Vacuoles Of Mesophyll Cells Research Articles

Bundle sheath and mesophyll cell differentiation in the C4 dicotyledon Atriplex rosea: quantitative ultrastructure

In leaves of most C4 species, both bundle sheath and mesophyll cells are derived from ground meristem, yet at maturity differ in photosynthetic enzyme complement and in cell size, shape, and subcellular ultrastructure. This quantitative ultrastructural study of bundle sheath and mesophyll cell differentiation in Atriplex rosea shows that while developmental pathways of bundle sheath and meosphyll cells are generally coordinated, the timing of developmental divergence differs among individual characteristics. For instance, bundle sheath cells are larger, with more chloroplasts and more and larger mitochondria by 8 days after leaf emergence, while differential growth of mesophyll cell chloroplast peripheral reticulum and increase in thylakoids per granum in bundle sheath chloroplasts do not develop until after 12 days. Multigroup principal components analysis (M-PCA) of the data emphasizes that the greatest source of variation is overall size change as both cell types expand. M-PCA also identifies patterns of allometry within the data; for instance, mesophyll cell vacuoles and chloroplast peripheral reticulum undergo greater relative growth than do bundle sheath microbody area and number. The greater structural specialization of bundle sheath cells is reflected in higher growth rates from the time of divergence, but developmental change in both cell types continues until leaf expansion is complete. Most structural changes occur substantially after the stage of cell-specific expression of C4 enzymes. Key words: bundle sheath, mesophyll, C4 photosynthesis, leaf development, Atriplex rosea, multigroup principal components analysis.

X‐Ray Microanalytical Studies on Mineral Concentrations in Vacuoles of Needle Tissues from Larix decidua (L.) Mill.

AbstractElemental concentrations in the vacuoles of mesophyll, endodermis and transfusion parenchyma cells were determined by means of electron probe microanalysis on bulk frozen hydrated needles of an outdoor‐grown larch tree. The investigations were done routinely throughout a life cycle of the deciduous larch needle, starting with bud burst in March and ending with leaf abscission in November. It could be shown that the endodermal vacuoles preferentially accumulate S, CI and Ca while the transfusion parenchyma vacuoles transiently store Mg, K and S. Between May and September the inorganic ion level of mesophyll cells is constantly lower than in the endodermal and transfusion parenchyma vacuoles. In autumn, when the degradation of chlorophylls becomes detectable, the elements Mg, S, CI and Ca are found increasingly in the vacuoles of the mesophyll cells. It is concluded that, in mature and intact larch needles, the vacuoles of the endodermis in cooperation with the transfusion parenchyma vacuoles act as efficient ion storage buffers which maintain a constant low ion level in the vacuoles of mesophyll cells.

Hydrogen peroxide scavenging systems in vacuoles of mesophyll cells of Vicia faba

Vacuoles isolated from mesophyll protoplasts of Vicia faba contained ascorbate in addition to 3,4-dihydroxyphenylalanine (DOPA) and peroxidase. When a low concentration of H 2O 2(0.25 mM) was added to the vauole fractions, a slow decrease in the level of ascorbate was observed. When 2.5 mM H 2O 2 was added to the vacuole fractions, a rapid decrease in the levels of ascorbate and formation of dopachrome were observed. The oxidation of ascorbate and the formation of dopachrome were inhibited by NaN 3 (10 mM), KCN (1 mM) and stimulated by tropolone (5 mM), suggesting the participation of peroxidase but not phenol oxidase in the reactions. Vacuolar peroxidase isolated from leaves of V. faba oxidized DOPA and ascorbate in the presence of H 2O 2. The oxidation of DOPA was nearly completely inhibited by ascorbate but the oxidation of ascorbate was stimulated by DOPA, suggesting the reduction of an oxidized intermediate of DOPA to DOPA by ascorbate. In fact, ascorbate reduced dopaquinone, a two electron oxidized DOPA.

Unusual patterns in the distribution of calcium oxalate in spruce needles and their possible relationships to the impact of pollutants.

The patterns of distribution of calcium oxalate crystals in spruce needles have been investigated in healthy needles and those subjected to either acid precipitation (ph 2.5) or various ozone fumigations. In the acid treatment, deposits of Ca oxalate were lacking in the outer walls of the epidermal cells. Under the impact of ozone, unusual complexes of Ca oxalate crystals embedded in a matrix of callose and cellulose projected from the walls inwards into the cells, filling the lumina of epidermal and hypodermal cells completely. In mesophyll cells, the complexes projected more or less from the walls into the vacuoles. In other cases, crystals appeared within the vacuoles of mesophyll cells. In dead cells, precipitates of Ca oxalate or phosphate apparently indicate destruction of the cytoplasm after a major influx of Ca2+ . It is speculated, that the physiological significance of enhanced calcium leaching has been overestimated. With regard to ozone impact, it seems that the major problem of thereby enhanced membrane permeabilities may not be an increased efflux of mineral elements, but rather an increased influx of Ca into the symplast and subsequent disturbances in metabolism.

The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles.

Soybean leaves contain three proteins (the vegetative storage proteins or VSPs) that respond to nitrogen status and are believed to be involved in the temporary storage of nitrogen. One of these proteins, with a molecular mass of 94 kD and termed vsp94, was microsequenced. Partial amino acid sequence indicated that vsp94 was highly homologous to the lipoxygenase protein family. Further evidence that vsp94 is a lipoxygenase was obtained by demonstrating that vsp94 cross-reacted with a lipoxygenase antibody. Also, a lipoxygenase cDNA coding region was able to detect changes in an mRNA that closely parallel changes in vsp94 protein levels resulting from alteration of nitrogen sinks. Extensive immunocytochemical data indicate that this vsp94/lipoxygenase is primarily expressed in the paraveinal mesophyll cells and is subcellularly localized in the vacuole. These observations are significant in that they suggest that plant lipoxygenases may be bifunctional proteins able to function enzymatically in the hydroperoxidation of lipids and also to serve a role in the temporary storage of nitrogen during vegetative growth.

The Soybean 94-Kilodalton Vegetative Storage Protein Is a Lipoxygenase That Is Localized in Paraveinal Mesophyll Cell Vacuoles

The Soybean 94-Kilodalton Vegetative Storage Protein Is a Lipoxygenase That Is Localized in Paraveinal Mesophyll Cell Vacuoles

Peroxidases in vacuoles of Vicia faba leaves

A basic peroxidase localized in vacuoles of mesophyll cells of Vicia faba was isolated by ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The peroxidase had a pH optimum at 5 and absorption maxima at 404, 510 and 635 nm in the oxidized form. Its absorption maxima in the presence of 0.1 mM H 2O 2 were 418, 523 and 558 nm. The M r of the enzyme was estimated to be 49 000 by sucrose density gradient centrifugation. The peroxidase oxidized flavonols and 3,4-dihydroxyphenylalanine (DOPA). The V max s were quercetin kaempferol > rutin (quercetin 3- O-rutinoside)>DOPA>robinin (kaempferol 3- O-rhamnogalactoside 7- O-rhamnoside). K m values were below 0.1 mM for flavonols and 6.7 mM for DOPA. The peroxidase could also oxidize flavonol glycosides which were contained in methanol extracts of the lower epidermis of Vicia faba leaves.

Low CO2 Prevents Nitrate Reduction in Leaves

The correlation between CO(2) assimilation and nitrate reduction in detached spinach (Spinacia oleracea L.) leaves was examined by measuring light-dependent changes in leaf nitrate levels in response to mild water stress and to artificially imposed CO(2) deficiency. The level of extractable nitrate reductase (NR) activity was also measured. The results are: (a) In the light, detached turgid spinach leaves reduced nitrate stored in the vacuoles of mesophyll cells at rates between 3 and 10 micromoles per milligram of chlorophyll per hour. Nitrate fed through the petiole was reduced at similar rates as storage nitrate. Nitrate reduction was accompanied by malate accumulation. (b) Under mild water stress which caused stomatal closure, nitrate reduction was prevented. The inhibition of nitrate reduction observed in water stressed leaves was reversed by external CO(2) concentrations (10-15%) high enough to overcome stomatal resistance. (c) Nitrate reduction was also inhibited when turgid leaves were kept in CO(2)-free air or at the CO(2)-compensation point or in nitrogen. (d) When leaves were illuminated in CO(2)-free air, activity of NR decreased rapidly. It increased again, when CO(2) was added back to the system. The half-time for a 50% change in activity was about 30 min. It thus appears that there is a rapid inactivation/activation mechanism of NR in leaves which couples nitrate reductase to net photosynthesis.

Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco

Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)(60), and Asn(90), but the third glycosylation site (Asn(202)) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.

Mobilisation of vacuolar amino acids in leaf cells as affected by ATP and the level of cytosolic amino acids: ATP regulates but appears not to energize vacuolar amino-acid release

The vacuoles of mesophyll cells are capable of storing amino acids which are mobilized on demand. An amino-acid carrier was characterized by studying the efflux of vacuolar amino acids from isolated mesophyll vacuoles using the silicon oil layer centrifugation technique. Efflux was slow in the absence of ATP. It was stimulated by a factor of more than 5 by the addition of ATP. A similar stimulation was brought about by adenylyl imidodiphosphate ( Ado PP [NH] P ), suggesting that efflux activation by ATP did not involve ATP hydrolysis. GTP, CTP, UTP and ADP were not effective, or were much less effective than ATP, in activating the amino-acid transport system. Neutral amino acids inhibited ATP-stimulated amino-acid efflux by an allosteric mechanism. External leucine, valine, phenylalanine and methionine were more effective in inhibiting efflux than alanine, arginine, lysine or glutamic acid when present outside the vacuole. Protein-modifying agents such as N -ethylmaleimide and p -chloromercuriphenylsulfonic acid activated amino-acid efflux from the vacuoles, whereas proteinase treatment led to an inhibition. Stimulation by ATP of amino-acid export from the vacuole and inhibition by specific amino acids may be part of a mechanism to stabilize cytoplasmic amino-acid levels during protein synthesis.

Active Transport of Sulfate into the Vacuole of Plant Cells Provides Halotolerance and Can Detoxify SO 2

Summary Barley seedlings grown hydroponically in the presence of high concentrations of potassium sulfate contained more sulfate in the mesophyll tissue of the leaves than in the epidermal tissue. Sulfate accumulated in the vacuoles of mesophyll cells. During 9 days of sulfate uptake by the seedlings the sulfate concentration in the vacuoles increased by a factor of about 10 or more, whereas the sulfate concentration in chloroplasts increased by a factor of less than 2. Maximal vacuolar sulfate levels were above l00 mol m −3 5 days after the sulfate concentration in the hydrophonic nutrient solution was increased to 300 mol m −3 . Higher sulfate levels were not tolerated by the seedlings. Isolated vacuoles did not leak much endogenous sulfate even under a diminished transtonoplast proton gradient as long as the membrane potential remained significant. Rapid loss of sulfate occurred when both the proton gradient and the membrane potential were dissipated. External tracer sulfate was taken up by isolated vacuoles more rapidly in the presence than in the absence of ATP. The rate of uptake increased nonlinearly with concentration at low sulfate concentrations, with and without ATP being present. At high concentrations, uptake was linear with concentration only in the presence of ATP. ATP-stimulated sulfate uptake resulted in the vacuolar accumulation of sulfate against its concentration gradient. ATP-stimulated sulfate uptake by isolated vacuoles was inhibited by agents which are known to inhibit vectorial proton translocation into the vacuoles by the tonoplast ATPase. pCMBS inhibited sulfate transport both in the presence and in the absence of ATP, and particularly pronounced inhibition was observed at sulfate concentrations below 1 mol m −3 , i.e. in the saturable range of the uptake kinetics. In the absence of a membrane potential, transfer of labelled sulfate across the tonoplast was inhibited significantly only by a large excess of chloride, nitrate, phosphate, or unlabelled sulfate, but not by malate. The main cation accompanying sulfate during net uptake into the vacuole was K + . It appeared to be taken up in exchange against H + . The transfer capacity of the tonoplast sulfate transporter is more than sufficient to account for temporary vacuolar storage of sulfate produced from SO 2 during exposure of barley leaves to air polluted with 140 or 400μg SO 2 m −3 . These concentrations are considered to be permissible, at least temporarily, according to Federal regulation. Vacuolar detoxification of the sulfuric acid formed from SO 2 appeared to be limited by the vacuolar capacity for proton storage, and not by that for sulfate storage.

Catabolites of chlorophyll in senescing barley leaves are localized in the vacuoles of mesophyll cells

Senescing barley leaves accumulate a series of pink pigments with the chemical properties of catabolites derived from chlorophyll. Levels of the major component of this group of pigments were quantified by HPLC and shown to be maximal in tissues exhibiting maximal rates of chlorophyll degradation. Protoplasts were isolated from senescent leaf tissue and fractionated to yield intact vacuoles and plastids. Although small but significant proportions both of total catabolites and of the dominant component of the series were recovered from the plastid fraction, the vast bulk of these compounds could be assigned to the vacuole. These observations suggest a role for the vacuole in the later stages of chlorophyll breakdown during senescence.

Accumulation and subcellular distribution of cations in relation to the growth of potassium‐deficient barley

Abstract Growth of barley (Hordeum vulgare L., cv. Georgie) was insensitive to soil K content above about 150 mg kg−1, but at lower levels it declined. The reduction in yield was greater in soils containing approximately 10 mg Na kg−1 than in soils with about 90 mg kg−1 of Na. Growth was unaffected by changes in shoot K concentration above 75 mol m−3, but declined at lower concentrations, and the decrease was less in plants grown in soils with high Na. Growth responses were not simply related to tissue K concentrations because plants grown in soils with extra Na had higher yields but lower K concentrations.When soil Na was low, plants accumulated Ca as tissue K declined, but when Na was provided this ion was accumulated. Plant Mg concentrations were generally low but increased as K decreased. The Ca and Mg were osmotically active. There were highly significant inverse linear relationships between yield and either the Ca or Mg concentrations in the shoots.X‐ray microanalysis was used to examine the compartmentation of cations in leaves from barley plants (cv. Clipper) grown in nutrient solutions with high and low K concentrations. In plants grown with 2.5 mol m−3 K, this was the major cation in both the cytoplasm and vacuole of mesophyll cells. However, in plants grown with 0.02 mol m−3 K it declined to undetectable levels in the vacuole, although it was still detectable in the cytoplasm. In all plants, Ca was mainly located in epidermal cells. The implication of the results for explaining responses to K. in terms of compartmentation of solutes is discussed.

The Compartmentation of Abscisic Acid and β-d-Glucopyranosyl Abscisate in Mesophyll Cells

beta-d-Glucopyranosyl abscisate (ABA-GE) is synthesized in Xanthium strumarium L. leaves during water stress. Following recovery from stress, the amount of ABA-GE does not decline. These observations led to the hypothesis that ABA-GE is sequestered in the vacuole where it is metabolically inert. The localization of abscisic acid (ABA) and ABA-GE was investigated by a dimethyl sulfoxide (DMSO) compartmentation method and by direct isolation of vacuoles.With the DMSO compartmentation method it was shown that in Xanthium mesophyll cells ABA was in a compartment not accessible to DMSO, presumably the chloroplast, whereas ABA-GE was in a compartment accessible to DMSO, presumably the vacuole. Neutral red, which accumulates in the vacuoles, showed a similar DMSO concentration dependence for its release from the cells as ABA-GE.Vacuoles isolated from Vicia faba L. leaf protoplasts contained 22% of the total ABA and 91% of the ABA-GE. Some of the ABA in the vacuole preparations was probably due to cytoplasmic contamination. These findings indicate that ABA-GE is sequestered in the vacuoles of mesophyll cells where the conjugated form of ABA is removed from the active ABA pool.

Mechanism of passive malic-acid efflux from vacuoles of the CAM plantKalanchoë daigremontiana

An analysis was carried out of the mechanism of malic-acid efflux from vacuoles of mesophyll cells of the crassulacean acid metabolism (CAM) plantKalanchoe daigremontiana. Following its accumulation in the vacuole as a result of nocturnal CO2 fixation, the malic acid is passively transported back across the tonoplast in the subsequent light period and is decarboxylated in the cytoplasm. Malic-acid efflux was studied using leaf slices in solution or by following malic-acid utilization (deacidification) in leaves of intact plants. Samples of leaf-cell sap were taken at different times during the day-night rhythm to establish the relation between cell-sap pH and malate content. From the empirically determined pK values for malic acid in the cell sap, it was then possible to calculate the proportion of malate existing as the undissociated acid (H2mal0) and in the anionic forms (Hmal1− and mal2−) for all times during the CAM rhythm. In leaf-slice experiments it has been found that the rate of malic-acid efflux increases exponentially with the malic-acid content of the tissue. This is shown to be related to the increasing amounts of H2mal0 present at high malic-acid contents. At low malic-acid contents (<65 mol m−3), when H2mal0 is not present in significant amounts, efflux must be in the form of Hmal−1 and/or mal2−. At high malic-acid contents it is suggested that efflux occurs predominantly in the form of passive, noncatalyzed diffusion of H2mal0 across the tonoplast by a ‘lipid-solution’ mechanism. This is supported by the fact that the slope of the curve relating efflux to H2mal0 concentration, when corrected for the presumed contributions from Hmal1− and mal2− transport and plotted on a log-log basis, approaches 1.0 at the highest malic-acid contents. Moreover, the permeability coefficient required to be consistent with such a mechanism\((P_{H_2 mal^0 } = 1.0to2.0 \times 10^{ - 8} m\sec ^{ - 1} )\) is similar to that estimated from a Collander plot, using the partition coefficient of malic acid between ether and water. We suggest that\(P_{H_2 mal^0 } \) may be important in determining the maximum amounts of malic acid that can be accumulated during the CAM rhythm.

Vacuolar Localization of Endoproteinases EP1 and EP2 in Barley Mesophyll Cells

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent Km of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg(2+) present, nor by protonophores and valinomycin (with K(+) present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.

Subcellular localization of acid proteinase in barley mesophyll protoplasts

Chloroplasts prepared from lysed protoplasts of barley mesophyll contain 2-8% of the total acid proteinase activity. This residual activity is not associated with intact chloroplasts isolated by means of density gradient centrifugation. Vacuoles isolated from lysed protoplasts contain 80-85% of the total acid proteinase activity, indicating that the enzyme(s) which is presumably responsible for the degradation of chloroplastic proteins is located largely in the central vacuoles of mesophyll cells.