Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters.

A major question in plant physiology is how the large amount of sucrose made in leaves is transported to the rest of the plant. Although physiological, biochemical, and anatomical investigations have been performed in this field, to date there have been very few genetic studies. Using a reverse genetic screen, we have identified mutant Arabidopsis plants containing transferred DNA insertions in the gene encoding a phloem-specific sucrose transporter, SUC2. SUC2 is thought to function in loading sugar from the apoplast into the conducting sieve tubes. In the homozygous state, these mutations resulted in stunted growth, retarded development, and sterility. The source leaves of mutant plants contained a great excess of starch, and radiolabeled sugar failed to be transported efficiently to roots and inflorescences. These data provide genetic proof that apoplastic phloem loading is critical for growth, development, and reproduction in Arabidopsis and that SUC2 is at least partially responsible for this step.

Modification of a Specific Class of Plasmodesmata and Loss of Sucrose Export Ability in the sucrose export defective1 Maize Mutant.

We report on the export capability and structural and ultrastructural characteristics of leaves of the sucrose export defective1 (sed1; formerly called sut1) maize mutant. Whole-leaf autoradiography was combined with light and transmission electron microscopy to correlate leaf structure with differences in export capacity in both wild-type and sed1 plants. Tips of sed1 blades had abnormal accumulations of starch and anthocyanin and distorted vascular tissues in the minor veins, and they did not export sucrose. Bases of sed1 blades were structurally identical to those of the wild type and did export sucrose. Electron microscopy revealed that only the plasmodesmata at the bundle sheath-vascular parenchyma cell interface in sed1 minor veins were structurally modified. Aberrant plasmodesmal structure at this critical interface results in a symplastic interruption and a lack of phloem-loading capability. These results clarify the pathway followed by photosynthates, the pivotal role of the plasmodesmata at the bundle sheath-vascular parenchyma cell interface, and the role of the vascular parenchyma cells in phloem loading.

Ultrastructure of and plasmodesmatal frequency in mature leaves of sugarcane.

Vascular bundles and contiguous tissues of leaf blades of sugarcane (Saccharum interspecific hybrid L62-96) were examined with light and transmission electron microscopes to determine their cellular composition and the frequency of plasmodesmata between the various cell combinations. The large vascular bundles typically are surrounded by two bundle sheaths, an outer chlorenchymatous bundle sheath and an inner mestome sheath. In addition to a chlorenchymatous bundle sheath, a partial mestome sheath borders the phloem of the intermediate vascular bundles, and at least some mestome-sheath cells border the phloem of the small vascular bundles. Both the walls of the chlorenchymatous bundlesheath cells and of the mestome-sheath cells possess suberin lamellae. The phloem of all small and intermediate vascular bundles contains both thick- and thin-walled sieve tubes. Only the thin-walled sieve tubes have companion cells, with which they are united symplastically by pore-plasmodesmata connections. Plasmodesmata are abundant at the Kranz mesophyll-cell-bundlesheath-cell interface associated with all sized bundles. Plasmodesmata are also abundant at the bundle-sheathcell-vascular-parenchyma-cell, vascular-parenchyma-cellvascular-parenchyma-cell, and mestome-sheath-cell-vascular-parenchyma-cell interfaces in small and intermediate bundles. The thin-walled sieve tubes and companion cells of the large vascular bundles are symplastically isolated from all other cell types of the leaf. The same condition is essentially present in the sieve-tube-companion-cell complexes of the small and intermediate vascular bundles. Although few plasmodesmata connect either the thin-walled sieve tubes or their companion cells to the mestome sheath of small and intermediate bundles, plasmodesmata are somewhat more numerous between the companion cells and vascular-parenchyma cells. The thick-walled sieve tubes are united with vascular-parenchyma cells by pore-plasmodesmata connections. The vascular-parenchyma cells, in turn, have numerous plasmodesmatal connections with the bundle-sheath cells.

Fine structure of plasmodesmata in mature leaves of sugarcane.

The fine structure of plasmodesmata in vascular bundles and contiguous tissues of mature leaf blades of sugarcane (Saccharum interspecific hybrid L62-96) was studied with the transmission electron microscope. Tissues were fixed in glutaraldehyde, with and without the addition of tannic acid, and postfixed in OsO4. The results indicate that the fine structure of plasmodesmata in sugarcane differs among various cell combinations in a cell-specific manner, but that three basic structural variations can be recognized among plasmodesmata in the mature leaf: 1) Plasmodesmata between mesophyll cells. These plasmodesmata possess amorphous, electron-opaque structures, termed sphincters, that extend from plasma membrane to desmotubule near the orifices of the plasmodesmata. The cytoplasmic sleeve is filled by the sphincters where they occur; elsewhere it is open and entirely free of particulate or spokelike components. The desmotubule is tightly constricted and has no lumen within the sphincters, but between the sphincters it is a convoluted tubule with an open lumen. 2) Plasmodesmata that traverse the walls of chlorenchymatous bundle-sheath cells and mestome-sheath cells. In addition to the presence of sphincters, these plasmodesmata are modified by the presence of suberin lamellae in the walls. Although the plasmodesmata are quite narrow and the lumens of the desmotubules are constricted where they traverse the suberin lamellae, the cytoplasmic sleeves are still discernible and appear to contain substructural components there. 3) Plasmodesmata between parenchymatous cells of the vascular bundles. These plasmodesmata strongly resemble those found in the roots of Azolla, in that their desmotubules are closed for their entire length and their cytoplasmic sleeves appear to contain substructural components for their entire length. The structural variations exhibited by the plasmodesmata of the sugarcane leaf are compared with those proposed for a widely-adopted model of plasmodesmatal structure.

The cell wall-plasmalemma interface in sieve tubes of barley.

Both thick- and thin-walled sieve tubes in leaf-blade veins of Hordeum vulgare L. exhibit a distinct, electron-opaque inner wall layer after fixation in glutaraldehyde-osmium tetroxide and staining with uranyl acetate and lead citrate. This inner wall layer is thickest at the sieve plates and lateral sieve areas where it is permeated by a labyrinth of tubules formed by the plasmalemma. Along the lateral walls between sieve areas the inner wall layer apparently is penetrated by numerous microvilli-like evaginations of the plasmalemma, giving the cell wall-plasmalemma interface the appearance of a brush border. It is suggested that a similar brush-border-like structure may occur at the cell wall-plasmalemma interface of sieve elements in a wide variety of vascular plants.

Loading and transport of assimilates in different maize leaf bundles : Digital image analysis of (14)C-microautoradiographs.

The loading and transport functions of vascular bundles in maize (Zea mays L.) leaf strips were investigated by microautoradiography after application of (14)CO2. The concentrations of (14)C-contents in thin-walled sieve tubes of individual bundles in the loading and transport regions were determined by digital image analysis of silver-grain density over the sieve tubes and compared. In the loading region, relatively high concentrations of (14)C-contents were found in the thin-walled sieve tubes of small bundles and in the small, thin-walled sieve tubes of the intermediate bundles; the concentration of (14)C-label in large bundles was very low. In the transport region, at a transport distance of 2 cm, all of the small bundles contained (14)C-assimilates, but generally less than the same bundles did in the loading region; by comparison, at that distance intermediate and large bundles contained two-to threefold more (14)C-assimilates than the same bundles in the loading region. The lateral transfer of assimilates from smaller to larger bundles via transverse veins could be demonstrated directly in microautoradiographs. A reverse transport from larger to smaller bundles was not found. At a transport distance of 4 cm, all large and intermediate bundles were (14)C-labeled, but many of the small bundles were not. Although all longitudinal bundles were able to transport (14)C-asimilates longitudinally down the blade, it was the large bundles that were primarily involved with longitudinal transport and the small bundles that were primarily involved with loading.

Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump.

Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H(+)-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H(+)-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump.

Plasmodesmatal distribution and frequency in vascular bundles and contiguous tissues of the leaf ofThemeda triandra.

Small and intermediate vascular bundles and contiguous tissues of the leaf blade ofThemeda triandra var.imberbis (Retz.) A. Camus were examined with transmission and scanning electron microscopes to determine the distribution and frequency of plasmodesmata between various cell types. Plasmodesmata are most abundant at the mesophyll/bundle-sheath cell and bundle-sheath/vascular parenchyma cell interfaces, and their numbers decrease with increasing proximity to both thick- and thin-walled sieve tubes. Among cells of the vascular bundles, the greatest frequency of plasmodesmata occurs between vascular parenchyma cells, followed by that of plasmodesmata between vascular parenchyma cells and companion cells, and then by the pore-plasmodesmata connections between companion cells and thin-walled sieve tubes (sieve tube-companion cell complexes). The sieve tube-companion cell complexes of theT. triandra leaf are not isolated symplastically from the rest of the leaf and, in this respect, differ from their counterparts in theZea mays leaf. However, the thick-walled sieve tubes, like their counterparts inZea mays, lack companion cells and are symplastically connected with vascular parenchyma cells that about the xylem.

Leaf vasculature in Zea mays L.

The vascular system of the Zea mays L. leaf consists of longitudinal strands interconnected by transverse bundles. In any given transverse section the longitudinal strands may be divided into three types of bundle according to size and structure: small, intermediate, large. Virtually all of the longitudinal strands intergrade structurally however, from one bundle type to another as they descend the leaf. For example, all of the strands having large-bundle anatomy appear distally as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. Only the large bundles and the intermediates that arise midway between them extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of longitudinal bundles at the base of the blade, both the total and mean cross-sectional areas of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.

Microautoradiographic studies of phloem loading and transport in the leaf of Zea mays L.

Microautoradiographs showed that [(14)C]sucrose taken up in the xylem of small and intermediate (longitudinal) vascular bundles of Zea mays leaf strips was quickly accumulated by vascular parenchyma cells abutting the vessels. The first sieve tubes to exhibit (14)C-labeling during the [(14)C]sucrose experiments were thick-walled sieve tubes contiguous to the more heavily labeled vascular parenchyma cells. (These two cell types typically have numerous plasmodesmatal connections.) With increasing [(14)C]sucrose feeding periods, greater proportions of thick- and thin-walled sieve tubes became labeled, but few of the labeled thin-walled sieve tubes were associated with labeled companion cells. (Only the thin-walled sieve tubes are associated with companion cells.) When portions of leaf strips were exposed to (14)CO2 for 5 min, the vascular parenchyma cells-regardless of their location in relation to the vessels or sieve tubes-were the most consistently labeled cells of small and intermediate bundles, and label ((14)C-photosynthate) appeared in a greater proportion of thin-walled sieve tubes than thick-walled sieve tubes. After a 5-min chase with (12)CO2, the thin-walled sieve tubes were more heavily labeled than any other cell type of the leaf. After a 10-min chase with (12)CO2, the thin-walled sieve tubes were even more heavily labeled. The companion cells generally were less heavily labeled than their associated thin-walled sieve tubes. Although all of the thick-walled sieve tubes were labeled in portions of leaf strips fed (14)CO2 for 5 min and given a 10-min (12)CO2 chase, only five of 72 vascular bundles below the (14)CO2-exposed portions contained labeled thick-walled sieve tubes. Moreover, the few labeled thick-walledsieve tubes of the "transport region" always abutted (14)C-labeled vascular parenchyma cells. The results of this study indicate that (1) the vascular parenchyma cells are able to retrieve at least sucrose from the vessels and transfer it to the thick-walled sieve tubes, (2) the thick-walled sieve tubes are not involved in long-distance transport, and (3) the thin-walled sieve tubes are capable themselves of accumulating sucrose and photosynthates from the apoplast, without the companion cells serving as intermediary cells.

Influence of Nitrate and Ammonia on Photosynthetic Characteristics and Leaf Anatomy of Moricandia arvensis.

The leaf anatomy and certain photosynthetic properties of nitrate- and ammonia-grown plants of Moricandia arvensis (L.) DC., a species previously reported to be a C(3)-C(4) intermediate, were investigated. Nitrate-grown plants had a high level of malate in the leaves while ammonia-grown plants had low levels of malate. In young leaves of nitrate-grown plants, there was a diurnal fluctuation of malate content, increasing during the day and decreasing during the night. Titratable acidity remained low in leaves of both nitrate- and ammonia-grown plants.In nitrate-grown plants, the activity of phosphoenolpyruvate (PEP) carboxylase was about 2-fold higher than in ammonia-grown plants, the latter having activity typical of C(3) species. Also, in nitrate-grown plants, the ratio of activities of ribulose 1,5-bisphosphate (RuBP) carboxylase/PEP carboxylase was lower than in ammonia-grown plants. Nitrate reductase activities were higher in nitrate- than in ammonia-grown plants and the greatest activity was found in younger leaves.With nitrate-grown plants, during a pulse-chase experiment the label in malate, as a percentage of the total labeled products, increased from about 7% after a 10-second pulse with (14)CO(2) up to 17% during a 5-minute chase with (12)CO(2). The pattern of (14)C labeling in various metabolites suggests the primary carboxylation is through RuBP carboxylase with a secondary carboxylation through PEP carboxylase. In similar experiments, with ammonia-grown plants, the percentage label in malate was only 0% to 4% with no increase in malate labeling during the chase period. The CO(2) compensation point was lower in nitrate-grown than ammonia-grown plants.There was no evidence of Kranz-like anatomy in either the nitrate or ammonia-grown plants. Mitochondria of bundle-sheath cells were strikingly positioned along the inner tangential wall. This might allow the chloroplasts of these cells to fix the mitochondrial photorespired CO(2) more effectively and contribute to the low CO(2) compensation point in the species. Chloroplasts of bundle-sheath cells and contiguous mesophyll cells were similar in size and structure in plants grown on different media, although chloroplast thylakoids and stromata of the ammonia-grown plants stained more intensely than those of nitrate-grown plants. In addition, irregular clusters of phytoferritin particles occurred in the chloroplasts of the ammonia-grown plants.The results indicate that the substantial activity of PEP carboxylase, incorporation of CO(2) into malate, the high malate content, and in part the relatively low CO(2) compensation point in Moricandia arvensis may be accounted for by metabolism of nitrate rather than by a state of C(3)-C(4) intermediacy.

Studies on the leaf of Amaranthus retroflexus (Amaranthaceae): ultrastructure, plasmodesmatal frequency, and solute concentration in relation to phloem loading.

Both the mesophyll and bundle-sheath cells associated with the minor veins in the leaf of Amaranthus retroflexus L. contain abundant tubular endoplasmic reticulum, which is continuous between the two cell types via numerous plasmodesmata in their common walls. In bundle-sheath cells, the tubular endoplasmic reticulum forms an extensive network that permeates the cytoplasm, and is closely associated, if not continuous, with the delimiting membranes of the chloroplasts, mitochondria, and microbodies. Both the number and frequency of plasmodesmata between various cell types decrease markedly from the bundle-sheath - vascular-parenchyma cell interface to the sicve-tube member - companion-cell interface. For plants taken directly from lighted growth chambers, a stronger mannitol solution (1.4 M) was required to plasmolyze the companion cells and sieve-tube members than that (0.6 M) necessary to plasmolyze the mesophyll, bundle-sheath, and vascular-parenchyma cells. Placing plants in the dark for 48 h reduced the solute concentration in all cell types. Judging from the frequency of plasmodesmata between the various cell types of the vascular bundles, and from the solute concentrations of the various cell types, it appears that assimilates are actively accumulated by the sieve-tube - companion-cell complex from the apoplast.

Leaf vasculature in sugarcane (Saccharum officinarum L.).

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.

Sucrose in the free space of translocating maize leaf bundles.

Following exposure of portions of mature maize (Zea mays L.) leaf strips to (14)CO(2), xylem exudate from the leaf strips contained [(14)C]sucrose. Sucrose was the only sugar in the xylem exudate which was obtained from the cut surface of the leaf strips by reducing the external pressure. The sucrose found in the xylem exudate apparently was obtained from the free space of the vascular bundles, its concentration amounting up to 0.25%. When [(14)C]glucose or [(14)C]fructose was supplied in the dark to one end of a maize leaf strip, each was taken up by the xylem, and transported to the opposite end. Xylem exudate from such leaf strips contained (14)C-labeled sucrose in addition to the (14)C-labeled hexose. The results of this study support the view that sucrose is loaded into the companion cell-sieve tube complexes from the apoplast of the vascular bundles in the maize leaf.

Leaf structure in relation to solute transport and phloem loading in Zea mays L.

Small and intermediate (longitudinal) vascular bundles of the Zea mays leaf are surrounded by chlorenchymatous bundle sheaths and consist of one or two vessels, variable numbers of vascular parenchyma cells, and two or more sieve tubes some of which are associated with companion cells. Sieve tubes not associated with companion cells have relatively thick walls and commonly are in direct contact with the vessels. The thick-walled sieve tubes have abundant cytoplasmic connections with contiguous vascular parenchyma cells; in contrast, connections between vascular parenchyma cells and thin-walled sieve tubes are rare. Connections are abundant, however, between the thin-walled sieve tubes and their companion cells; the latter have few connections with the vascular parenchyma cells. Plasmolytic studies on leaves of plants taken directly from lighted growth chambers gave osmotic potential values of about-18 bars for the companion cells and thin-walled sieve tubes (the companion cell-sieve tube complexes) and about-11 bars for the vascular parenchyma cells. Judging from the distribution of connections between various cell types of the vascular bundles and from the osmotic potential values of those cell types, it appears that sugar is actively accumulated from the apoplast by the companion cell-sieve tube complex, probably across the plasmalemma of the companion cell. The thick-walled sieve tubes, with their close spatial association with the vessels and possession of plasmalemma tubules, may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream. The transverse veins have chlorenchymatous bundle sheaths and commonly contain a single vessel and sieve tube. Parenchymatic elements may or may not be present. Like the thick-walled sieve tubes of the longitudinal bundles, the sieve tubes of the transverse veins have plasmalemma tubules, indicating that they too may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream.

Tubular extensions of the plasmalemma in leaf cells of Zea mays L.

Leaf tissues of Zea mays were examined with a transmission electron microscope and a high-voltage electron microscope. Tubular extensions (invaginations) of the plasmalemma were found in vascular parenchyma cells and thick-walled, lateformed sieve elements of intermediate and small veins, and in epidermal, mesophyll, and sheath cells of all leaves examined. No continuity seems to exist between the tubules and other cellular membranes.

Distribution and structure of the plasmodesmata in mesophyll and bundle-sheath cells of Zea mays L.

In leaf blades of Zea mays L. plasmodesmata between mesophyll cells are aggregated in numerous thickened portions of the walls. The plasmodesmata are unbranched and all are characterized by the presence of electron-dense structures, called sphincters by us, near both ends of the plasmodesmatal canal. The sphincters surround the desmotubule and occlude the cytoplasmic annulus where they occur. Plasmodesmata between mesophyll and bundle-sheath cells are aggregated in primary pit-fields and are constricted by a wide suberin lamella on the sheath-cell side of the wall. Each plasmodesma contains a sphincter on the mesophyll-cell side of the wall. The outer tangential and radial walls of the sheath cells exhibit a continuous suberin lamella. However, on the inner tangential wall only the sites of plasmodesmatal aggregates are consistently suberized. Apparently the movement of photosynthetic intermediates between mesophyll and sheath cells is restricted largely or entirely to the plasmodesmata (symplastic pathway) and transpirational water movement to the cell walls (apoplastic pathway).

Effect of auxin-cytokinin interaction on organogenesis in haploid callus of Pelargonium hortorum.

Bud differentiation from haploid anther callus of geranium was achieved on Murashige and Skoog medium supplemented with 0.5 mg per 1 NAA and 2.5 mg per 1 kinetin. At concentrations higher than 5 mg per 1 kinetin, malformation and tissue senescence were evident.

Sieve-element ultrastructure in Platycerium bifurcatum and some other polypodiaceous ferns: The nucleus.

Sieve elements of various ages were examined in Platycerium bifurcatum (Cav.) C. Chr. and Phlebodium aureum (L.) J. Sm., only older ones in Polypodium schraderi Mett. and Microgramma lycopodioides (L.) Copel. Early in sieve-element differentiation small crystalloids arise in the matrix of the sieve-element nuclei in Platycerium. As differentiation continues, the crystalloids increase in size and eventually may occupy up to a third of the cross-sectional area of the nucleus and extend almost its entire length. At the time of nuclear degeneration the crystalloids are liberated into the cytoplasm. Nuclear degeneration during sieve-element development in Phlebodium is essentially similar to that in Platycerium, with the exception that no nuclear inclusions exist in the sieve-element nuclei in Phlebodium. Stacking of endoplasmic reticulum against the nuclear envelope occurs in both Platycerium and Phlebodium. In the final stages of degeneration, the nuclear envelope ruptures and the contents of the nucleus mix with the cytoplasm. At maturity the sieve elements of all four species are devoid of nuclei, although occasional remnants of chromatin persist along the walls of some mature cells.