Sucrose carrier RcSCR1 is involved in sucrose retrieval, but not in sucrose unloading in growing hypocotyls of Ricinus communis L

The transport of assimilates from source to sink tissues is mediated by the phloem. Along the vascular system the phloem changes its physiological function from loading phloem to transport and unloading phloem. Sucrose carrier proteins have been identified in the transport phloem, but it is unclear whether the physiological role of these transporters is phloem unloading of sucrose or retrieval of apoplasmic sucrose back into the sieve element/companion cell complex. Here, we describe the dynamic expression of the Ricinus communis sucrose carrier RcSCR1 in the hypocotyl at different sink strengths. Our results indicate that phloem unloading in castor bean is not catalysed by the phloem loader RcSCR1. However, this sucrose carrier represents the molecular basis of the sucrose retrieval mechanism along the transport phloem, which is dynamically adjusted to the sink strength. As a consequence, we assume that other release carrier(s) exist in sink tissues, such as the hypocotyl, in R. communis.     

FACTS AND MECHANISMS: A COMPARATIVE SURVEY

1. This review aims to survey the process of translocation of solutes in the phloem, including the experimental observations of the process, hypothetical mechanisms with their consequences, and the compatibility of these mechanisms with the experimental information. 2. Some properties of the sieve elements are summarized. The characteristic constituent of the sieve elements is a fibrillar protein, P-protein, of 60–120 A. filaments, whose function and distribution in intact sieve elements are still the subject of debate. 3. Apart from the very high levels of sucrose (0.3–0.9 m) and of specific amino acids and amides (10–100 mm), the contents of the sieve elements are characterized by close regulation of the ionic content; thus K (20–85 ^mM) and Mg (2.3–23 mM) are very high relative to Na (0.06–0.3 mM) and Ca (0.25–0.5 mM) respectively; the pH is also very high. 4. Convective movement (mass flow) is demanded by the very high rates of mass transfer. The longitudinal sucrose flux is about 2.5 times 10⁶ pmoles cm.^-2 sec.^-1 in petioles, and several times higher in fruits or trees; this is about 10⁵ times any reasonable transmembrane flux, and demands very large loading areas for each file of sieve elements. It also renders unlikely any mechanism demanding an associated trans-membrane flux of any solute which approaches within several orders of magnitude of the sucrose flow. 5. The evidence from tracer measurements (of ¹⁴C or of heat) favour a mass flow of some kind in the sieve tube, with only restricted exchange between the flowing stream and other sucrose pools in the phloem (or out of it). It is not consistent with ready equilibration with a large stationary reservoir of sucrose, or with reverse flows. There is close correspondence between the input and output kinetics of a length of the trans-location path, or of build-up curves at different distances; hence lateral exchange from the moving stream is relatively minor. 6. Tracer measurements show that loading into the translocation stream is relatively slow, and is the main determining factor in the time course of appearance of tracer down the stem, or in the profile of radioactivity against distance in the stem. This applies not only to the initial steep front of radioactivity in the stem, but also to the error function profiles found at longer times in some plants; those do not arise as has been suggested, by exchange in a two-way system of transcellular strands, but are a reflexion of the loading kinetics. 7. The evidence for or against bidirectional movement is equivocal. In conditions in which there is a strong source/sink gradient imposed, the movement of both labelled carbon and heat is consistent with a one-way system, and is difficult to reconcile with two-way movement. However, in the absence of any strong gradient there is evidence for bidirectional movement. It is suggested that the pattern of flow, as well as the direction and rate of flow, may be controlled by the source/sink relations along the path. 8. Electro-osmosis as a mechanism for translocation seems to be ruled out by a number of theoretical difficulties. The most basic of these is the fact that an electro-osmotic mechanism is inherently incapable of the transport of both anions and cations, whereas the phloem can do both. There are further quantitative difficulties. The ratio of sucrose to potassium in the sieve elements is about 10, and if potassium provides the current a longitudinal potassium flux of about 2.5 times 10⁶ pmoles cm.^-2 sec.^-l would therefore be required in petioles, and considerably more in fruits or trees. This raises very great difficulties of potassium circulation to provide a complete current loop, in the path of recirculation, the size of the transmembrane fluxes required, and the energetics of pumping enough potassium to maintain the driving force for electro-osmosis. 9. Possibilities of activated mass flow, by a mechanism similar to that involved in protoplasmic streaming are discussed. Experimental work on streaming in Nitella and in the slime mould Physarum is reviewed, including the evidence that in both these systems, fibrils, made up of 50–70 Å. filaments, are responsible for the production of the motive force, and that these fibrils are akin to actomyosin. 10. Possible ways in which fibrillar P-protein might be organized in the sieve elements to produce translocation are discussed. The force generated by Nitella-type filaments at the density of P-protein in phloem exudate would be more than adequate for the observed rates of flow. Alternatively the fibrillar arrangement in the slime mould is capable of producing volume flows as large as those in phloem. This hypothesis provides a function for P-protein, and is also consistent with the curious ionic concentrations characteristic of sieve elements. 11. It is suggested that the control by the source/sink relations of the pattern, rate and direction of flow in the phloem might be achieved by the orientation of force-generating microfilaments by a Münch-type flow. Such a flow is inevitable if sucrose is pumped in at one end of the path and removed at the other; it seems to be inadequate to explain the rates of mass transfer, but it might be responsible for inducing the correct orientation and polarity in the motive force.     

Analysis of the driving forces of phloem transport in Ricinus seedlings: sucrose export and volume flow are determined by the source

The aim of this study was to reveal the factors determining sucrose export and volume flow through the sieve tubes in Ricinus communis L. seedlings. The cotyledons take up sucrose from the apoplasm in vivo, and export most of it to the growing sinks, hypocotyl and root. This simple source-sink system allowed sucrose uptake and export to be studied under controlled conditions with respect to apoplasmic sucrose concentrations. From the additional knowledge of the sucrose concentrations in the mesophyll and the sieve tubes, transmembrane concentration differences were calculated. The volume flow rate along the sieve tubes could be calculated from the export rate and the sucrose concentration in the sieve tubes. While the export rate exhibited saturation kinetics, the volume flow rate decreased at high external sucrose concentrations. The export rate correlated with the sucrose uptake rate, the volume flow rate correlated with the sucrose concentration (osmotic pressure) difference across the sieve-tube plasma membrane, the driving force for transmembrane water flux. From these data it can be concluded that sucrose export and the volume flow through the sieve tubes are determined by activities of the source. Export out of Ricinus cotyledons was considerably higher than export out of green source leaves of different species. The concomitant comparatively low sucrose concentration in the sieve-tube sap of the seedlings can thus be attributed to a very high water flux into and along the sieve tubes associated with the high sucrose flux. Received: 28 November 1997 / Accepted: 4 April 1998     

The Movement of Sugars into the Sieve Elements of Bark Strips of Willow: II. EVIDENCE FOR TWO PATHWAYS FROM THE BATHING SOLUTION

It is suggested that when ¹⁴C-labelled sucrose, or its constituenthexoses, are applied to the cambial surface of a bark strip,the sugars can move into the sieve elements by two pathways.The first is a direct one which probably involves the companioncells; in it the labelled sugars do not mix with pools of unlabelledsugars before entry into the sieve elements. Entry by this pathwayleads to activity in a wide range of compounds in the sieve-tubeexudate. The second pathway is an indirect one, involving thestorage parenchyma of the phloem, where considerable metabolicchanges take place involving the sugar applied to the strip,but the products of these changes, apart from sucrose, are unableto move into the sieve elements. These results are discussed in relation to published work onthe cytology of angiosperm phloem, and the results of otherinvestigators on the movement of sugars into plant cells.     

Application of a single-solute non-steady-state phloem model to the study of long-distance assimilate transport

A mass-balanced, finite-difference solution to Münch's osmotically generated pressure-flow hypothesis is developed for the study of non-steady-state sucrose transport in the phloem tissue of plants. Major improvements over previous modeling efforts are the inclusion of wall elasticity, nonlinear functions of viscosity and solute potential, an enhanced calculation of sieve pore resistance, and the introduction of a slope-limiting total variation diminishing method for determining the concentration of sucrose at node boundaries. The numerical properties of the model are discussed, as is the history of the modeling of pressure-driven phloem transport. Idealized results are presented for a sharp, fast-moving concentration front, and the effect of changing sieve tube length on the transport of sucrose in both the steady-state and non-steady-state cases is examined. Most of the resistance to transport is found to be axial, rather than radial (via membrane transport), and most of the axial resistance is due to the sieve plates. Because of the sieve plates, sieve tube elasticity does not provide a significant enhancement to conductivity at high pressure, as previously suspected. The transit time of sucrose through a sieve tube is found to be inversely proportional to the square of the sieve tube's length; following that observation, it is suggested that 20 1-m-long sieve tubes could transport sucrose 20 times faster than a single 20 m sieve tube. Short sieve tubes would be highly sensitive to differentials between loading and unloading rate, and would require close cooperation with adjacent companion cells for proper function.     

A symplasmic flow of sucrose contributes to phloem loading in Ricinus cotyledons

External sucrose, supplied by the endosperm in vivo, is the physiological source of sucrose for Ricinus communis L. seedlings. It is taken up by the cotyledons and exported via the sieve tubes to the growing hypocotyl and root. Two parallel pathways of external sucrose to the sieve tubes, directly via the apoplasm and indirectly after transit through the mesophyll, have already been established (G. Orlich and E. Komor, 1992). In this study, we analysed whether a symplasmic flow of sucrose contributes to phloem loading. Uptake of external sucrose into the mesophyll and into the sieve tubes, and export of total sucrose were measured with intact and exuding seedlings in the presence of p-chloromercuribenzenesulfonic acid (PCMBS). Sucrose uptake into the mesophyll and into the sieve tubes was inhibited by 80–90%. Consequently, export of total sucrose slowed down. However, after the addition of PCMBS, sucrose was transiently exported in such a high amount that could not be accounted for by the residual uptake activity nor by the amount of sucrose confined to the sieve element-companion cell complex (seccc). From the results, we conclude that most of the sucrose exported transiently had moved to the sieve tubes from a symplasmic domain larger than the seccc, comprising at least all the cells of the bundle including the bundle sheath. We suggest that the symplasmic flow of sucrose observed is a mass flow driven by a turgor pressure. As a structural prerequisite for a symplasmic flow, plasmodesmata interconnect all the cells from the bundle sheath to the sieve tubes and also occur between the bundle sheath and the mesophyll. The phloem loading pathway of Ricinus cotyledons can thus be classified as a combination of three different routes. Received: 17 October 1997 / Accepted: 9 March 1998     

PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major

Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters. For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias. For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions. Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family. The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose. In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago. Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues. Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed.     

A new method for the concentration of micro protein samples

In many cases before a protein sample can be subjected to analysis by gel filtration, electrophoresis, or immunoelectrophoresis, concentration may be necessary. Some of the commonly used techniques for sample concentration are adsorption on columns, ultrafiltration through cellophane membranes, evaporation of water, absorption by hydrophilic gels, precipitation by salts or organic solvents, and rechromatography. Except for evaporation, all of the other procedures are practical only when the sample size exceeds a few milliliters. Although there is no lower limit on the sample size when evaporation is employed, the resulting increase in salt concentration of the final product is a problem.In this communication we describe a simple technique that is capable of concentrating, severalfold, samples of proteins 25–250 μl in volume. The technique is based on the same principle as ultrafiltration through cellophane membranes.     

A Reanalysis of Particle Motion in Sieve Tubes of Heracleum

From an analysis of the Brownian motion of particles in sieveelements of Heracleum mantegazzianum and Heracleum sphondylium,Barclay and Johnson have suggested that the in situ viscosityof sieve tube sap is four to six times higher than has previouslybeen assumed. In particular, they obtained a value for the sapviscosity of about 10^–2 Pa s, which compares with a valueof 2 x 10^–3 Pa s for a 20 per cent (w/v) sucrose solution.The present paper describes a reanalysis of their data. It isargued that Barclay & Johnson underestimated the Brownianmotion of sieve element particles and so overestimated the sapviscosity. An exact correction was not possible, but it is concludedthat the in situ viscosity of Heracleum sieve tube sap mustbe less than 3 x 10^–3 Pa s, which corresponds to a sucroseconcentration of less than 29 per cent. Hence it may not beunreasonable to suppose that the viscosity of sieve tube sapis determined primarily by the concentration of sucrose, ashas been assumed in theoretical analyses of the Munch hypothesis.It is also concluded that the sieve tubes studied by Barclayand Johnson were not functional, in the sense that they didnot exhibit an axial bulk flow of sap. Heracleum, sieve tubes, Brownian motion, viscosity, Munch hypothesis     

LIGHT MICROSCOPE INVESTIGATION OF SIEVE-ELEMENT ONTOGENY AND STRUCTURE IN ULMUS AMERICANA

At maturity the sieve elements of Ulmus americana L. contain a parietal network of very fine strands of slime which is continuous from one sieve element to the next through the sieve-plate pores. Upon injury this parietal network, which is derived from the slime bodies of immature sieve elements, sometimes becomes distorted into longitudinally oriented strands. Some of these strands frequently extend the length of the cells and often are continuous from one sieve element to the next through the sieve-plate pores. At times past such strands have erroneously been interpreted as normal constituents of the mature sieve-element protoplast. Many mature sieve elements of U. americana contain nuclei, which apparently persist for the life of the sieve elements. In addition, some evidence has been found in mature sieve elements for the presence of a membrane which delimits the parietal layer of cytoplasm, including its network of slime strands, from the vacuolar region of the cell.     

Studies on the Movement of Solutes between the Sieve Tubes and Surrounding Tissues3: I. INTERFERENCE BETWEEN SOLUTES AND RATE OF TRANSLOCATION MEASUREMENTS

Certain aspects of the secretion of solutes into, and removalfrom, the sieve tubes of isolated stem segments and rooted cuttingsof Salix viminalis have been studied. Sieve-tube sap was obtainedeither as honeydew from whole individuals or via the severedstylets of the aphid Tuberolachnus salignus (Gmelin). It was shown that interference occurred between the chemicallyunrelated solutes, sucrose and the cations potassium and rubidium.On raising the potassium concentration in the sieve-tube sapby passing a solution of this ion through the xylem, the sucroseconcentration declined. When the sucrose concentration fellover a period of days due to respiratory loss of carbohydratesfrom an isolated stem segment, a concomitant rise in eitherthe potassium or rubidium level in the sap occurred. When a solution of sodium was passed through the xylem, theconcentration of this ion in the sieve-tube sap rose, whilstthat of potassium fell at first, but later rose higher thanits initial value, indicating that both antagonism and synergycan occur between these ions. On introducing both these cationsinto the xylem simultaneously, more sodium than potassium wastaken up by the segment, though the increase in the sodium concentrationin the sieve-tube sap was less than that of the potassium. Perfusingthe xylem with a calcium solution had no effect upon the concentrationof potassium in the sieve tube. It has been shown that the rate of translocation of a solutealong the sieve tube, as measured by the two colony technique,depends upon the rate of removal of this solute from the sievetube. The amount of such lateral loss from the sieve tube isrelated to the potential gradient for a solute between the sievetube and surrounding cells.     

Sieve-plate pores,open or occluded? A critical review

Abstract It is widely believed that there is a growing body of evidence that sieve plate pores in functioning phloem are open and quite free from obstruction. The arguments used to establish this idea are critically examined one by one and the conclusion is reached that it has very little foundation in published work. On the contrary, the view that sieve plate pores are delicately-occluded with P-protein in the angiosperms has much more evidence to substantiate it. Since ‘open’ and ‘occluded’ are physiologically defined this means that the pressure-flow hypothesis must be regarded as seriously in doubt.     

蔗糖诱导拟南芥幼苗胚轴维管束细胞数量增多

为揭示蔗糖能否引起植物胚轴维管束细胞数量增多，将拟南芥播种于添加88mmol·L-1蔗糖和不添加糖的MS培养基上，对生长在不同培养基上的幼苗胚轴横切，显微镜下统计切片上维管束细胞数量。结果显示，与不加糖相比，加糖条件下萌发4d后幼苗维管束细胞总数增加约70％，维管薄壁细胞和导管分子都增加100％以上，筛管分子增加约90％，中柱鞘细胞数量不变。显然，蔗糖不仅使维管束薄壁细胞数量增多，也使筛管分子和导管分子数量增多。因此认为，添加蔗糖对拟南芥幼苗胚轴维管束具有双重效应，既引起维管薄壁细胞增殖，又促进维管薄壁细胞分化，从而使导管分子和筛管分子数量增多。     

Long-distance trafficking of macromolecules in the phloem

The fact that macromolecules such as proteins and mRNAs overcome the symplastic barriers between various tissue domains was first evidenced by the movement of plant viruses. We have recently demonstrated that viral infection disengages the symplastic restriction present between the sieve element-companion cell complex and neighboring cells in tobacco plants. As a result, green fluorescent protein, which was produced in mesophyll and bundle sheath cells, could traffic into the sieve tube and travel long distances within the vascular system. In this addendum we discuss the likely existence of a novel plant communication network in which macromolecules also act as long-distance trafficking signals. Plasmodesmata interconnecting sieve elements and companion cells as well as plasmodesmata connecting the sieve tube with neighboring cells may play a central role in establishing this communication network.Key words: companion cells, cucumber mosaic virus, Cucumis melo, plasmodesmata, movement protein, sieve-elementsTranslocation of photoassimilates from the source (site of synthesis) to various sink organs is governed, in part, by short-distance intercellular transfer of assimilates to the loading region of the phloem and long-distance transport within the plant vascular system. Sucrose, which is synthesized in the leaf mesophyll, moves cell-to-cell symplastically through plasmodesmata until it reaches the boundary of the sieve element (SE)-companion cell (CC) complex. In many plant species, the connection between phloem parenchyma (PP)/bundle sheath (BS) cells and CCs is characterized by a sparseness of plasmodesmata (e.g., Solanaceae), and sucrose is exported out of the cells to the apoplast. This type of plants (apoplastic loaders) uses sucrose proton symporters to load the sucrose into the vasculature.¹ Cucurbits are considered one of the model plants for symplastic phloem loading.² This type of plant is characterized by abundant plasmodesmata interconnecting the intermediary cells, which are specialized CCs, with the neighboring BS cells. It is generally accepted that in these plants, phloem loading includes intercellular movement of sucrose through the plasmodesmata, along the entire pathway from the mesophyll cell to the SE-CC complex.Interestingly, the existence of plasmodesmata interconnecting the SE-CC complex and neighboring cells is evident in all plant species that are characterized by an apoplastic phloem-loading mechanism. Moreover, microinjection experiments have indicated that plasmodesmata interconnecting the PP-CC are functional, in that they allow the exchange of small membrane-impermeable fluorescent probes.³ Virus movement through plasmodesmata from the mesophyll into the SEs further supports the notion that the symplastic communication between the CC-SE complex and the neighboring cells is functional.⁴One can assume that in apoplastic-loading plants, it would be an advantage to maintain the SE-CC complex as an isolated domain, with no functional plasmodesmata interconnecting it to the neighboring tissue. Symplastic continuity between the two domains could result in leakage of sucrose out of the vasculature and a significant reduction in the efficacy of sucrose loading. The fact that the two domains are interconnected suggests that any back-leakage of sucrose that might occur is insignificant relative to the likely efficacy of this communication route.What might the advantage be for symplastic communication between the SE-CC complex and the neighboring tissue? Accumulated evidence suggests that at the tissue/organ level, cell-to-cell trafficking of information molecules allows for noncell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway, delivering a wide range of macromolecules to enable the plant to function as a whole organism.^5–8 We advanced the hypothesis that plasmodesmata interconnecting the CCs and PP/BS cells play a pivotal role in controlling the long-distance trafficking of putative signaling molecules.     

Stimulation of sugar loading into sieve elements of willow by potassium and sodium salts

Potassium as the chloride, nitrate or sulphate or sodium as the chloride, were applied at a concentration of 50 mM either to the xylem of stem segments or to the cambial surface of bark strips of willow. Potassium chloride increased the concentration of sucrose in sieve tube exudate collected via severed aphid stylets, without significantly affecting the volume flow rate, or the concentration of potassium in the exudate. The increase in the sucrose level in the sieve tube sap was shown to be due to a stimulation of loading, rather than to an enhancement of longitudinal transport. Potassium nitrate and sulphate or sodium chloride, were not as effective as potassium chloride in stimulating the loading of sucrose. It is suggested that uptake of the cation into cells supplying sugars to the sieve tube is linked to the rate of release of sugars by the supplying cells.     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Diurnal changes in assimilate concentrations and fluxes in the phloem of castor bean (Ricinus communis L.) and tansy (Tanacetum vulgare L.)

Reports about diurnal changes of assimilates in phloem sap are controversial. We determined the diurnal changes of sucrose and amino acid concentrations and fluxes in exudates from cut aphid stylets on tansy leaves (Tanacetum vulgare), and sucrose, amino acid and K(+) concentrations and fluxes in bleeding sap of castor bean pedicel (Ricinus communis). Approximately half of the tansy sieve tubes exhibited a diurnal cycle of sucrose concentrations and fluxes in phloem sap. Data from many tansy plants indicated an increased sucrose flux in the phloem during daytime in case of low N-nutrition, not at high N-nutrition. The sucrose concentration in phloem sap of young Ricinus plants changed marginally between day and night, whereas the sucrose flux increased 1.5-fold during daytime (but not in old Ricinus plants). The amino acid concentrations and fluxes in tansy sieve tubes exhibited a similar diurnal cycle as the sucrose concentrations and fluxes, including their dependence on N-nutrition. The amino acid fluxes, but not the concentrations, in phloem sap of Ricinus were higher at daytime. The sucrose/amino acid ratio showed no diurnal cycle neither in tansy nor in Ricinus. The K(+)-concentrations in phloem sap of Ricinus, but not the K(+) fluxes, decreased slightly during daytime and the sucrose/K(+)-ratio increased. In conclusion, a diurnal cycle was observed in sucrose, amino acid and K(+) fluxes, but not necessarily in concentrations of these assimilates. Because of the large variations between different sieve tubes and different plants, the nutrient delivery to sink tissues is not homeostatic over time.     

Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases

Rat liver contains alpha-D-mannosidases in lysosomes, Golgi membranes, and cytosol. The lysosomal enzyme has now been purified approximately 30,000-fold over the crude extract and is free of at least 13 other lysosomal hydrolases. The enzyme has an apparent molecular weight of 335,000 by molecular sieve chromatography and 200,000 by sucrose density centrifugation. It is a glycoprotein, as evidenced by its binding to a concanavalin A affinity column and by a positive periodic acid-Schiff stain. The enzyme has a pH optimum near 4.6. Although it is generally insensitive to a large variety of inorganic salts, chelating agents, and sulfhydryl reagents, prolonged exposure to ethylenediaminetetraacetic acid caused loss of activity, which could be restored by the addition of ZnSO4. Substrate specificity studies were performed on the purified lysosomal alpha-D-mannosidase, as well as on the purified Golgi and cytosolic alpha-D-mannosidases. The three enzymes exhibited only very limited activity on native glycoproteins, but were found to be active on glycopeptides and oligosaccharides, hydrolyzing 1 yields 2 and 1 yields 3 linkages, except that the Golgi enzyme had negligible activity towards the latter linkage. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related. The alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolated from rat liver.     

Evolution of viral structure

Viruses vastly outnumber their host cells and must present a huge selective pressure. It is also becoming evident that only a small percent of the eukaryotic genome codes for molecules involved in cellular structures and functions, and that much of the remainder may have a viral origin. Viruses clearly play a central role in the biosphere, but how is this viral world organized? Classification was originally based on virus morphology and the particular host infected, but now there is an increasing trend to rely on sequence information. The type of genome (e.g., RNA or DNA, single- or double-stranded) provides fundamental classification criteria, while sequence comparisons can provide fine mapping for closely related viruses. However, it is currently very difficult to identify long-range evolutionary relationships. We present here a different approach, based on the idea that each virus has an innate "self." When the structures and functions characteristic of this "self" are identified, then they uncover relationships beyond those accessible from sequence information alone. The new approach is illustrated by sketching some possible viral lineages. We propose that urviruses were present before the division of cellular life into its current domains, and that the viral world has lineages that can be traced back to the root of the universal tree of life.     

Types and meaning of pollen carbohydrate reserves

During pollen development, soluble carbohydrates of sporophytic origin may be consumed immediately, polymerized to form starch reserves or intine, or transformed into other molecules. Disregarding intine, in mature pollen there are three different types of carbohydrates: (1) polysaccharides such as starch in amyloplasts or polysaccharides in cytoplasmic vesicles, (2) disaccharides such as sucrose and (3) monosaccharides such as glucose and fructose. At dispersal, pollen may be partly or slightly dehydrated, or not dehydrated at all. Partly dehydrated pollen has the capacity to lose or acquire water within limits without detriment to its viability. Slightly and non-dehydrated pollen is vulnerable to water loss and quickly becomes inviable. In partly dehydrated of pollen the carbohydrates consist of cytoplasmic polysacharides and sucrose; in slightly and non-dehydrated pollen these are absent or in low concentrations but there may be reserves of cytoplasmic callose. Starch, glucose and fructose are found in both types. It is postulated that cytoplasmic carbohydrates and sucrose are involved in protecting pollen viability during exposure and dispersal.