Isolation of mesophyll and secretory cell protoplasts of the halophyte Ceratostigma plumbaginoides (L.): a comparison of ATPase concentration and activity.

A method is described for isolating mesophyll protoplasts from leaves and secretory cell protoplasts from salt glands of the facultative halophyte, Ceratostigma plumbaginoides (L.). Rates of ATP hydrolysis in both cell types were determined, and levels in secretory cell protoplast preparations were fourfold higher than those in mesophyll protoplast preparations, based on total protein. The rate of ATP hydrolysis was sensitive to azide and vanadate, and stimulated by Triton-X-100. Additionally, immunoblot procedures using an antibody to the plasma membrane H(+)/ATPase was used to compare ATPase levels of the mesophyll and secretory cell protoplasts. Results indicate that secretory cells have a higher concentration of H(+)/ATPase than mesophyll cells, consistent with their putative function in salt glands.

The Impact of Chlorophyll-Retention Mutations, d1d2 and cyt-G1, during Embryogeny in Soybean.

The ultrastructural, physiological, and molecular changes in developing and mature seeds were monitored in a control line (Glycine max [L.] Merr., cv Clark) that exhibited seed degreening and two mutant lines (d1d2 and cyt-G1) that retained chlorophyll upon seed maturation. Ultrastructural studies showed that the control line had no internal membranes, whereas stacked thylakoid membranes were detected in the green seed from the mutant lines. Pigment analyses indicated that total chlorophyll was lowest in the mature seeds of the control line. Mature d1d2 and cyt-G1 seed had elevated Chl a and Chl b levels, respectively. In both control and mutant lines, Lhcb1, Lhcb2, and RbcS mRNAs were abundant in embryos prior to cotyledon filling, declined after the onset of storage protein accumulation, and were barely detectable or undetectable in all later stages of seed development. Therefore, the chlorophyll-retention phenotype must be a result of the alteration of a process that occurs after translation of photosynthesis-related mRNAs to stabilize apoprotein and pigment levels. Furthermore, different elements controlling either the synthesis or turnover of Chl a and Chl b must be impaired in the d1d2 and cyt-G1 lines. No reproducible differences in total leaf, embryonic, and chloroplast protein profiles and plastid DNAs could be correlated with the mutations that induced chlorophyll retention.

Advantages of the use of intact plant tissues in freeze-fracture electron microscopy.

The use of whole, intact plant tissue for freeze-fracture electron microscopy provides important information that cannot be obtained from the use of isolated biological membranes or of artificial (phospholipid) membrane preparations. This is not to imply that these examinations of such preparations are not useful, since it would be difficult to interpret our observations of intact cells and tissues without the analysis of these model systems. Analysis of intact tissue and cells reveals the relative densities of membrane proteins of the different membranes within a cell; the three-dimensional organization of various organelles, especially the endoplasmic reticulum; changes in intramembranous particle (IMP) distribution due to stress or injury; and, in conjunction with the use of filipin, membrane sterol content and relative distribution. It is our intention that this survey of freeze-fracture images of intact plant tissues will illustrate the uniqueness of the information gained from an analysis of whole plant tissues compared to isolated membrane fractions.

Freeze-fracture observations on membranes of dry and hydrated pollen from Collomia, Phoenix and Zea.

Pollen from Collomia grandiflora Dougl. ex Lindl., Phoenix dactylifera L. and Zea mays L. was examined by freeze-fracture electron microscopy. Particular attention was paid to the organization of the cell membranes in the naturally dehydrated, as compared to the fully hydrated, state. All membranes examined had a normal bilayer organization similar to that seen in the hydrated cells of these and other plants. This organization of dry pollen membranes is discussed as it relates to physiological studies (e.g., leakage of ions during hydration), and to biophysical properties of biological and model membranes under various conditions of hydration and dehydration.

Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons.

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.

The ultrastructure of Cynodon salt glands: secreting and nonsecreting.

Experimental conditions were established in which the salt glands in the leaves of Bermuda grass could be identified as secreting or nonsecreting. These glands consist of two cells, an outer cap cell and an inner basal cell, and at the ultrastructural level, three primary differences existed between secreting and nonsecreting cells. In the secreting glands, these were a tubular elaboration of the so-called partitioning membranes of the basal cell, an expansion of the overarching cuticle of the glands, and an increase in vacuolation within the cap cell. All of these changes may relate to the secretion process.

Freeze fracture of intact plant tissues.

A procedure for dissolving and handling replicas of cutinized leaves and other plant tissues is described. This technique yields comparatively large replicas which can include vascular tissue, epidermal cells and the cuticle. Dissolution of the tissue involves the sequential use of alcoholic KOH, sulfuric acid and chromic acid. The use of clean, uncoated grids for transfer of the tissue and replicas results in rapid, easy handling with a minimum of breakage or loss. The procedure is more efficient than previous methods and produces large replicas. This allows tissue orientation and more positive identification of cell types for better correlation of freeze fracture results with thin sections of similar material.

The ultrastructure of Cynodon salt glands: the apoplast.

The salt glands of Bermuda grass were found to be similar to those of many dicotyledonous plants with respect to the relatively large quantities of chloride that accumulate in the subcuticular collecting chamber of secreting glands. The salt glands of grasses lack the suberized or cuticularized zone that is present in the walls of the glands of dicotyledonous plants that has been hypothesized to prevent the apoplastic backflow of accumulated salts from the collecting chamber to the leaf mesophyll. An ionic lanthanum solution was used to determine whether or not the apoplastic pathway is blocked between the collecting chamber and the leaf mesophyll. The results of the lanthanum tracer study revealed that this pathway is significantly restricted. It was determined that the restriction of the apoplastic pathway was due to the highly lignified condition of the lateral cell walls in the outer portion of the basal gland cell. The lanthanum tracer study also revealed that an apoplastic continuum exists between the leaf mesophyll and a system of membranous extracellular channels that occur in the basal cell. Based on this finding, it was suggested that the extracellular channels may function in the absorption of solutes from the apoplast.

Lipid phase separations and intramembranous particle movements in the yeast tonoplast.

The tonoplast of Saccharomyces cerevisiae contains regions depleted of intramembranous particles as the cells enter stationary phase. Freeze-fracture studies on intact cells from this growth stage show that a dispersed particle distribution predominates if the cell temperature is raised to 40 degrees C but that particle-depleted areas prevail at or below the cell growth temperature of 30 degrees C. Tonoplasts of isolated vacuoles also contain particle-depleted regions. Differential thermal analysis of lipids extracted from isolated vacuoles show an endothermic transition which encompasses the cell growth temperature. These results suggest that the tonoplast at this stage contains patches of gel-phase lipid and that these patches correspond to the intramembranous particle-depleted areas of the freeze-fractured tonoplast.

Chloroplast division in spinach leaves examined by scanning electron microscopy and freeze-etching.

Spinach leaf disks were cultured for 5 days in low-intensity green light and then were transferred to high-intensity white light. Harvests over the next 16 h established that cell area increased by about 80% and chloroplast number per cell increased by about 65%, while the percentage of dumbbell-shaped chloroplasts per cell decreased by 65%. Freeze-etch replicas of fixed and unfixed leaf disks, as well as scanning electron-microscope preparations of fixed material, contained dumbbell-shaped chloroplasts constricted to various degrees. Freeze-etch replicas of unfixed cells from young leaf bases, in which the number of chloroplasts per cell is known to be rapidly increasing, also contained many constricted chloroplasts. It is concluded that dumbbell-shaped chloroplasts occur in vivo and represent a stage in the division of chloroplasts.

Ultrastructural transitions associated with the development of the bladder cells of the trichomes of Atriplex.

Early in development, bladder cells are characterized by the absence of a vacuole or vacuoles, the presence of autophagic vesicles, and numerous, unaggregated ribosomes. With the formation and expansion of the central vacuole, the ribosomes become aggregated and elements of rough endoplasmic reticulum become apparent. This developmental transition is probably related to the production of proteins involved in ion accumulation in the vacuole. Throughout expansion, invaginations of the tonoplast and membraneous structures are associated with the vacuole. These may be indicative of a continued lytic function for this compartment. Also, dictyosomes are continuously present and dictyosome vesicles are associated with both the plasmalemma and tonoplast, which suggest that they contribute to both membrane systems during expansion of the cell and vacuole.

Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain.

Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K(+)-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots.

Effects of lanthanum and ethylenediaminetetraacetate on leaf movements of mimosa.

Lanthanum and ethylenediaminetetraacetate (EDTA) profoundly affect the rapid leaf movements of Mimosa pudica L. Lanthanum, which mimics calcium but does not penetrate the plasmalemma, inhibits the closing response but does not affect reopening. A low concentration of EDTA retards the reopening process while a higher EDTA concentration prevents the closing movement. There is evidence that the EDTA effects result from chelation of calcium ions rather than chelation of other cations. These results are discussed with regard to the role of calcium in leaf movements.

Effect of lanthanum on ion absorption in corn roots.

Short term (10 min) influx of (86)Rb-labeled potassium into corn (Zea mays L. WF9 x M14) root segments was inhibited by La (NO(3))(3) or LaCl(3). Half maximal inhibition of K(+) influx from 0.25 mm KCl was obtained with 0.025 mm La(3+). Kinetic analysis indicated the inhibition to be of a competitive nature. With absorption periods exceeding one hour, La(3+) no longer inhibited, but rather stimulated K(+) influx rates. La(3+) was not an inhibitor of (36)Cl or (32)P absorption. Separated cortex and stele absorbed labeled potassium (and phosphate) at comparable rates, and La(3+) inhibited potassium influx in both tissues. The effects of La(3+) on ion absorption were similar to those of Ca(2+), suggesting that the two polyvalent cations act at the same site. Based on this and the observation that La(3+) does not seem to penetrate the plasma membrane, it was concluded that La(3+) and Ca(2+) affect changes in ion transport without entering cells.

The casparian strip as a barrier to the movement of lanthanum in corn roots.

The effectiveness of the Casparian strip as a barrier to apoplastic movement of solutes from cortex to stele of corn roots was investigated by using lanthanum in combination with electron microscopy. Lanthanum deposits were found only in cell walls and on the outside of the plasma membrane of epidermal, cortical, and endodermal cells up to the Casparian strip. Lanthanum was completely absent from the stele, indicating that the Casparian strip provides an effective barrier to apoplastic movement of solutes. Inhibitory effects of trivalent lanthanum ions on the absorption of potassium ions are discussed in relation to the nature of the lanthanum ion binding site on membranes.

Plastid differentiation, acyl lipid, and Fatty Acid changes in developing green maize leaves.

Plastid differentiation, acyl lipid, and fatty acid composition have been followed in successive 2-cm sections from the base (youngest tissue) to the tip (oldest tissue) of green Zea mays (maize) leaves grown under a normal diurnal light regime. Although the youngest cells (0-4 cm from the leaf base) had only proplastids with one or two grana, they contained chlorophylls a and b, monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. In the more mature sections, the plastids increased in size 5-fold, and differentiation into mesophyll and bundle-shealth chloroplasts had occurred. Concomitantly, the levels of all the lipids increased with the exception of phosphatidylcholine and phosphatidylethanolamine which decreased. With increasing cell maturity, the percentage of linolenic acid increased in all the individual acyl lipids, but palmitic acid remained constant in phosphatidylcholine, phosphatidylethanolamine, and sulfolipid. The Delta(3t)-hexadecenoic acid was only detectable in the phosphatidylglycerol of the most mature maize tissue.