A morphometric analysis of the phloem-unloading pathway in developing tobacco leaves

A morphometric analysis of developing leaves of Nicotiana tabacum L. was conducted to determine whether imported photoassimilates could be unloaded by symplastic transport and whether interruption of symplastic transport could account for termination of import. Five classes of veins were recognized, based on numbers of cells in transverse section. Photoassimilate is unloaded primarily from Class III veins in tissue nearing the end of the sink phase of development. Smaller veins (Class IV and V) do not transport or unload photoassimilate in sink tissue because the sieve elements of these veins are immature until after the tissue stops importing. In Class III veins the sieve element-companion cell (SE-CC) complexes are surrounded by phloem parenchyma which abuts the bundle sheath. Along the most obvious unloading route, from SE-CC complex to phloem parenchyma to bundle sheath to mesophyll cells, the frequency of plasmodesmata at each interface increases. To determine whether this pattern of plasmodesmatal contact is consistent with symplastic unloading we first demonstrated, by derivation from Fick's law that the rate of diffusion from a compartment is proportional to a number N which is equal to the ratio of surface area to volume of the compartment multiplied by the frequency of pores (plasmodesmata) which connect it to the next compartment. N was calculated for each compartment within the vein which has the SE-CC complex as its center, and was shown to be statistically the same in all cases except one. These observations are consistent with a symplastic unloading route. As the leaf tissue matures and stops importing, plasmodesmatal frequency along the unloading route decreases and contact area between cells also decreases as intercellular spaces enlarge. As a result, the number of plasmodesmata between the SE-CC complex and the first layer of mesophyll cells declines in nonimporting tissue to 34% of the number found in importing tissue, indicating that loss of symplastic continuity between the phloem and surrounding cells plays a role in termination of photoassimilate unloading.Abbreviation SE-CC sieve element-companion cell     

Geographic distributions and physiological characteristics of co-existing Flaveria species in south-central Mexico

The genus Flaveria consists of 23 species with significant variation in photosynthetic physiologies. We tested whether photosynthetic pathway variation in seven co-existing Flaveria species corresponds to geographic distributions or physiological performance in C₃, C₄, and intermediate species growing under natural conditions in south-central Mexico. We found that Flaveria pringlei (C₃) was the most widely distributed species with multiple growth habits. Numerous populations of Flaveria kochiana (C₄), a recently described species with a previously unknown distribution, were located in the Mixtec region of Oaxaca. Flaveria cronquistii (C₃) and Flaveria ramosissima (C₃-C₄) were only located in the Tehuacán Valley region while Flaveria trinervia (C₄) was widely distributed. Only one population of Flaveria angustifolia (C₃-C₄) and Flaveria vaginata (C₄-like) were located near Izúcar de Matamoros. Midday leaf water potential differed significantly between Flaveria species, but did not vary according to growth habit or photosynthetic pathway. The quantum yield of photosystem II did not vary between species, despite large differences in leaf nitrogen content, leaf shape, plant size and life histories. We did not find a direct relationship between increasing C₄ cycle characteristics and physiological performance in the Flaveria populations examined. Furthermore, C₃ species were not found at higher elevation than C₄ species as expected. Our observations indicate that life history traits and disturbance regime may be the primary controllers of Flaveria distributions in south-central Mexico.     

The Use of Fluorescent Tracers to Characterize the Post-Phloem Transport Pathway in Maternal Tissues of Developing Wheat Grains

Various polar fluorescent tracers were used to characterize the pathways for apoplastic and symplastic transport in the "crease tissues" (i.e. the vascular strand, chalaza, nucellus, and adjacent pericarp) of developing wheat (Triticum aestivum L.) grains. With mostly minor exceptions, the results strongly support existing views of phloem unloading and post-phloem transport pathways in the crease. Apoplastic movement of Lucifer yellow CH (LYCH) from the endosperm cavity into the crease was virtually blocked in the chalazal cell walls before reaching the vascular tissue. However, LYCH could move slowly along the cell wall pathway from the chalaza into the vascular parenchyma. Slow uptake of LYCH into nucellar cell cytoplasm was observed, but no subsequent symplastic movement occurred. Carboxyfluorescein (CF) imported into attached grains moved symplastically from the phloem across the chalaza and into the nucellus, but was not released from the nucellus. In addition, CF moved in the opposite direction (nucellus to vascular parenchyma) in attached grains. Thus, the post-phloem symplastic pathway can accommodate bidirectional transport even when there is an intense net assimilate flux in one direction. When fresh sections of the crease were placed in fluorochrome solutions (e.g. LYCH or pyrene trisulfonate), dye was rapidly absorbed into intact cells, apparently via unsealed plasmodesmata. Uptake was not visibly reduced by cold or by respiratory inhibitors, but was greatly reduced by plasmolysis. Once absorbed, the dye moved intercellularly via the symplast. Based on this finding, a size-graded series of fluorescein-labeled dextrans was used to estimate the size-exclusion limits (SEL) for the post-phloem symplastic pathway. In most, and perhaps all, cells of the crease tissues except for the pericarp, the molecular diameter for the SEL was about 6.2 nm. The SEL in much of the vascular parenchyma may be smaller, but it is still at least 3.6 nm. Channel diameters would likely be about 1 nm larger, or about 4.5 to 7.0 nm in the vascular parenchyma and 7.0 nm elsewhere. These dimensions are substantially larger than those for "conventional" symplastic connections (about 3 nm), and would have a greater than proportionate effect on the per channel diffusive and hydraulic conductivities of the pathway. Thus, relatively small and probably ultrastructurally undetectable adjustments in plasmodesmatal structure may be sufficient to account for assimilate flux through the crease symplast.     

Le vie fotosintetiche C3, C4 e CAM nel contesto ecologico

Abstract

Ecological aspects of C₃, C₄ and CAM photosynthetic pathways. - Three different photosynthetic CO₂ fixation pathways are known to occur in higher plants. However all three pathways ultimately depend on the Calvin-Benson cycle for carbon reduction. The oxygenase activity of RuBP carboxilase is responsible for photorespiratory CO₂ release. Both C₄ and CAM pathways behave as a CO₂ concentrating mechanism which prevent photorespiration. The CO₂-concentrating mechanism in C₄ plants is based on intracellular symplastic transport of C₄ dicarboxylic acids from mesophyll-cells to the adjacent bundle-sheath cells. On the contrary in CAM plants the CO₂-concentrating mechanism is based on the intracellular transport of malic acid into and out of the vacuole.

The C₄ photosynthetic pathway as compared to the C₃ pathway permits higher rates of CO₂ fixation in high light and high temperature environments at low costs in terms of water loss, given the stability of the photosynthetic apparatus under such conditions.

CAM is interpreted as an adaptation to arid environments because it enables carbon assimilation to take place at very low water costs during the night when the evaporative demand is low. Nevertheless many aquatic species of Isoetes and some relatives are CAM, suggesting the adaptive role of CAM to environments which become depleted in CO₂.

The photosynthetic carbon fixation pathway certainly contributes to the ecological success of plants in different environments. However the distribution of plants may also reflect their biological history. On the other hand plants with different photosynthetic pathways coexist in many communities and tend to share resources in time. In any case some generalizations are possible: C₄ plants enjoy an ecological advantage in hot, moist, high light regions while the majority of species in desert environments are C₃; CAM plants are more frequent in semiarid regions with seasonal rainfall, coastal fog deserts, and in epiphytic habitats in tropical rain forests.     

Fine structure,distribution and frequency of plasmodesmata and pits in the cortex ofLaminaria hyperborea andL. saccharina

Prior to a long-distance transport of photoassimilate in the sieve elements ofLaminaria, a parenchyma transport across the cortex must occur. It is suggested that this transport is a symplastic one. The structural basis for this statement, continuous cytoplasmic interconnections of cells along the transport pathway, is demonstrated here forL. hyperborea andL. saccharina. The distribution, size, and frequency of pit fields in cell walls of all planes were determined. The data suggest that the conductivity for assimilate transport in the cortex is highest in the long axis of the thallus, not radially across the cortex. The fine structure, arrangement and number of plasmodesmata in pit fields were studied. The estimated flux rates and the anatomical findings clearly point to a symplastic parechyma transport of photoassimilate in the cortex ofLaminaria.     

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.     

Intercellular Symplastic Connection and Isolation of the Unloading Zone in Flesh of the Developing Grape Berry

The uhrastructure and intercellular connection of the sugar unloading zone (i. e. the phloem in the dorsal vascular bundle and the phloem-surrounding the assimilate sink-cells) of grape ( Vitis vinifera x V. labrusca cv. Jingchao) berry was observed via transmission electron microscopy. The results showed that during the early developmental stages of grape berry, numerous plasmodesmata were found in the phloem between sieve element (SE) and companion cell (CC), between SE/CC complexes, between SE/CC complex and phloem parenchyma cell and in between phloem parenchyma cells, which made the phloem a symplastic integration, facilitating sugar unloading from sieve elements into both companion cells and phloem parenchyma cells via a symplastic pathway. On the contrary, there was almost no plasmodesma between phloem and its surrounding flesh photoassimilate sink-cells, neither in between the flesh photoassimilate sink-cells giving rise to a symplastic isolation both between phloem and its surrounding flesh photoassimilate sink-cells, as well as among the flesh photoassimilate sink-cells. This indicated that both the sugar unloading from phloem and pestphloem transport of sugars should be mainly via an apoplastic pathway. Dining the ripening stage, most of the plasmodesmata between SE/CC complex and the surrounding phloem parenchyma cells were shown to be blocked by the electron-opaque globules, and a phenomenon of plasmolysis was found in a number of companion cells, indicating a symplastic isolation between SE/CC complex and its surrounding parenchynm cells during this phase. The symplastic isolation between the whole phloem and its surrounding photoassimilate sink-cells during the early developmental stages shifted to a symplastic isolation within the phloem during the ripening phase, and thus the symplastic pathway of sugar unloading from SE/CC complex during the early development stages should be replaced by a dominant apoplastic unloading pathway from SE/CC complex in concordance.     

Intracellular calcium puffs in osteoclasts

We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.     

Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter

Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.     

Evaluating the use of pairwise dissimilarity metrics in paleoanthropology

Questions of alpha taxonomy are best addressed by comparing unknown specimens to samples of the taxa to which they might belong. However, analysis of the hominin fossil record is riddled with methods that claim to evaluate whether pairs of individual fossils belong to the same species. Two such methods, log se_m and the related STET method, have been introduced and used in studies of fossil hominins. Both methods attempt to quantify morphological dissimilarity for a pair of fossils and then evaluate a null hypothesis of conspecificity using the assumption that pairs of fossils that fall beneath a predefined dissimilarity threshold are likely to belong to the same species, whereas pairs of fossils above that threshold are likely to belong to different species. In this contribution, we address (1) whether these particular methods do what they claim to do, and (2) whether such approaches can ever reliably address the question of conspecificity. We show that log se_m and STET do not reliably measure deviations from shape similarity, and that values of these measures for any pair of fossils are highly dependent upon the number of variables compared. To address these issues we develop a measure of shape dissimilarity, the Standard Deviation of Logged Ratios (s_LR). We suggest that while pairwise dissimilarity metrics that accurately measure deviations from isometry (e.g., s_LR) may be useful for addressing some questions that relate to morphological variation, no pairwise method can reliably answer the question of whether two fossils are conspecific.     

Phloem unloading in the potato tuber. Pathways and sites of ATPase

Summary Potential pathways for sucrose unloading in the potato tuber were examined by light and electron microscopy. Abundant plasmodesmata connected sieve elements with surrounding parenchyma elements and also sieve elements with companion cells. Plasmodesmata were rarer, however, between companion cells and parenchyma elements. These observations suggest that sucrose may leave the sieve elements and enter the storage parenchyma cells directly via the symplast and that transport through the companion cell may not be a prerequisite for unloading. Plasmodesmata, grouped together in primary pit fields, were also abundant between storage cells, and isolated storage cells, separated enzymically, showed considerable variation in plasmodesmatal distribution between cells and also on different faces of a single cell. Deposition of starch was found to occur in the tuber cortex while an endodermis with Casparian strip was present external to the phloem, suggesting that assimilates initially enter the cortical storage cells by an entirely symplastic pathway. The possible involvement of ATPase in the unloading process was examined cytochemically, using a lead-salt precipitation method. By contrast with previous findings for phloem no evidence was found for ATPase activity that was unique to the sieve element-companion cell complex. The present observations favour the view that phloem unloading in the potato tuber is a symplastic and passive process.     

The contributions of apoplastic,symplastic and gas phase pathways for water transport outside the bundle sheath in leaves

Water movement from the xylem to stomata is poorly understood. There is still no consensus about whether apoplastic or symplastic pathways are more important, and recent work suggests vapour diffusion may also play a role. The objective of this study was to estimate the proportions of hydraulic conductance outside the bundle sheath contributed by apoplastic, symplastic and gas phase pathways, using a novel analytical framework based on measurable anatomical and biophysical parameters. The calculations presented here suggest that apoplastic pathways provide the majority of conductance outside the bundle sheath under most conditions, whereas symplastic pathways contribute only a small proportion. The contributions of apoplastic and gas phase pathways vary depending on several critical but poorly known or highly variable parameters namely, the effective Poiseuille radius for apoplastic bulk flow, the thickness of cell walls and vertical temperature gradients within the leaf. The gas phase conductance should increase strongly as the leaf centre becomes warmer than the epidermis – providing up to 44% of vertical water transport for a temperature gradient of 0.2 K. These results may help to explain how leaf water transport is influenced by light absorption, temperature and differences in leaf anatomy among species.     

The neck constriction in plasmodesmata

Simple plasmodesmata between mesophyll and bundle sheath cells in actively expanding leaves of Salsola kali L. and roots of Epilobium hirsutum L. are shown to possess specialized structures, called sphincters, around their neck regions. The sphineters are made visible by the combined effects of tannic acid and heavy metal staining; they are localized just outside that area of the plasmalemma, which forms the collar around the entrance to each plasmodesmos. This localization corresponds to a very active area of the plasmodesmos/olasmalemma complex (i.e. enzyme activity and/or presence of strongly reducing substances).Evidence is presented that these ring structures are structural equivalents to hypothetical sphincters performing some valve function; i.e. participating in the control of rates and directions of symplastic transport of solutes through plasmodesmata. The middle layer of the plasmalemma in the neck region is composed of closely-packed, globular subunits appearing in negative contrast. Apparently, these subunits correspond to particle clusters observed at the plasmodesmatal entrance in freeze-fracture preparations. They appear similar to particle clusters in animal tight junctions, and their possible function in providing electrical coupling via low resistance junctions between plant cells is discussed.     

Dynamic continuity of cytoplasmic and membrane compartments between plant cells

Fluorescence photobleaching was employed to examine the intercellular movement of fluorescein and carboxyfluorescein between contiguous soybean root cells (SB-1 cell line) growing in tissue culture. Results of these experiments demonstrated movement of these fluorescent probes between cytoplasmic (symplastic) compartments. This symplastic transport was inhibited with Ca2+ in the presence of ionophore A23187, and also with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both of these agents have previously been demonstrated to inhibit gap junction-mediated cell-cell communication in animal cells. In a companion experiment, a fluorescent phospholipid analogue, N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylcholine (NBD-PC), was incorporated into soybean cell membranes to examine whether dynamic membrane continuity existed between contacting cells, a transport route not existing between animal cells. Photobleaching single soybean cells growing in a filamentous strand demonstrated that phospholipid did exchange between contiguous cells.     

Searching for the molecular arrangement of transmembrane ceramide channels

Ceramides have been implicated in the initiation of apoptosis by permeabilizing the mitochondrial outer membrane to small proteins, including cytochrome c. In addition, ceramides were shown to form large metastable channels in planar membranes and liposomes, indicating that these lipids permeabilize membranes directly. Here we analyze molecular models of ceramide channels and test their stability in molecular dynamics simulations. The structural units are columns of four to six ceramides H-bonded via amide groups and arranged as staves in either a parallel or antiparallel manner. Two cylindrical assemblies of 14 columns (four or six molecules per column) were embedded in a fully hydrated palmitoyloleoyl-phosphatidylcholine phospholipid bilayer, and simulated for 24 ns in total. After equilibration, the water-filled pore adopted an hourglass-like shape as headgroups of ceramides and phospholipids formed a smooth continuous interface. The structure-stabilizing interactions were both hydrogen bonds between the headgroups (including water-mediated interactions) and packing of the hydrocarbon tails. Ceramide's essential double bond reduced the mobility of the hydrocarbon tails and stabilized their packing. The six-column assembly remained stable throughout a 10-ns simulation. During simulations of four-column assemblies, pairs of columns displayed the tendency of splitting out from the channels, consistent with the previously proposed mechanism of channel disassembly.     

Photosynthetic and morphological functional types for native species from mixed prairie in Southern Saskatchewan, Canada

Photosynthetic pathways (e.g. C₃, C₄) and morphological functional types (e.g. trees, shrubs, high perennial grasses, perennial forbs) were identified for the native species from the Saskatchewan mixed prairie, using the data from references published between 1950 and 2003. Of the total 219 identified species in 145 genera and 45 families, 208 species in 137 genera and 44 families were found with C₃ photosynthesis, and most of these species are dominants (e.g. Agropyron dasystachyum Hook. and Stipa spartea var. curtiseta Hitchc.). 11 species in 10 genera and 3 families were identified with C₄ photosynthesis (e.g. Atriplex argentea Nutt., Andropogon scoparius Michx., Boutelou gracilis Lag., Calamovilfa longifolia Hook.). The amount of total identified C₄ species in the region is much less than that from the South Dakota mixed prairie (27 species). Gramineae is the leading family with C₄ photosynthesis (8 species), Chenopodiaceae ranks the second (2 species). Relatively less forb types [50 % perennial forbs (PEF) and 12 % annual forbs (ANF)] and more graminoid types (25 %) composition suggested that the rangelands in the region are relatively stable. Lacking of the knowledge on the optimal traits for PFTs classification in the region, further studies (e.g. C₃ and C₄ plant identification and optimal trait selection) are needed to explore the relationships between PFTs and vegetation variations, as well as land-use and climate changes.     

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades—namely, the leaf sheath, stem, scale leaf, and constituents of the spike—also have this unique anatomy and the C₄ pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C₄-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C₄ anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C₃ and C₄ enzymes revealed that all the organs exhibited essentially the same C₄ pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C₄ pathway operation.     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).