar sequestration syndrome involves enhanced vacuolation. It involves vesicular trafficking in HM detoxification. The present review provides an updated account of molecular aspects that contribute to the vacuolar compartmentalization of HMs.

Effect of sulphur deprivation on osmotic potential components and nitrogen metabolism in oilseed rape leaves: identification of a new early indicator.

Identification of early sulphur (S) deficiency indicators is important for species such as Brassica napus, an S-demanding crop in which yield and the nutritional quality of seeds are negatively affected by S deficiency. Because S is mostly stored as SO4 (2-) in leaf cell vacuoles and can be mobilized during S deficiency, this study investigated the impact of S deprivation on leaf osmotic potential in order to identify compensation processes. Plants were exposed for 28 days to S or to chlorine deprivation in order to differentiate osmotic and metabolic responses. While chlorine deprivation had no significant effects on growth, osmotic potential and nitrogen metabolism, Brassica napus revealed two response periods to S deprivation. The first one occurred during the first 13 days during which plant growth was maintained as a result of vacuolar SO4 (2-) mobilization. In the meantime, leaf osmotic potential of S-deprived plants remained similar to control plants despite a reduction in the SO4 (2-) osmotic contribution, which was fully compensated by an increase in NO3 (-), PO4 (3-) and Cl(-) accumulation. The second response occurred after 13 days of S deprivation with a significant reduction in growth, leaf osmotic potential, NO3 (-) uptake and NO3 (-) reductase activity, whereas amino acids and NO3 (-) were accumulated. This kinetic analysis of S deprivation suggested that a ([Cl(-)]+[NO3 (-)]+[PO4 (3-)]):[SO4 (2-)] ratio could provide a relevant indicator of S deficiency, modified nearly as early as the over-expression of genes encoding SO4 (2-) tonoplastic or plasmalemmal transporters, with the added advantage that it can be easily quantified under field conditions.

Flue gas desulfurization gypsum by-products alters cytosolic Ca2 + distribution and Ca2+-ATPase activity in leaf cells of oil sunflower in alkaline soil

Soil salinization in arid zones is a major factor that resulted in the reduction in the yield and quality of many important crops in Northwestern China. In this study, the potential mechanism of flue gas desulfurization gypsum by-product (FGDB) mediated amendment of alkaline soils was investigated in an oil sunflower model by accessing the Ca2+ distribution and Ca2+-ATPase activity in leaf cells. Our results demonstrated an increased calcium concentration, as well as intact chloroplast structure with increasing calcium precipitates in the cell wall, intercellular space, and vacuole of leaf cells in the plants grown in alkaline soils supplied with FGDB or CaSO4. Additionally, a dose-dependent Ca2+-ATPase activity was detected in the plasma membrane and tonoplast of leaf cells from the plants grown in FGDB or CaSO4 supplemented soils. These results implied that the Ca2+-ATPase activity cause cytosolic Ca2+ efflux. The Ca2+ influx is through the Ca2+-channels, and increasing cytosolic Ca2+ concentration might benefit the stability and integrity of cell membrane and cell wall, sequentially alleviated the injury of oil sunflower against alkali stress.

Plant homeostasis of foliar manganese sinks: specific variation in hyperaccumulators

Plant manganese (Mn) hyperaccumulation provides unusual insight into homeostasis of this essential micronutrient, in particular its excessive storage in shoot tissues. The compartmentation of hyperaccumulated foliar Mn appears exceptional among metal hyperaccumulators, since it occurs via specific microdistribution patterns. Here, three associated Mn hyperaccumulators, Virotia neurophylla, Maytenus fournieri, and Garcinia amplexicaulis exhibiting distinctly different Mn detoxification strategies were examined. Non-invasive sample preparation in conjunction with cryo scanning electron microscopy (SEM) was used to obtain in vivo quantitative microprobe X-ray and anatomical data from fully hydrated cells. Highly vacuolated large palisade mesophyll cells in V. neurophylla leaves were found to contain around 650mM Mn. The large non-photosynthetic hypodermal cells of M. fournieri leaves, also with high vacuolar content, and the main site for Mn disposal, had an estimated mean vacuolar Mn concentration of around 600mM. Previous qualitative X-ray mapping had shown Mn to be almost evenly sequestered across the entire leaf cross section of G. amplexicaulis. However, quantitative data obtained here showed a marked variation in localised concentrations that ranged between~15 and>800mM. Notable among these were mean values of>600mM in spongy mesophyll cells, and~800mM within cells of a narrow sub epidermal layer preceding the palisade mesophyll. This study demonstrated the extraordinary Mn carrying capacities of different types of leaf cell vacuoles.

Moving magnesium in plant cells

Magnesium (Mg) is among the most abundant mineral elements in plants, yet the knowledge of which genes control its accumulation in specific tissues and organelles lags behind that of many other mineral elements. Only in recent years has identification of important molecular play ers begun to take shape. In this issue of New Phytologist, Conn et al. (pp. 583–594) shed additional light on two Mg transporters that play important roles in accumulation of Mg in leaf cell vacuoles. Using subcellular-level ion measurements on leaves, gene expression measurements after single-cell sampling, a genetic approach, and clever use of calcium (Ca) and Mg supply to plants or detached leaves, Conn et al. have demonstrated that vacuoles of mesophyll rather than epidermal or bundle sheath cell types of Arabidopsis leaves are the main sites of Mg accumulation. They have also shown the effects of mutations in MRS2-1 and MRS2-5 genes on this accumulation and a role for these

Prophylactively parking sodium in the plant

Prophylactively parking sodium in the plant

Responses of Seidlitzia rosmarinus to salt stress

The responses of the salt-accumulating halophyte Seidlitzia rosmarinus to salt stress (500 mM NaCI) were studied. Observations were carried out over the span of 72 h. Three phases of responses were identified. Parallel observations of physiological parameters and cell ultrastructure allowed us to conclude that S. rosmarinus is capable of resisting salt stress owing to changes in its osmoregulatory systems (ions or organic osmolytes). In addition, these observations showed that salt ions are localized in the central vacuole of leaf cells (via pinocytosis) and also in small cytoplasmic vacuoles of leaf and root cells.

PH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves.

After opening the stomata in CO(2)-free air, darkened leaves of several plant species were titrated with CO(2) at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO(2) for times ranging between 4.4 and 300 s, fast CO(2) release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO(2) was released with time constants of about 5, 15 and approximately 300 s. With CO(2) as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit(-1 )in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit(-1), i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO(2) reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO(2)-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO(2) minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO(2)-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO(2) titration with and without added buffer was somewhat below 4 mM pH-unit(-1). Thus the apoplastic and cytosolic pH responses to additions of CO(2 )indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast.

Redox-state of intactNitella cells: dependency on intracellular pH and photosynthesis

The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (ERED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, ERED values of+237±93mVand+419±51 mV with respect to a normal H+-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state ERED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of ERED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic ERED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic ERED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10μM 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), ERED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.

Flux of SO2 into Leaf Cells and Cellular Acidification by SO2

A comparison of fluxes of SO(2) from the atmosphere into leaves with fluxes across biomembranes revealed that, apart from the cuticle, the main barrier to SO(2) entry into leaves are the stomates. SO(2) fluxes into leaves can be calculated with an accuracy sufficient for many purposes on the assumption that the intracellular SO(2) concentration is zero. SO(2) entering green leaf cells is trapped in the cytoplasm. In the light, the products formed in its reaction with water are processed particularly in the chloroplasts. Flux of SO(2) to the acidic central vacuole of leaf cells is insignificant. Intracellular acidification of barley mesophyll protoplasts by SO(2) was measured by the uptake of (14)C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. The measured acidification was similar to the acidification calculated from known buffer capacities and the rate of SO(2) influx when the H(+)/SO(2) ratio was assumed to be 2. A comparison of photosynthesis inhibition by SO(2) with calculated acidification revealed different mechanisms of inhibition at low and at high concentrations of SO(2). At very low concentrations, inhibition by SO(2) was even smaller than expected from calculated acidification. The data suggest that, if acidification cannot be compensated by pH-stabilizing cellular mechanisms, it is a main factor of SO(2) toxicity at low SO(2) levels. At high levels of SO(2), anion toxicity and/or radical formation during oxidation of SO(2) to sulfate may play a large role in inhibition.

A comparative study of the leaf anatomy of olive trees growing in the city and the country

The leaf anatomy, airspace system volume and other leaf parameters of an olive tree growing in the centre of the city of Thessaloniki, Northern Greece, were investigated by light and electron microscopy and other techniques and compared with samples from a rural environment. Urban leaves were found to be harder, thicker, and with a smaller leaf volume airspace system in comparison to that for rural leaves (23.6 and 31.9%, respectively). Morphometric cytological assessments revealed differences between urban and rural leaf chloroplasts; palisade chloroplasts of urban leaves contained a more extensive internal membrane system than those of rural leaves. However, no significant differences were found for spongy chloroplasts. In general, spongy chloroplasts had a larger internal membrane system volume relative to that for palisade chloroplasts. Starch grain number was greater in urban leaves, but their volume was not significantly different in the two specimens. In vacuoles of urban leaf cells dense osmiophilic substances, localized on the vacuolar side of the tonoplast, were frequently encountered; they were rarely observed in the rural leaf cell vacuoles. All differences observed might be attributed to the polluted environment of the city centre.

Symplastic Transport in Ipomea tricolor Source Leaves

A novel method for the delivery of the fluorescent dye Lucifer Yellow CH to the cytosol of a source leaf mesophyll cell was devised which utilized a preencapsulation of the dye in phospholipid vesicles (liposomes). The liposomes were easily injected into the vacuoles of leaf cells of Beta vulgaris or Ipomea tricolor, where fusion with the tonoplast resulted in the release of the dye into the cytosol. Subsequent cell-to-cell movement of the dye was readily followed by fluorescence microscopy. Using this liposome technique symplastic continuity from the the mesophyll to the minor veins of the source leaf of Ipomea tricolor was demonstrated. This agreed with ultrastructural studies which demonstrated the presence of plasmodesmata between all cells from the mesophyll to the minor veins. The symplastic movement of dye from the injected mesophyll cell to the minor veins was unaffected by pretreatment of the leaf tissues with 2 millimolar p-chloromercuribenzenesulfonic acid. Pretreatment of the leaf tissues at alkaline pH (3-[N-morpholino] propanesulfonic acid-KOH, pH 8.0) had no apparent effect on dye movement between adjacent mesophyll cells but inhibited the movement of dye into and along the minor veins. Thus, although there were no apparent barriers to symplastic solute movement in this leaf, symplastic barriers could be imposed by the experimental conditions used.