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.     

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.     

Raffinose oligosaccharide concentrations measured in individual cell and tissue types in Cucumis melo L. leaves: implications for phloem loading

Raffinose, stachyose, and galactinol are synthesized in intermediary cells (specialized companion cells) of the minor-vein phloem of cucurbits. To better understand the role of these carbohydrates and the regulation of their synthesis and transport, we measured the concentrations of each of the components of the raffinose oligosaccharide synthetic pathway in mesophyll and sieve element-intermediary cell complexes (SE-ICCs) in the leaves of melon (Cucumis melo L. cv. Hale's Best Jumbo). These concentrations are consistent with a polymer-trapping mechanism for phloem loading, with sucrose diffusing from mesophyll into intermediary cells and being made into raffinose and stachyose, which are too large to diffuse back to the mesophyll. To determine carbohydrate concentrations, we developed a method involving microdissected tissues. Blind endings of areoles, and mesophyll surrounding these veins, were separately removed from lyophilized leaf tissue. Carbohydrates were quantitated by high-performance liquid chromatography with pulsed amperometric detection. A small amount of mesophyll remained attached to the blind endings; the carbohydrate contribution of these cells to the vein sample was eliminated by subtraction, based on the amount of chlorophyll. Volumes of cells and subcellular compartments were calculated by morphometric analysis and were used to calculate carbohydrate concentrations. Assuming no subcellular compartmentation, the additive concentration of sugars in the SE-ICCs of minor veins is about 600 mM. Stachyose and raffinose concentrations are about 330 mM and 70 mM, respectively, in SE-ICCs; concentrations of these sugars are much lower in mesophyll (0.2 and 0.1 mM). This is consistent with the view that stachyose and raffinose are unable to pass through the plasmodesmata between intermediary cells and bundle-sheath cells. Sucrose levels appear to be higher in the SE-ICC (about 130mM) than in the mesophyll (about 10 mM), but if compartmentation is taken into account the gradient for sucrose is probably downhill from mesophyll to intermediary cells. Flux through plasmodesmata between the bundle sheath and intermediary cells was calculated and was found to be within the range of values of flux through plasmodesmata reported in the literature.Abbreviations BS-IC bundle sheath-intermediary cell - PC plasmodesmatal channel - SE-ICC sieve element-intermediary cell complex - SEL size exclusion limit We would like to thank Gayle Volk, Philip Laible, Canan Inan, Esther Gowan, Richard Medville, Nathan Wilson, Jessica Plant, and Steven Boese for their help, Thomas Owens, M.V. Parthasarathy, and Ian Merwin for use of equipment, and Nancy Haritatos for suggestions. This research was supported by U.S. Department of Agriculture Competitive Grant 94-37306-0351 (R.T.), the Swiss National Foundation (F.K.), and a NSF/DOE/USDA Cornell Plant Science Center fellowship (E.H.).     

LEAF OF SPINACIA OLERACEA (SPINACH): ULTRASTRUCTURE,AND PLASMODESMATAL DISTRIBUTION AND FREQUENCY,IN RELATION TO SIEVE-TUBE LOADING

Minor veins and contiguous tissues of the Spinacia oleracea leaf were analyzed by electron microscopy to determine the characteristics of the component cells and the structure, distribution, and frequency of plasmodesmata between the various cell types of the leaf. Mesophyll and bundle-sheath cells contain components typical of photosynthetic cells although the latter cell type contains smaller chloroplasts and fewer mitochondria and microbodies than the mesophyll cells. In addition, the mesophyll cells contain numerous invaginations of the plasmalemma bordering the chloroplasts and evaginations of the outer membrane of the opposing chloroplast envelope. In places, these membranes appear continuous with each other. The minor veins consist of tracheary elements, xylem parenchyma cells, sieve-tube members, companion and phloem parenchyma cells, and other cells simply designated vascular parenchyma cells. The companion and phloem parenchyma cells are typically larger than the sieve-tube members with the companion cells containing a much denser cytoplasm that the phloem parenchyma. Cytoplasmic connections occur along all possible routes from the mesophyll to the sieve-tube members and consist of either simple or branched plasmodesmata between parenchymatic elements or pore-plasmodesmata between the sieve-tube members and parenchyma cells. The highest frequency of plasmodesmata occurs between the sieve-tube members and companion cells, although the value is essentially the same as between the various parenchymatic elements of the phloem. Compared to several previously studied species, the frequency of plasmodesmata between cell types of the spinach leaf is low. These results are discussed in relation to apoplastic vs. symplastic solute transport and sieve-tube loading in this species.     

Ultrastructure,plasmodesmatal frequency,and solute concentration in green areas of variegated Coleus blumei Benth. leaves

The photosynthetic tissue of green portions of variegated Coleus blumei leaves consists primarily of palisade and spongy parenchyma cells as well as bundle-sheath cells. The moderate numbers of plasmodesmata connecting these cells may be sufficient to provide a symplastic pathway for assimilates moving toward the minor veins. The minor veins, however, are unusual in having two sets of phloem-loading cells which have little symplastic continuity with one another: one consisting of large, peripherally located intermediary cells, and a second set made up of smaller, usually more internal companion cells, both sets having their associated sieve-tube members. The intermediary cells are connected to vascular-parenchyma and bundle-sheath cells by unique branched plasmodesmata which are particularly abundant at the bundle-sheath interface. In addition, numerous plasmodesmata-pore connections occur between the intermediary cells and their associated sieve-tube members. Neither the intermediary cells nor their sieve-tube members plasmolyze when treated with 1.4 M mannitol, whereas mesophyll and vascular-parenchyma cells plasmolyze at 0.5 M and bundle-sheath cells at 0.6 M mannitol. By contrast, the companion cells and their associated sieve-tube members are symplastically isolated from the bundle-sheath cells and the sieve-tube-intermediary-cell complexes, and share few plasmodesmata with the vascular-parenchyma cells. Moreover, the companion cells plasmolyze at 1.1 M mannitol and their sieve tubes at 1.3 M. The intermediary-cell-sieve-tube complex thus appears to be structurally equipped to load assimilates entirely via the symplast, while the sieve-tube-companion-cell complex is probably loaded from the apoplast.Abbreviation ER endoplasmic reticulum     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): ULTRASTRUCTURE,PLASMODESMATAL FREQUENCY,AND SOLUTE CONCENTRATIONS

The minor veins and contiguous tissues of mature leaves of Populus deltoides Bartr. ex Marsh. were examined with the electron microscope to determine the ultrastructural characteristics of the component cells and to determine the structure, distribution, and frequency of plasmodesmata between the various cell types. In addition, plasmolytic studies were carried out to determine the solute concentrations of the various cell types of the minor veins and contiguous tissues. The cells comprising the mesophyll and bundle sheath contain all the components typical of photosynthetic cells. Paraveinal mesophyll cells and bundle-sheath cells have fewer microbodies and smaller chloroplasts than do palisade parenchyma cells. Vascular parenchyma and companion cells tend to intergrade with one another structurally but can be distinguished from one another by their characteristic plastids. The mature, enucleate sieve-tube member is lined by a parietal layer of cytoplasm consisting of plasmalemma, endoplasmic reticulum, mitochondria, plastids, and P-protein. Plasmodesmata occur along all possible routes from the palisade parenchyma cells to the sieve tubes of the minor veins, and their frequency increases with increasing proximity to the sieve-tube members. Plasmolytic studies revealed that the paraveinal mesophyll cells had a higher C₅₀ (estimated mannitol concentration plasmolyzing, on the average, 50% of a given cell type) than any other cell type of the leaf. Concentration gradients existed along the palisade cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route as well as along the paraveinal mesophyll cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route. Considering the frequency of plasmodesmata along these routes, it is conceivable that photosynthate diffuses from palisade cells to the companion cells along concentration gradients. Within the minor veins, the C₅₀ was higher for sieve-tube members than for either companion cells or vascular parenchyma cells, indicating that loading of the sieve tubes is an active, energy-dependent process.     

Closure of plasmodesmata in maize (Zea mays) at low temperature: a new mechanism for inhibition of photosynthesis

Background and Aims

Photosynthesis is one of the processes most susceptible to low-temperature inhibition in maize, a tropical C4 crop not yet fully adapted to a temperate climate. C4 photosynthesis relies on symplasmic exchange of large amounts of photosynthetic intermediates between Kranz mesophyll (KMS) and bundle sheath (BS) cells. The aim of this study was to test the hypothesis that the slowing of maize photosynthesis at low temperature is related to ultrastructural changes in the plasmodesmata between KM and BS as well as BS and vascular parenchyma (VP) cells.

Methods

Chilling-tolerant (CT) KW 1074 and chilling-sensitive (CS) CM 109 maize (Zea mays) lines were studied. The effect of moderate chilling (14 °C) on the rate of photosynthesis, photosynthate transport kinetics, and the ultrastructure of plasmodesmata linking the KMS, BS and VP cells were analysed. Additionally, the accumulation of callose and calreticulin was studied by the immunogold method.

Key Results

Chilling inhibited photosynthesis, photosynthate transfer to the phloem and photosynthate export from leaves in both lines. This inhibition was reversible upon cessation of chilling in the CT line but irreversible in the CS line. Simultaneously to physiological changes, chilling induced swelling of the sphincters of plasmodesmata linking KMS and BS cells and a decreased lumen of the cytoplasmic sleeve of plasmodesmata at the BS/VP interface in the CS line but not in the CT line. Accumulation of calreticulin, which occurred near the neck region of the closed plasmodesmata was observed after just 4 h of chilling and over-accumulation of callose at the KMS/BS and BS/VP interfaces occurred after 28 h of chilling.

Conclusions

Stronger chilling sensitivity of the CM 109 maize line compared with the KW 1074 line, shown by decreased photosynthesis and assimilate export from a leaf, is related to changes in the ultrastructure of leaf plasmodesmata at low temperature. The chain of reactions to chilling is likely to include calreticulin action resulting in rapid and efficient closure of the plasmodesmata at both KMS/BS and BS/VP interfaces. Callose deposition in a leaf was a secondary effect of chilling.     

A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.     

Ultrastructural effects in potato leaves due to antisense-inhibition of the sucrose transporter indicate an apoplasmic mode of phloem loading

To study the export of sugars from leaves and their long-distance transport, sucrose-proton/co-transporter activity of potato was inhibited by antisense repression of StSUT1 under control of either a ubiquitously active (CaMV 35S ) or a companion-cell-specific (rolC) promotor in transgenic plants. Transformants exhibiting reduced levels of the sucrose-transporter mRNA and showing a dramatic reduction in root and tuber growth, were chosen to investigate the ultrastructure of their source leaves. The transformants had a regular leaf anatomy with a single-layered palisade parenchyma, and bicollateral minor veins within the spongy parenchyma. Regardless of the promoter used, source leaves from transformants showed an altered leaf phenotype and a permanent accumulation of assimilates as indicated by the number and size of starch grains, and by the occurrence of lipid-storing oleosomes. Starch accumulated throughout the leaf: in epidermis, mesophyll and, to a smaller degree, in phloem parenchyma cells of minor veins. Oleosomes were observed equally in mesophyll and phloem parenchyma cells. Companion cells were not involved in lipid accmulation and their chloroplasts developed only small starch grains. The similarity of ultrastructural symptoms under both promotors corresponds to, rather than contradicts, the hypothesis that assimilates can move symplasmically from mesophyll, via the bundle sheath, up to the phloem. The microscopical symptoms of a constitutively high sugar level in the transformant leaves were compared with those in wild-type plants after cold-girdling of the petiole. Inhibition of sugar export, both by a reduction of sucrose carriers in the sieve element/companion cell complex (se/cc complex), or further downstream by cold-girdling, equally evokes the accumulation of assimilates in all leaf tissues up to the se/cc complex border. However, microscopy revealed that antisense inhibition of loading produces a persistently high sugar level throughout the leaf, while cold-girdling leads only to local patches containing high levels of sugar. Received: 4 March 1998 / Accepted: 7 April 1998     

Starch and apparent rate of photosynthesis of leaves of Lolium perenne grown at different temperatures

Summary Leaves from Lolium perenne plants grown at day/night temperatures of 15°/10° had slower rates of apparent photosynthesis at 15° than leaves from plants grown at 25°/20°. Electron microscopy showed a higher starch concentration in chloroplasts from low- compared with the higher temperature-grown plants. However, all differences in apparent photosynthesis were negatively associated with differences in size of mesophyll cells. It is suggested that the presence of starch per se had no effect on photosynthesis rate and that temperature during growth influences subsequent rates because leaves from the higher temperature have smaller cells and chloroplasts than those from the lower one.     

Ultrastructure of minor veins inCucurbita pepo leaves

Summary The minor veins ofCucurbita pepo leaves were examined as part of a continuing study of leaf development and phloem transport in this species. The minor veins are bicollateral along their entire length. Mature sieve elements are enucleate and lack ribosomes. There is no tonoplast. The sieve elements, which are joined to each other by sieve plates, contain mitochondria, plastids and endoplasmic reticulum as well as fibrillar and tubular (190–195 diameter) P-protein. Fibrillar P-protein is dispersed in mature abaxial sieve elements but remains aggregated as discrete bodies in mature adaxial sieve elements. In both abaxial and adaxial mature sieve elements tubular P-protein remains undispersed. Sieve pores in abaxial sieve elements are narrow, lined with callose and are filled with P-protein. In adaxial sieve elements they are wide, contain little callose and are unobstructed. The intermediary cells (companion cells) of the abaxial phloem are large and dwarf the diminutive sieve elements. Intermediary cells are densely filled with ribosomes and contain numerous small vacuoles and many mitochondria which lie close to the plasmalemma. An unusually large number of plasmodesmata traverse the common wall between intermediary cells and bundle sheath cells suggesting that the pathway for the transport of photosynthate from the mesophyll to the sieve elements is at least partially symplastic. Adaxial companion cells are of approximately the same diameter as the adaxial sieve elements. They are densely packed with ribosomes and have a large central vacuole. They are not conspicuously connected by plasmodesmata to the bundle sheath.     

宁夏枸杞果实韧皮部及其周围细胞超微结构研究

应用透射电镜技术研究了宁夏枸杞果实韧皮部细胞的超微结构变化。结果表明:(1)随着枸杞果实的发育成熟,果实维管组织中的韧皮部筛分子筛域逐渐变宽,筛孔大而多,通过筛孔的物质运输十分活跃;筛分子和伴胞间有胞间连丝联系,伴胞属传递细胞类型,与其相邻韧皮薄壁细胞和果肉薄壁细胞连接处的细胞界面发生质膜内突,整个筛分子/伴胞复合体与韧皮薄壁细胞之间形成共质体隔离,韧皮部糖分的卸载方式主要以质外体途径进行。(2)韧皮薄壁细胞间的胞间连丝较多,而韧皮薄壁细胞与果肉薄壁细胞的胞间连丝相对较少,但果肉薄壁细胞间几乎无胞间连丝;果肉薄壁细胞之间胞间隙较大,细胞壁和质膜内突间形成较大的质外体空间,为质外体的糖分运输创造了条件。(3)筛管、伴胞、韧皮薄壁细胞和果肉薄壁细胞中丰富的囊泡以及活跃的囊泡运输现象,暗示囊泡也参与了果实糖分的运输过程。研究推测,枸杞果实韧皮部同化物的卸载方式以及卸载后的同化物运输主要以质外体途径为主。     

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).Abbreviation ER endoplasmic reticulum     

The mechanism of phloem loading in rice (Oryza sativa)

Carbohydrates, mainly sucrose, that are synthesized in source organs are transported to sink organs to support growth and development. Phloem loading of sucrose is a crucial step that drives long-distance transport by elevating hydrostatic pressure in the phloem. Three phloem loading strategies have been identified, two active mechanisms, apoplastic loading via sucrose transporters and symplastic polymer trapping, and one passive mechanism. The first two active loading mechanisms require metabolic energy, carbohydrate is loaded into the phloem against a concentration gradient. The passive process, diffusion, involves equilibration of sucrose and other metabolites between cells through plasmodesmata. Many higher plant species including Arabidopsis utilize the active loading mechanisms to increase carbohydrate in the phloem to higher concentrations than that in mesophyll cells. In contrast, recent data revealed that a large number of plants, especially woody species, load sucrose passively by maintaining a high concentration in mesophyll cells. However, it still remains to be determined how the worldwide important cereal crop, rice, loads sucrose into the phloem in source organs. Based on the literature and our results, we propose a potential strategy of phloem loading in rice. Elucidation of the phloem loading mechanism should improve our understanding of rice development and facilitate its manipulation towards the increase of crop productivity.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Current perspectives on plasmodesmata: structure and function

Recent studies on plasmodesmata have shown that these important intercellular passages for communication and transport are much more sophisticated in both structure and regulatory abilities than previously imagined. A complex, but not well understood, substructure has been revealed by a variety of increasingly reliable ultrastructural techniques. Proteinaceous particles are seen within the cytoplasmic sleeve surrounding the desmotubule. Dye-coupling studies have provided experimental evidence for the physical pathway of solute movement, supporting conclusions about substructural dimensions within plasmodesmata drawn from the ultrastructural studies. Calcium has been identified as a major factor in the regulation of intercellular communication via plasmodesmata. Evidence from studies on virus movement through plasmodesmata suggests a direct interaction between virallycoded movement proteins and plasmodesmata in the systemic spread of many viruses. There is increasing evidence, albeit indirect, that in some plant species phloem loading may involve transport of photoassimilate entirely within the symplast from mesophyll cells to the sieve element-companion cell complexes of minor veins.     

Cell specialization within the parenchymatous bundle sheath of barley

Abstract. Structural and physiological aspects of the parenchymatous bundle sheath (PBS) were studied in cultivars of Hordeum distichum L. The PBS of intermediate, lateral and midrib veins consisted of a single layer of cells closely appressed to the mestome sheath. These cells were large, vacuolate and approximately cylindrical in shape, extending parallel to the vein. Mean PBS cell volume was 4 × 10⁻⁵mm³ compared to 1.23 × 10⁻⁵mm³ for mesophyll cells. Transverse sections revealed three cell types within the PBS, cells with small chloroplasts (S-type), cells with large chloroplasts (L-type) and structural cells. The majority of cells were S-type, containing chloroplasts of approximately a third of the volume of mesophyll chloroplasts; they were able to reduce tetranitro blue-tetrazolium and synthesize starch. Structural cells interrupted the phloem and xylem are of the sheath in lateral veins and the midrib, whilst between one and four PBS cells within the phloem are of each vein type contained chloroplasts similar in volume and starch content to those of the mesophyll. Only these L-type cells contained noticeable starch grains at the end of an 8-h dark period, a further 4 h darkness being required for complete mobilization of starch. Starch deposition within S-type and structural cells was detectable after 4 h illumination but was only appreciable in leaves excised from the plant and illuminated for 9–12 h. The role of S-type PBS cells in assimilate transport is discussed in relation to these findings.     

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     

Carbohydrate distribution and cellular injuries in acid rain and cold-treated spruce needles

Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation.