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The extracellular matrix (ECM) of many charophytes, the assemblage of green algae that are the sister group to land plants, is complex, produced in large amounts, and has multiple essential functions. An extensive secretory apparatus and endomembrane system are presumably needed to synthesize and secrete the ECM, but structural details of such a system have not been fully characterized. Penium margaritaceum is a valuable unicellular model charophyte for studying secretion dynamics. We report that Penium has a highly organized endomembrane system, consisting of 150–200 non-mobile Golgi bodies that process and package ECM components into different sets of vesicles that traffic to the cortical cytoplasm, where they are transported around the cell by cytoplasmic streaming. At either fixed or transient areas, specific cytoplasmic vesicles fuse with the plasma membrane and secrete their constituents. Extracellular polysaccharide (EPS) production was observed to occur in one location of the Golgi body and sometimes in unique Golgi hybrids. Treatment of cells with brefeldin A caused disruption of the Golgi body, and inhibition of EPS secretion and cell wall expansion. The structure of the endomembrane system in Penium provides mechanistic insights into how extant charophytes generate large quantities of ECM, which in their ancestors facilitated the colonization of land., A layered endomembrane system consisting of linear rows of Golgi bodies and a peripheral vesicle network fuel the secretion of the complex extracellular matrix of the unicellular charophyte, Penium.

Upon plasmolysis, protoplast ER becomes more persistently cisternal without changing its actin association or internal flow, while wall-associated ER is tubular, associated with altered microtubules, and has reduced flow., Plasmolysis of hypocotyl cells of transgenic Arabidopsis thaliana and Nicotiana benthamiana diminishes the dynamics of the remodeling of the endoplasmic reticulum (ER) in the central protoplast, namely that withdrawn from the cell wall, and more persistent cisternae are formed, yet little change in the actin network in the protoplast occurs. Also, protein flow within the ER network in the protoplast, as detected with fluorescence recovery after photobleaching (FRAP), is not affected by plasmolysis. After plasmolysis, another network of strictly tubular ER remains attached to the plasma membrane-wall interface and is contained within the Hechtian strands and reticulum. FRAP studies indicate that protein flow within these ER tubules diminishes. Actin is largely absent from the Hechtian reticulum and the ER becomes primarily associated with altered, branched microtubules. The smaller volume of the central protoplast is accompanied by decreased movement rates of tubules, cisternae, and spheroid organelles, but this reduced movement is not readily reversed by the increase in volume that accompanies deplasmolysis.

This paper examines the significance of the recent demonstration of polar auxin transport (PAT) in the green macroalga Chara (Charophyceae: Charales) and, especially, options for explaining some features of PAT in the Charales. The occurrence of PAT in the Charales shows that PAT originated in the algal ancestors of the embryophytes (liverworts, mosses, hornworts, and vascular plants), although it is not yet known if PAT occurs elsewhere in the Charophyceae or in other algae. While in the embryophytes PAT occurs in parenchymatously constructed structures which commonly also have xylem and phloem (or their bryophyte analogues) as long-distance transport processes in parallel to PAT, in Chara corallina PAT shares the pathway for long-distance transport of nutrients though the parenchymatously constructed nodal complexes and the single giant cells of the internode. The speed of auxin movement of PAT is much more rapid than that attributable to diffusion and of the same order as the rate of cytoplasmic streaming in the giant internodal cells, yet complete inhibition of streaming by the inhibitor cytochalasin H does not slow down auxin transport. Explanations for this phenomenon are sought in the operation of other mechanochemical motors, dynein-tubulin and kinesin-tubulin, as alternatives to the myosin-actin system which powers cytoplasmic streaming. Experiments in which microtubules are disrupted, for example by colchicine, could show if one of the tubulin-based motors is involved. If these motors are involved, some mechanism is needed to amplify the speeds known for the motors to explain the order of magnitude higher speeds seen for auxin transport.

In higher plants, cell-to-cell polar auxin transport (PAT) of the phytohormone auxin, indole-3-acetic acid (IAA), generates maxima and minima that direct growth and development. Although IAA is present in all plant phyla, PAT has only been detected in land plants, the earliest being the Bryophytes. Charophyta, a group of freshwater green algae, are among the first multicellular algae with a land plant-like phenotype and are ancestors to land plants. IAA has been detected in members of Charophyta, but its developmental role and the occurrence of PAT are unknown. We show that naphthylphthalamic acid (NPA)-sensitive PAT occurs in internodal cells of Chara corallina. The relatively high velocity (at least 4-5 cm/h) of auxin transport through the giant (3-5 cm) Chara cells does not occur by simple diffusion and is not sensitive to a specific cytoplasmic streaming inhibitor. The results demonstrate that PAT evolved early in multicellular plant life. The giant Chara cells provide a unique new model system to study PAT, as Chara allows the combining of real-time measurements and mathematical modelling with molecular, developmental, cellular, and electrophysiological studies.

It has recently been found that among the 17 Arabidopsis myosins, six (XIC, XIE, XIK, XI-I, MYA1, and MYA2) have a major role in the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum. Here, the same dominant negative tail fragments were also found to arrest the movement of Gogi bodies when transiently expressed in Arabidopsis plants. However, when a Golgi marker was transiently expressed in plants knocked out in these myosins, its movement was dramatically inhibited only in the xik mutant. In addition, a tail fragment of myosin XIK could inhibit the movement of several post-Golgi organelles, such as the trans-Golgi network, pre-vacuolar compartment, and endosomes, as well as total cytoplasmic streaming, suggesting that myosin XIK is a major player in cytoplasm kinetics. However, no co-localization of myosin tails with the arrested organelles was observed. Several deletion truncations of the myosin XIK tail were generated to corroborate function with localization. All deletion mutants possessing an inhibitory effect on organelle movement exhibited a diffuse cytoplasmic distribution. Point mutations in the tail of myosin XIK revealed that Arg1368 and Arg1443 are essential for its activity. These residues correspond to Lys1706 and Lys1779 from mouse myosin Va, which mediate the inhibitory head–tail interaction in this myosin. Therefore, such an interaction might underlie the dominant negative effect of truncated plant myosin tails and explain the mislocalization with target organelles.

The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.

Tracheary element (TE) differentiation represents a unique system to study plant developmental programmed cell death (PCD). TE PCD occurs after deposition of the secondary cell walls when an unknown signal induces tonoplast rupture and the arrest of cytoplasmic streaming. TE PCD is tightly followed by autolysis of the protoplast and partial hydrolysis of the primary cell walls. This review integrates TE differentiation, programmed cell death (PCD), and autolysis in a biological and evolutionary context. The collective evidence from the evolutionary and molecular studies suggests that TE differentiation consists primarily of a programme for cell death and autolysis under the direct control of the transcriptional master switches VASCULAR NAC DOMAIN 6 (VND6) and VND7. In this scenario, secondary cell walls represent a later innovation to improve the water transport capacity of TEs which necessitates transcriptional regulators downstream of VND6 and VND7. One of the most fascinating features of TEs is that they need to prepare their own corpse removal by expression and accumulation of hydrolases that are released from the vacuole after TE cell death. Therefore, TE differentiation involves, in addition to PCD, a programmed autolysis which is initiated before cell death and executed post-mortem. It has recently become clear that TE PCD and autolysis are separate processes with separate molecular regulation. Therefore, the importance of distinguishing between the cell death programme per se and autolysis in all plant PCD research and of careful description of the morphological, biochemical, and molecular sequences in each of these processes, is advocated.

Root hairs are excellent cells for the study of the exocytotic process that leads to growth in higher plants, because the exocytotic event takes place locally and because the cells are directly accessible for signals, drugs, fixatives, microinjection, and microscopic observation. Well-characterized lipochitooligosaccharides, signal molecules excreted by Rhizobium bacteria, induce root hair growth which can be recorded microscopically in a root hair deformation assay developed for Vicia sativa L. Root hair deformation is a morphogenetic process involving swelling of the hair tip and subsequent new hair outgrowth from that swelling. This response to the signal occurs at a specific developmental stage, namely when hairs are terminating growth. Thus, since polar growth can be triggered intentionally, the system allows the study of growth phenomena in higher plants at the cellular level. Furthermore, important advances are being made with molecular genetics that will allow the unravelling of the signal transduction pathways in root hair morphogenesis leading to growth. This paper first discusses cytological phenomena involved in the process of polar growth, such as cytoplasmic polarity, cytoplasmic streaming and the organization of actin filaments, the location of a spectrin-like antigen, the distribution of intracellular calcium, cortical microtubules and cell wall texture, endocytosis by means of coated pits, and physical aspects of the incorporation of exocytotic vesicles into the plasma membrane. In the second part, changes are discussed that occur in some of these phenomena when growth is influenced by growth regulators and mutations.

Four different fura-2 compounds were tested for the application in Characean cells (fura-AM ; fura-C 18 ; fura-K 5 ; fura-dextran ; MW = 10 kDa). It is demonstrated that Characean cells impose special problems when cytosolic pCa has to be measured with fluorescent ratio dyes. Fluorescence (λ ex =340 nm) from the dye which had diffused from the cytosol to the huge central vacuole with milimolar Ca 2+ concentrations overrides the signal from the cytosol and makes Ca 2+ -quantification difficult. This can be avoided by pressure injection of fura-dextran. Because of inhomogeneities in dye concentration or in thickness of the cytoplasmic layer, cytoplasmic streaming causes high noise or pretend oscillations in pCa if data are obtained by subsequent image grabbing. In addition, vesicles filled with high concentrations of dye may sometimes be expelled into the vacuole during the loading procedure enhance this effect. These sources of inhomogeneities can be minimized by loading fura-dextran via the neighbouring cell. The slow loading procedure through the plasmodesmata takes 1-10 h. It results in a more homogeneous distribution of the dye. The operation of the new method is illustrated by the measurement of Ca 2+ -transients during action potentials, the temperature dependence of the fluorescence signal in vivo and in vitro and the butyrate-induced elevation of [Ca 2+ ] c . Fura-AM was found not to be well suited for use in algal cells. Fura-C 18 has toxic effects and induces clotting of the cytoplasm. In addition, some aspects of the properties of dextran-derivates are discussed.

Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

Although organelle movement in higher plants is predominantly actin-based, potential roles for the 17 predicted Arabidopsis myosins in motility are only just emerging. It is shown here that two Arabidopsis myosins from class XI, XIE, and XIK, are involved in Golgi, peroxisome, and mitochondrial movement. Expression of dominant negative forms of the myosin lacking the actin binding domain at the amino terminus perturb organelle motility, but do not completely inhibit movement. Latrunculin B, an actin destabilizing drug, inhibits organelle movement to a greater extent compared to the effects of AtXIE-T/XIK-T expression. Amino terminal YFP fusions to XIE-T and XIK-T are dispersed throughout the cytosol and do not completely decorate the organelles whose motility they affect. XIE-T and XIK-T do not affect the global actin architecture, but their movement and location is actin-dependent. The potential role of these truncated myosins as genetically encoded inhibitors of organelle movement is discussed.

The morphology and the movement of organelles, in particular of the endoplasmic reticulum (ER), have been studied using video-enhanced microscopy in living pollen tubes of Lilium longiflorum Thunb. The patterns of the cytoplasmic streaming in the pollen grains and tubes generally correspond with the descriptions by Iwanami (1956). We found, however, that the organelles move as single and independent elements reflecting highly individual characteristics. The apical zone of the pollen tube, the so-called 'clear' zone, is populated by numerous intensively vibrating organelles of small size, most probably Golgi vesicles. The migration of vesicles to the apical plasma membrane of the growing pollen tube is not a smooth flow of particles, but a turbulent process. Spherical bodies with a high contrast, probably polysaccharide (p-) particles and/or lipid drops, and some mitochondria are also visible in the extreme tip. Their penetration into the apex seems to be inhibited by the dense mass of agitated vesicles. The small vacuoles located in the central zone are very motile; they change their shape continuously. The tonoplast, therefore, must be very plastic and, to a certain extent, mechanically resistant. The vegetative nucleus and the generative cell maintained a stable shape despite constant collisions with the organelles of the vigorously streaming cytoplasm. The thin cytoplasmic layer in the vacuolate zone contains tubular ER (diameter about 0-3 /urn) arranged in complex closed networks. The tubules are permanently moving. Gentle undulations alternated with sudden dramatic distortions. Rapid fusions or divisions of tubules are common. This dynamic behaviour is taken as proof of the plasticity of the ER tubules and the high fluidity of the membranes of the ER. Organelles preferentially move along the ER tubules in a saltatory manner. It is assumed that the ER tubules are associated with cytoskeletal elements.

The correlation between the velocities of cytoplasmic streaming and of translocation of 14C photosynthate and 32P-phosphate associated radioactivity has been investigated in whole plants of the green freshwater alga Chara hispida L. Tracer was supplied to the plant's rhizoid system in a split-chamber. The velocity of cytoplasmic streaming of 52 ± 3-3 pm s""1 compares with 57 ± 10 pm s~' found for 14C-transport and 32 + 20 pm s_1 found for 32P-transport. There was no indication of intercellular translocation at a velocity faster than visible streaming. Cytochalasin B inhibited the translocation of 32P and cytoplasmic streaming. Cytochalasin B becomes fully effective in inhibiting streaming and transport after an incubation time of at least 5 h.

The effect of streaming speed on intercellular transport of chloride has been studied using pairs of internodal cells of Chara. The rate of transport was measured by that fraction of the chloride that entered one internode which was transported out of it into the cells of the node and the next internode. The speed of cytoplasmic streaming was altered by treating the first cell with cytochalasin B. The relative rate of intercellular transport depended markedly on the streaming speed at all speeds up to those found in untreated cells. The chloride influx into the treated cell did not depend on the streaming speed. It is concluded that the rate of intercellular transport of low molecular weight solutes in Chara will be normally limited by the rate at which cytoplasmic streaming brings solute to the plasmodesmata, rather than by the diffusion permeability of the plasmodesmata. This conclusion may well apply to other charophyte plants, and could in principle apply to higher plants.

The ionic currents across the plasmalemma of Nitellopsis obtusa were measured in voltage clamp experiments. Depolarization of the cell by 30-100 mV from the level of the resting potential resulted in (1) a rapid inward current, (2) a subsequent slow inward current, and (3) a stationary outward current. The first current component changed sign at —20 to —30 mV. The second component decreased to a minimum at this clamped level. With increasing depolarizing steps some slow transient current component reappeared without changing sign. This transient inward current occurred also when the potential was clamped either at large depolarizing (+80 mV) or at large hyperpolarizing (—300 mV) potentials. In cases when the slow inward current component was evident cessation of protoplasmic streaming was observed. The ATPase inhibitor dicyclo hexylcarbodiimide (DCCD) at a concentration of 2 x 10~5 M in the external medium inhibited the slow transient inward current without affecting the first rapid current component. It is suggested that the irreversible slow transient current component reflects the onset of some active ion-transport system in the plasmalemma during cell excitation.

At slow stepwise insertion of the glass micro-electrode into the cell of Nitellopsis obtusa and Nitella translucens four potential jumps were recorded which corresponded to the penetration of the micro-electrode across the cell wall, the plasmalemma, the supposed chlorolemma and the tonoplast, respectively. The photo-electric depolarization appeared after the third jump only, which was supposed to be the penetration of a membrane layer between the plasmalemma and the streaming part of the cytoplasm. The vibration of this chlorolemma, caused probably by the protoplasmic streaming, resulted in large (up to 100 mV) hyperpolarizing and depolarizing potential fluctuations when the micro-electrode tip was near to this membrane. During cell excitation the potential difference (p.d.) had changed also across the chlorolemma. It was shown that the potential changes across the chlorolemma, caused by local illumination on the isolated part of the cell, were measurable also at a considerable distance (more than 1-4 cm) from the illuminated and isolated sequence. The lightand electron microphotographs obtained showed that the chloroplasts might really form a very compact layer with tight connections between the neighbouring chloroplasts and that there existed at least one continuous membrane layer which separated the immobile chloroplasts from the streaming cytoplasm. It was concluded that the p.d. measured between the streaming cytoplasm and the external medium was the sum of the p.d.s across the cell wall, the plasmalemma and the chlorolemma, respectively.

At high external pH, the Chara membrane is known to switch to a new state in which the membrane potential is highly negative; it has been characterized as a passive diffusion potential for H+ (or OH"). DCCD is shown to inhibit the increased conductance and the highly negative membrane potential associated with this state. DCCD also inhibits the placmalemma H+-ATPase, as well as cytoplasmic streaming. The alkaline state of the membrane is shown to involve a decreased permeability to K+; this enhances the selectivity for H+ (or OH") which results from the increased permeability to H+ (or OH"). Altering the cytoplasmic pH affects the membrane potential at both neutral and alkaline pH. It can also affect the ability of the cell to make the transition to the alkaline state.

Movements of nC and 14C in N. translucens occur at rates two to five times greater than those of visually observed streaming. Like cytoplasmic streaming, longitudinal tracer movement is stopped reversibly by action potentials, irreversibly by iV-ethylmaleimide (NEM) and is unaffected by colchicine (colch), suggesting that both processes share a common mechanism. Colchicine causes an increased loading delay time. In both N. translucens and C. corallina the activity of nC and 14C at the nodes shows a sharp discontinuity: it is low on the feed side of the node and high at the node itself. This suggests that transport across plasmodesmata may be active.

The role of calmodulin in Chara was investigated using the antipsychotic drug trifluoperazine (TFP), which is known to bind to the calmodulin/Ca++ complex preventing it from carrying out its normal functions. At low concentration (4-0 ,uM), TFP had profound and irreversible effects on the electrophysiology of Chara plasmalemma. The resting p.d. depolarized and the membrane conductance decreased suggesting an inhibition of the proton pump. After several hours of exposure the membrane became leaky, as cells deteriorated. The excitation was also affected by TFP. Spontaneous repetitive firing was observed. The excitation increased in duration and the action potential peak depolarized to ~ + 20 mV. The cytoplasmic streaming was unaffected by TFP; the streaming rate at the resting potential remained unchanged when TFP was added to the medium, stoppage occurred at the time of excitation and the streaming slowly resumed.

The time courses of axial movement in Nitella translucens of a number of 14C-labelled compounds and of 36C1 and 22Na have been determined together with simultaneous micro scopical observations of the rates of visible protoplasmic streaming. In general, the trans location profiles suggest the irreversible absorption of tracer though there are examples of reversible absorption. The coupling between visible streaming and the front of the axial transport is weak. Light, DCMU (dichlorophenyldimethylurea), and temperature affect the translocation but visible streaming is affected only by temperature and the passage of an action potential. There is evidence, from a number of compounds, of a faster-than-streaming translocation of small amounts of tracer in the form of 'pulses' which are completely detached from the main body of tracer. Either vesicles or microperistaltic packets are thought to account for this rapid moiety. Observations of transnodal transfer suggest that an active mechanism is involved in the transference of carbonate, urea, acetate, and chloride. The influxes of the various compounds have been calculated and shown to be in good agreement with existing published values. Action potentials reduce the influx of carbonate, chloride, and possibly acetate, as do lack of light and DCMU. It is suggested that the influx of these anions is coupled with the active anion pump and with loading sites on the plasma membrane which become inoperative during and immediately following an action potential.

Measurements of intra- and extracellular pH during the excitation of Nitellopsis obtusa were carried out with antimony microelectrodes at conditions of dark adaptation and of continuous illumination. The pH of the vacuolar sap of both dark-adapted and illuminated cells increased during cell excitation by 0-1-0-15 units. H+ ions which had entered the cytoplasm during excitation in dark-adapted cells were extruded back into the vacuole across the tonoplast. After cell excitation in the light H+ ions were extruded from the cytoplasm also into the external medium probably across the light-stimulated active H+-channels. Protoplasmic streaming ceased during excitation in the dark for 1-3 min, and during excitation in the light—for 5-20 s.

A microscope mount was designed so that specimen temperatures could be monitored and con trolled without impairing phase contrast optics and used to measure rates of protoplasmic streaming between 0 and 25 °C in trichome cells of Lycopersicon esculentum, Lycopersicon hirsutum, Citrullus vulgaris, Tradescantia albiflora, Digitalis purpurea, and Veronica persica. Between 10 and 20 °C the rates of streaming varied from 2-6 pm s-1 depending on the temperature, and differences between the species were small. The temperature coefficient of streaming rates was found to increase as the temperature was lowered so that the plot of log rate against temperature had a steeper slope at the lower temperatures. The largest temperature cofficients were for the warmth-requiring L. esculentum (tomato) and C. vulgaris (water melon), and the smallest for the temperate-zone plants V. persica (speedwell) and D. purpurea (foxglove). The changes in rate always occurred over a range of temperature; no 'critical temperature' was observed below which streaming abruptly stopped and above which it was active, although the amount of streaming as well as the rate decreased as the lowest temperatures were approached. The temperatures experienced by the specimens during the experiment did not affect the recovery of normal streaming rates between about 10 and 20 °C. In a population of a wild tomato, Lycopersicon hirsutum Humb. and Bonpl., collected from different altitudes in Peru and Ecuador, i.e. from locations of different environmental temper ature, the rate of protoplasmic streaming at 5 °C was greatest in the varieties collected from the highest altitudes. The results suggest that streaming rates correlate with genetic adaptation to low temperature in the species examined.

A new cell-extraction medium was developed, under conditions such that physical injury was minimized, for the simultaneous isolation of plant nuclei, mitochondria, and chloroplasts in morphological forms closely resembling those in living cells. Comparisons were made of organelles isolated in buffered NaCl, sucrose, and specially developed formulations with the synthetic high-molecular weight polymers Ficoll (polymerized form of sucrose, MW 400,000) and Dextran 40 (MW 26,000). The finally developed medium contained : sucrose (osmoticum) ; Tris-acetate (buffer); Ficoll, Dextran 40, and bovine serum albumin (structure preservatives) ; glutathione, cysteine, ascorbic acid, or mercaptoethanol (reducing agents); and magnesium acetate and manganese acetate (activity preservatives). The properties of the SFDP medium are such that nuclei, chloroplasts, and mito chondria resemble their forms in living cells; mitochondria exhibit dynamic pleomorphic changes in form similar to those occurring in living cells and hair cells immersed in the medium can be cut open while cytoplasmic streaming con tinues during and after the incision.

The protoplasmic-streaming theory of phloem transport has been extended to include a transcellular streaming concept. Phloem exudate, concentration gradients, and the respiratory energy requirement are interpreted in terms of this concept. Physiological and cytological experiments are considered, and it appears that none of the unequivocal results contradict the proposed model. Reconciliation of apparently conflicting evidence is possible because transcellular streaming is primarily a fluid flow phenomenon and is directly dependent on respiration for its motive force energy. As in other theories, it is difficult to understand how the measured rates of mass transfer occur through the phloem. The present model may be able to meet quantitative demands because of increased efficiency due to the direct linear pathways provided by transcellular strands, and the increased cross-section of sieve-tube cytoplasm. More facts are required before the mass transfer problem can be resolved.