Vacuoles Of Mesophyll Cells Research Articles

Cellular-level distribution of manganese in Macadamia integrifolia, M. ternifolia, and M. tetraphylla from Australia.

Macadamia integrifolia and M. tetraphylla, unlike M. ternifolia, are known for their edible nuts. All three species over-accumulate the trace metal nutrient manganese (Mn) in their shoots. This study seeks to examine tissue- and cellular-level distribution of Mn and other plant nutrients in the three Macadamia species. The distribution of Mn, calcium, iron, and potassium were investigated in whole leaves and cross-sections of roots, petioles, and leaves using synchrotron-based X-ray fluorescence microscopy (XFM) in M. integrifolia, M. tetraphylla, and M. ternifolia. The results show Mn sequestration primarily in the leaf and midrib palisade mesophyll cells of all three species. Leaf interveinal regions, root cortical cells, and phloem cells were also found to be Mn loaded. The current study confirms earlier findings but further reveals that Mn is concentrated in the vacuoles of mesophyll cells owing to the exceptional resolution of the synchrotron XFM data, and the fact that fresh hydrated samples were used. New insights gained here into Mn compartmentalization in these highly Mn-tolerant Macadamias expand knowledge about potentially toxic over-accumulation of an essential micronutrient, which ultimately stands to inform strategies around farming edible species in particular.

Back to the Wild: On a Quest for Donors Toward Salinity Tolerant Rice.

Salinity stress affects global food producing areas by limiting both crop growth and yield. Attempts to develop salinity-tolerant rice varieties have had limited success due to the complexity of the salinity tolerance trait, high variation in the stress response and a lack of available donors for candidate genes for cultivated rice. As a result, finding suitable donors of genes and traits for salinity tolerance has become a major bottleneck in breeding for salinity tolerant crops. Twenty-two wild Oryza relatives have been recognized as important genetic resources for quantitatively inherited traits such as resistance and/or tolerance to abiotic and biotic stresses. In this review, we discuss the challenges and opportunities of such an approach by critically analyzing evolutionary, ecological, genetic, and physiological aspects of Oryza species. We argue that the strategy of rice breeding for better Na+ exclusion employed for the last few decades has reached a plateau and cannot deliver any further improvement in salinity tolerance in this species. This calls for a paradigm shift in rice breeding and more efforts toward targeting mechanisms of the tissue tolerance and a better utilization of the potential of wild rice where such traits are already present. We summarize the differences in salinity stress adaptation amongst cultivated and wild Oryza relatives and identify several key traits that should be targeted in future breeding programs. This includes: (1) efficient sequestration of Na+ in mesophyll cell vacuoles, with a strong emphasis on control of tonoplast leak channels; (2) more efficient control of xylem ion loading; (3) efficient cytosolic K+ retention in both root and leaf mesophyll cells; and (4) incorporating Na+ sequestration in trichrome. We conclude that while amongst all wild relatives, O. rufipogon is arguably a best source of germplasm at the moment, genes and traits from the wild relatives, O. coarctata, O. latifolia, and O. alta, should be targeted in future genetic programs to develop salt tolerant cultivated rice.

Cellular distribution of cadmium in two amaranth (Amaranthus mangostanus L.) cultivars differing in cadmium accumulation.

Differences in cellular cadmium (Cd) distribution between Cd-tolerant and Cd-sensitive lines of amaranth (Amaranthus mangostanus L.) may reveal mechanisms involved in Cd tolerance and hyperaccumulation. We compared the cellular distribution and accumulation of Cd in roots, stems, and leaves between a low-Cd accumulating cultivar (Zibeixian, L-Cd) and a high-Cd accumulating cultivar (Tianxingmi, H-Cd) in a hydroponic experimental system. In all treatments, H-Cd grew better than L-Cd and accumulated more Cd. As the Cd concentration increased, the H-Cd plants grew normally and their biomass increased, except in the 60 μM Cd treatment. The biomass of L-Cd decreased with increasing Cd concentrations. The highest Cd concentration in the roots, stems, and leaves of H-Cd was 950 mg/kg, 305 mg/kg, and 205 mg/kg, respectively, compared with 269 mg/kg, 62.9 mg/kg, and 74.8 mg/kg, respectively, in L-Cd. The Cd distribution differed between the two cultivars. Scanning and transmission electron microscopy and energy-dispersive spectrometry analyses showed that Cd was distributed across the entire cross section of H-Cd roots but largely restricted to the epidermal cells and the exodermis of L-Cd roots. The main Cd storage sites were the root apoplast, cell walls, and intercellular spaces in H-Cd and the root epidermal cells and the exodermis in L-Cd. In H-Cd leaves, Cd accumulated mainly in vacuoles of epidermal cells and, at high external Cd concentrations, in the vacuoles of mesophyll cells.

Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading.

The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellular metabolite distributions as it minimizes the risk of metabolite interconversions or redistribution during the process. The principle of NAF is the separation of small subcellular particles, which are obtained by homogenization, lyophilization, and sonication, in a nonaqueous density gradient. Due to the varying composition-dependent density of the fragments, their segregation reflects compartmental distributions throughout the gradient. By determining marker enzymes for chloroplast stroma, cytosol, and vacuole in gradient fractions the proportions of each subcellular compartment in each gradient fraction can be analyzed. The measured distribution of marker enzymes and of metabolites in each fraction of the gradient can be used to calculate the subcellular distribution of the metabolites.

Na+ extrusion from the cytosol and tissue-specific Na+ sequestration in roots confer differential salt stress tolerance between durum and bread wheat.

The progress in plant breeding for salinity stress tolerance is handicapped by the lack of understanding of the specificity of salt stress signalling and adaptation at the cellular and tissue levels. In this study, we used electrophysiological, fluorescence imaging, and real-time quantitative PCR tools to elucidate the essentiality of the cytosolic Na+ extrusion in functionally different root zones (elongation, meristem, and mature) in a large number of bread and durum wheat accessions. We show that the difference in the root's ability for vacuolar Na+ sequestration in the mature zone may explain differential salinity stress tolerance between salt-sensitive durum and salt-tolerant bread wheat species. Bread wheat genotypes also had on average 30% higher capacity for net Na+ efflux from the root elongation zone, providing the first direct evidence for the essentiality of the root salt exclusion trait at the cellular level. At the same time, cytosolic Na+ accumulation in the root meristem was significantly higher in bread wheat, leading to the suggestion that this tissue may harbour a putative salt sensor. This hypothesis was then tested by investigating patterns of Na+ distribution and the relative expression level of several key genes related to Na+ transport in leaves in plants with intact roots and in those in which the root meristems were removed. We show that tampering with this sensing mechanism has resulted in a salt-sensitive phenotype, largely due to compromising the plant's ability to sequester Na+ in mesophyll cell vacuoles. The implications of these findings for plant breeding for salinity stress tolerance are discussed.

SOS1, HKT1;5, and NHX1 Synergistically Modulate Na+ Homeostasis in the Halophytic Grass Puccinellia tenuiflora.

Puccinellia tenuiflora is a typical salt-excluding halophytic grass with excellent salt tolerance. Plasma membrane Na+/H+ transporter SOS1, HKT-type protein and tonoplast Na+/H+ antiporter NHX1 are key Na+ transporters involved in plant salt tolerance. Based on our previous research, we had proposed a function model for these transporters in Na+ homeostasis according to the expression of PtSOS1 and Na+, K+ levels in P. tenuiflora responding to salt stress. Here, we analyzed the expression patterns of PtSOS1, PtHKT1;5, and PtNHX1 in P. tenuiflora under 25 and 150 mM NaCl to further validate this model by combining previous physiological characteristics. Results showed that the expressions of PtSOS1 and PtHKT1;5 in roots were significantly induced and peaked at 6 h under both 25 and 150 mM NaCl. Compared to the control, the expression of PtSOS1 significantly increased by 5.8-folds, while that of PtHKT1;5 increased only by 1.2-folds in roots under 25 mM NaCl; on the contrary, the expression of PtSOS1 increased by 1.4-folds, whereas that of PtHKT1;5 increased by 2.2-folds in roots under 150 mM NaCl. In addition, PtNHX1 was induced instantaneously under 25 mM NaCl, while its expression was much higher and more persistent in shoots under 150 mM NaCl. These results provide stronger evidences for the previous hypothesis and extend the model which highlights that SOS1, HKT1;5, and NHX1 synergistically regulate Na+ homeostasis by controlling Na+ transport systems at the whole-plant level under both lower and higher salt conditions. Under mild salinity, PtNHX1 in shoots compartmentalized Na+ into vacuole slowly, and vacuole potential capacity for sequestering Na+ would enhance Na+ loading into the xylem of roots by PtSOS1 through feedback regulation; and consequently, Na+ could be transported from roots to shoots by transpiration stream for osmotic adjustment. While under severe salinity, Na+ was rapidly sequestrated into vacuoles of mesophyll cells by PtNHX1 and the vacuole capacity became saturated for sequestering more Na+, which in turn regulated long-distance Na+ transport from roots to shoots. As a result, the expression of PtHKT1;5 was strongly induced so that the excessive Na+ was unloaded from xylem into xylem parenchyma cells by PtHKT1;5.

A magic red coat on the surface of young leaves: anthocyanins distributed in trichome layer protect Castanopsis fissa leaves from photoinhibition.

The presence of anthocyanins in young leaves plays an important role in mitigation against photodamage and allows leaves to grow and develop normally. Many studies have reported that foliar anthocyanins are distributed within the vacuoles of mesophyll cells, so we explored the novel defence style of anthocyanin-coated young leaves of Castanopsis fissa, a dominant subtropical forest tree species, via removable trichomes. Anthocyanins were distributed in C. fissa leaf trichomes, which produced a red coating for the young leaves. As young leaves developed and then matured, the thickness and density of the anthocyanin trichomes progressively decreased, the coating finally disappearing, allowing greater utilization of light by mature leaves. In addition to anthocyanins, the trichomes contained a remarkably high amount of phenolics, which enable the red coating to be more efficient in screening ultraviolet light. Compared with mature leaves, the young leaves exhibited lower photosynthetic ability, which was attributable to the reduced chlorophyll and Rubisco contents. Removal of the red coating had little effect on the photosynthetic capacity of young leaves. However, the young leaves without the coating suffered greater light-induced photoinhibition due to greater excess light entering the chloroplast and the production of H2O2 Our results suggest that the anthocyanin coating is photoprotective and this anthocyanin defence style may be a metabolically cost-effective way of adjusting the anthocyanin content in response to demand.

Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: An excess light stress affair?

The experiment was conducted using Fraxinus ornus plants grown outside under full sunlight irradiance, and supplied with 100% (well-watered, WW), 40% (mild drought, MD), or 20% (severe drought, SD) of the daily evapotranspiration demand, with the main objective of exploring the effect of excess light stress on the activity of antioxidant enzymes and phenylpropanoid biosynthesis. Net CO2 assimilation rate at saturating light and daily assimilated CO2 were significantly smaller in SD than in WW and MD plants. Xanthophyll-cycle pigments supported nonphotochemical quenching to a significantly greater extent in SD than in MD and WW leaves. As a consequence, the actual efficiency of PSII (ΦPSII) was smaller, while the excess excitation-energy in the photosynthetic apparatus was greater in SD than in WW or MD plants. The concentrations of violaxanthin-cycle pigments relative to total chlorophyll (Chltot) exceeded 200mmolmol−1 Chltot in SD leaves at the end of the experiment. This leads to hypothesize for zeaxanthin a role not only as nonphotochemical quencher, but also as chloroplast antioxidant. Reductions in ascorbate peroxidase and catalase activities, as drought-stress progressed, were paralleled by greater accumulations of esculetin and quercetin 3-O-glycosides, both phenylpropanoids having effective capacity to scavenge H2O2. The drought-induced accumulation of esculetin and quercetin 3-O-glycosides in the vacuoles of mesophyll cells is consistent with their putative functions as reducing agents for H2O2 in excess light-stressed leaves. Nonetheless, the concentration of H2O2 and the lipid peroxidation were significantly greater in SD than in MD and WW leaves. It is speculated that vacuolar phenylpropanoids may constitute a secondary antioxidant system, even on a temporal basis, activated upon the depletion of primary antioxidant defences, and aimed at keeping whole-cell H2O2 within a sub-lethal concentration range.

A comprehensive picture of phloem loading strategies

Mechanisms of phloem loading in the minor veins of leaves are known for only a few species. We propose that there are a limited number of loading strategies for the primary photoassimilates, sucrose and sugar alcohols. These strategies can be predicted based on thermodynamic and anatomical considerations and identified by autoradiography of veins following uptake of (14)C-labeled compounds, analysis of leaf solute composition and concentrations, and plasmodesmatal counting. Experiments on 45 dicotyledonous species identified the predicted loading patterns. Over 50-fold differences in concentrations of sucrose and sugar alcohols in leaves were measured. The cumulative concentrations of transport compounds in leaves correlated with loading mechanisms, a previously unrecognized association. Comparisons of solute concentrations and osmotic potentials of whole leaves suggest that sucrose and sugar alcohols are more concentrated in the cytosol than in the vacuoles of mesophyll cells, thus increasing the driving force for passive loading in species that employ this strategy. Passive loading is more widespread than previously thought, especially in trees. The results indicate that plants have exploited all thermodynamically feasible and structurally compatible loading strategies and that these strategies can be identified with straightforward protocols.

Mesophyll distribution of 'antioxidant' flavonoid glycosides in Ligustrum vulgare leaves under contrasting sunlight irradiance.

Flavonoids have the potential to serve as antioxidants in addition to their function of UV screening in photoprotective mechanisms. However, flavonoids have long been reported to accumulate mostly in epidermal cells and surface organs in response to high sunlight. Therefore, how leaf flavonoids actually carry out their antioxidant functions is still a matter of debate. Here, the distribution of flavonoids with effective antioxidant properties, i.e. the orthodihydroxy B-ring-substituted quercetin and luteolin glycosides, was investigated in the mesophyll of Ligustrum vulgare leaves acclimated to contrasting sunlight irradiance. In the first experiment, plants were grown at 20 % (shade) or 100% (sun) natural sunlight. Plants were exposed to 100 % sunlight irradiance in the presence or absence of UV wavelengths, in a second experiment. Fluorescence microspectroscopy and multispectral fluorescence microimaging were used in both cross sections and intact leaf pieces to visualize orthodihydroxy B-ring-substituted flavonoids at inter- and intracellular levels. Identification and quantification of individual hydroxycinnamates and flavonoid glycosides were performed via HPLC-DAD. Quercetin and luteolin derivatives accumulated to a great extent in both the epidermal and mesophyll cells in response to high sunlight. Tissue fluorescence signatures and leaf flavonoid concentrations were strongly related. Monohydroxyflavone glycosides, namely luteolin 4'-O-glucoside and two apigenin 7-O-glycosides were unresponsive to changes in sunlight irradiance. Quercetin and luteolin derivatives accumulated in the vacuoles of mesophyll cells in leaves growing under 100 % natural sunlight in the absence of UV wavelengths. The above findings lead to the hypothesis that flavonoids play a key role in countering light-induced oxidative stress, and not only in avoiding the penetration of short solar wavelengths in the leaf.

Lead accumulation in the aquatic fern Azolla filiculoides

In this study, we characterized lead (Pb 2+) accumulation and storage by the aquatic fern Azolla filiculoides. Lead precipitates were detected in the vacuoles of mesophyll cells of Azolla plants cultured for 6 d in rich growth medium containing 20 mg l –1 Pb 2+. Energy dispersive spectroscopy (EDS) analysis of the relative element content of leaves collected from these plants revealed a 100% increase in the levels of P, S, Na and Ca and a 40% decrease in Mg and Cl compared to the untreated plants. Both Azolla whole plants and isolated apoplasts were incubated for 6 d in 20 mg l –1 Pb 2+. Lead content in the whole plant composed 0.37%, 2.3% and 1.8% of the dry weight after 2, 4 and 6 d of growth, respectively, while the isolated Azolla apoplast contained 0.125%, 1.22% and 1.4% Pb 2+, respectively. Lead content in Azolla whole plant increase by 200%, 100% and 22% after 2, 4 and 6 d of growth, respectively, when compared to Azolla apoplast. Dark, electron dense deposits of lead were observed in light and transmission electron microscope in leaf cells treated with lead. All the observed lead deposits were localized in vacuoles while larger lead deposits were found in mature leaves than in young leaves. No lead deposits were found in cells of the cyanobiont Anabaena when the plants were exposed to similar conditions. Activity and content of V-H +-ATPase were studied in Azolla plants grown in the presence of 20, 40 and 80 mg l –1 of lead for a period of 4 d. Activity of V-H +-ATPase was increased by 190%, 210% and 220%, respectively, but the content of V-H +-ATPase was reduced by all lead concentrations.

Accumulation and distribution of aluminium and other elements in tea (Camellia sinensis) leaves

Tea plants (Camellia sinensis) are well known for their ability to accumulate high concentrations of aluminium (Al). Aluminium distribution in tea leaves has been previously estimated on a qualitative basis using energy dispersive X-ray microanalysis (EDXMA). However, no comprehensive studies have been carried out to quantitatively determine the distribution of Al, especially in young leaves which are used for the manufacture of commercial tea. Cuttings of C. sinensis var. sinensis grown in acidic soil were collected and young and old leaves were used for analysis. Total Al concentrations in young and old leaves were 380 and 6866 µg·g -1 , respectively. The leaves were freeze-fractured and the quantitative distribution of Al and other elements such as Mg, Ca and K was determined using EDXMA. In the young and old leaves Al was found to be preferentially accumulated in the upper epidermis. In particular, Al was accumulated in the cell walls, which had significantly higher levels than the vacuoles of epidermal cells. The Al concentrations in the vacuole of mesophyll cells was found not to change significantly between young and old leaves. In contrast, a large increase in Al concentration was observed in the epidermal cell walls. Calcium was present in higher levels on the cell walls and also present as crytals in the spongy mesophyll tissue. In contrast, K and Mg were evenly distributed across the leaf. aluminium / Camelia sinensis / tea / accumulation / EDXMA

Atmospheric CO 2 enrichment influences the synthesis and mobilization of putative vacuolar storage proteins in sour orange tree leaves

Concentrations of three soluble proteins with molecular masses of 33, 31 and 21 kDa were measured weekly for a period of 1 year in leaves of sour orange ( Citrus aurantium L.) trees that had been grown for 6 years at atmospheric CO 2 concentrations of 400 and 700 ppm. Abundances of the proteins were generally lower in CO 2-enriched leaves than in ambient-treatment leaves during the central portion of the year. Over the early and latter parts of the year, however, they typically were much greater in leaves of the CO 2-enriched trees. The decrease from their high wintertime levels in the CO 2-enriched trees possibly provided a source of nitrogen required for the enhanced new branch growth observed in the spring on the trees growing in CO 2-enriched air. The hypothesis that they are vegetative storage proteins (VSPs) is also supported by the N-terminal amino acid sequence obtained for the 21-kDa protein, which has homology with sporamin B, an implicated storage protein in sweet potato tubers. In addition, immunoelectron microscopy demonstrated the presence of these proteins within amorphous material in the vacuoles of mesophyll cells, where VSPs are commonly located. The fact that elevated CO 2 had little impact on the amount of leaf rubisco suggests that enhanced branch and fruit growth observed in the CO 2-enriched trees was not correlated with an increased rate of breakdown of this major protein, another potential source of the nitrogen. The 33-, 31- and 21-kDa proteins appear to be specific to citrus species, as immunologically related proteins were detected in a variety of orange, grapefruit, lemon, tangelo and kumquat trees, but were not found in a large number of herbaceous plants and unrelated woody species.

KCO1 is a component of the [formula omitted]low- [formula omitted]acuolar (SV) ion channel

The Arabidopsis double pore K + channel KCO1 was fused to green fluorescent protein and expressed in tobacco protoplasts. Microscopic analysis revealed a bright green fluorescence at the vacuolar membrane. RT-PCR experiments showed that KCO1 is expressed in the mesophyll. Vacuoles from Arabidopsis wild-type and kco1 knockout plants were isolated for patch-clamp analyses. Currents mediated by s low-activating v acuolar (SV) channels of mesophyll cell vacuoles were significantly smaller in kco1 plants compared to the wild-type. This shows that KCO1 is involved in the formation of SV channels.

Vacuoles of Mesophyll Cells as a Transient Reservoir for Assimilates

We studied the efflux of radioactive photosynthetic products from the central vacuole into the cytosol of protoplasts isolated from the mesophyll tissue of the sugar beet (Beta vulgaris L.) after their darkening and subsequent cessation of photosynthesis. Among the products accumulated in the vacuole were the 14C-labelled sugars malate and alanine, small amounts of citric, glutamic, and aspartic acids, and some other amino acids. During the initial 20–30 min of darkness, there was no substantial utilization of photoassimilates accumulated in the vacuole during the preceding light period. An efflux of assimilates occurred later, after 30–40 min of darkness. A decrease in the vacuolar 14C-sucrose occurred not only due to its exit into the cytosol but also because of its conversion into 14C-monosaccharides by the vacuolar invertase. In fact, this decrease in the sucrose content correlated well with the accumulation of monosaccharides. Immediately after photosynthesis ceased, the chloroplastic 14C-starch was utilized for the maintenance of cytoplasmic metabolism. After 30-min darkness, the content of starch in the chloroplasts decreased by several times. We believe that the vacuoles of sugar-beet mesophyll cells are transient reservoirs for assimilates and the products of their conversion (glucose and fructose), which can rapidly leave the vacuole to maintain homeostasis in the cytosol under varying environmental conditions.

Modulation of the vacuolar H+-ATPase by adenylates as basis for the transient CO2-dependent acidification of the leaf vacuole upon illumination

Using tonoplast vesicles, we have investigated the activity of the vacuolar H+-ATPase which is the dominant proton pump at the tonoplast of mesophyll cells. Bafilomycin-sensitive ATP hydrolysis or acidification of tonoplast vesicles in the presence of ATP were measured at varying ATP, ADP and Pi concentrations, and in the presence of oxidized or reduced glutathione. Increased ATP/ADP ratios as reported for the extrachloroplast cytoplasm during the induction phase of photosynthesis at high or low CO2 (P. Gardeström, Biochim. Biophys. Acta 1183 (1993) 327–332) increased the activity of the V-ATPase in simulation experiments with vesicles. Depending on reported subsequent decreases in cytoplasmic ATP/ADP ratios in the presence of high or low CO2, the ATPase activity of tonoplast vesicles changed in simulation experiments to lower values. More than 10 mM phosphate was required to decrease the ATPase activity in vesicles significantly at ATP/ADP ratios of 3 or higher, indicating that ATPase activity is controlled more by ratios of ATP to ADP than by phosphorylation potentials (ATP)/(ADP)(Pi). Oxidized glutathione was inhibitory. The results permit interpretation of the observation that on illumination of previously darkened leaves the pH of the vacuoles of mesophyll cells decreases indicating energized transport of protons across the tonoplast into acidic vacuoles, and that the extent of vacuolar acidification depends on the CO2 concentration of the surrounding air (Z.-H. Yin, S. Neimanis, U. Heber, Planta 182 (1990) 253–261). We conclude that short term control of tonoplast ATPase activity in leaves during dark/light transients can essentially be understood on the basis of reported changes in cytoplasmic ATP/ADP ratios, with a possible participation of redox modulation.

Endodermal Ca and Sr partitioning in needles of the European larch ( Larix decidua (L.) Mill.)

Summary The aim of this study was to find out whether the needle endodermis of young larch trees contributes to the regulation of needle Ca ++ content as well as the retention of toxic minerals such as Sr ++ from the surrounding mesophyll. For this, plants were hydroponically grown under controlled conditions in nutrient solutions without (‹control›) and with Sr ++ (Ca ++ /Sr ++ ratio: 1; ‹mixed-treatment›). EDX-analyses of deep frozen needle segments revealed that in control plants the endodermal Ca-concentrations were much higher than in the vacuoles of mesophyll cells but they coincided with the mean Ca-concentration found in the whole needle. The discrepancy between endodermal and mesophyll Ca-concentrations can be explained by the presence of intracellular Ca-oxalate crystals localized in the parenchyma tissue of the vascular bundle. When Ca ++ and Sr ++ were supplied in equal concentrations in the nutrient solution, Ca ++ translocation into the needles occurred in preference to Sr ++ . In contrast, more Sr ++ than Ca ++ was accumulated in the vacuoles of the endodermis cells. Furthermore, endodermal Sr ++ concentrations were much higher than in the mesophyll. More than 19 % of the total needle Sr ++ was stored in the endodermis, which occupies only 3–4% of the needle volume. A function for retaining harmful substances in the endodermal vacuoles is discussed.

The vacuolar targeting signal of the 2S albumin from Brazil nut resides at the C terminus and involves the C-terminal propeptide as an essential element.

Genetic constructs in which different N- and C-terminal segments of Brazil nut (Bertholletia excelsa H.B.K.) 2S albumin were fused to secretory yeast invertase were transformed into tobacco (Nicotiana tabacum) plants to investigate the vacuolar targeting signal of the 2S albumin. None of the N-terminal segments, including the complete precursor containing all propeptides, was able to direct the invertase to the vacuoles. However, a short C-terminal segment comprising the last 20 amino acids of the precursor was sufficient for efficient targeting of yeast invertase to the vacuoles of the transformed tobacco plants. Further analyses showed that peptides of 16 and 13 amino acids of the C-terminal segment were still sufficient, although they had slightly lower efficiency. When segments of 9 amino acids or shorter were analyzed, a decrease to approximately 30% was observed. These segments included the C-terminal propeptide of four amino acids (Ile-Ala-Gly-Phe). When the 2S albumin was expressed in tobacco, it was also localized to the vacuoles of mesophyll cells. If the C-terminal propeptide was deleted from the 2S albumin precursor, all of this truncated 2S albumin was secreted from the tobacco cells. These results indicate that the C-terminal propeptide is necessary but not sufficient for vacuolar targeting. In addition, an adjacent segment of at least 12 amino acids of the mature protein is needed to form the complete signal for efficient targeting.

How Plants Dispose of Chlorophyll Catabolites: DIRECTLY ENERGIZED UPTAKE OF TETRAPYRROLIC BREAKDOWN PRODUCTS INTO ISOLATED VACUOLES

During the yellowing of leaves the porphyrin moiety of chlorophyll is cleaved into colorless linear tetrapyrrolic catabolites, which eventually are deposited in the central vacuoles of mesophyll cells. In senescent cotyledons of rape, Brassica napus, three nonfluorescent chlorophyll catabolites (NCCs), accounting for practically all the chlorophyll broken down, were found to be located in the vacuoles (vacuoplasts) prepared from protoplasts. Transport of catabolites across the tonoplast was studied with vacuoles isolated from barley mesophyll protoplasts in conjunction with a radiolabeled NCC, Bn-NCC-1, prepared from senescent rape cotyledons. The uptake of Bn-NCC-1 into vacuoles was against a concentration gradient and strictly dependent on MgATP and it followed saturation kinetics with a Km of approximately 100 microM. Although the hydrolysis of ATP was required, transport was apparently independent of the vacuolar proton pumps: accumulation of the NCC occurred both in the presence of the H+-ATPase inhibitor bafilomycin and after destroying the DeltapH between the vacuolar sap and the medium. ATP could be replaced by GTP or UTP, and the transport was inhibited in the presence of vanadate. Chlorophyll catabolites isolated from senescent barley leaves competed with the rape-specific substrate for uptake into the vacuoles. Compounds such as the glutathione conjugate of N-ethylmaleimide and taurocholate, which are known to be transported across the tonoplast in a primary active mode, did not significantly inhibit uptake of Bn-NCC-1. Although the heme catabolites biliverdin and bilirubin inhibited the uptake of the NCC, this effect is caused by unspecific binding to the vacuolar membrane rather than to the specific inhibition of carrier-mediated transport. Taken together, the results demonstrate that barley mesophyll vacuoles are constitutively equipped with a directly energized carrier that transports tetrapyrrolic catabolites of chlorophyll into the vacuole.

Light-dependent proton transport into mesophyll vacuoles of leaves of C3 plants as revealed by pH-indicating fluorescent dyes: a reappraisal

Esculin, a pH-sensitive fluorescent dye, was used to indicate light-dependent pH changes in leaves of Spinacia oleracea L. and Pelargonium zonale L. Shortly after its introduction into the leaves via the transpiration stream, esculin was localized mainly in the symplasm. An increase in its blue fluorescence on illumination with red actinic light indicated that the cytosolic pH had increased. A similar light-dependent alkalinization was seen when the green fluorescence of pyranine was used to monitor changes in the cytosolic pH. After esculin had been transferred into the vacuoles, a light-dependent vacuolar acidification was indicated by a decrease in its blue fluorescence. Since the pK of esculin is close to neutrality, it is suitable as an indicator of proton transport into vacuoles provided the vacuolar sap is only moderately acidic. In leaf cells with very acidic vacuoles, esculin therefore responds only to cytosolic pH changes as long as it remains in the cytosol. The observations made with esculin after it had entered the vacuoles confirmed earlier conclusions on light-dependent proton transport into the vacuoles of mesophyll cells. Previous measurements had been made with 5-carboxy-2′,7′-dichlorofluoresceine (CDCF), which has a pK of 4.8. In contrast to esculin, CDCF can, in principle, record pH changes in very acidic vacuoles. However, earlier conclusions made on the basis of observed CDCF fluorescence are now recognized to have no unambiguous basis because new measurements, reported here, show that CDCF fluorescence is influenced not only by pH changes but also by changes in light scattering. The latter are, like pH changes, light-dependent and originate from the thylakoid system of chloroplasts. They indicate both the formation of a large transthylakoid proton gradient and the dissipation of excess light energy as heat. Decreased green fluorescence of leaves which had been fed CDCF may therefore, depending on conditions, indicate vacuolar acidification or the dissipation of excess light energy absorbed by the pigment system of chloroplasts, or both. Pyranine fluorescence was found to be much less influenced by light scattering than CDCF fluorescence.