Supercooling of xylem ray parenchyma cells in tropical and subtropical hardwood species

 The freezing behavior of xylem ray parenchyma cells in several woody species, Ficus elastica, F. microcarpa, Mangifera indica, Hibiscus Rosa-sinensis, and Schefflera arboricola, that are native to non-frost tropical and subtropical zones, was investigated by differential thermal analysis (DTA), cryo-scanning electron microscopy (cryo-SEM) and freeze-replica electron microscopy. Although profiles after DTA did not exhibit clear evidence of supercooling in the xylem ray parenchyma cells, electron microscopy revealed that the majority of xylem ray parenchyma cells in all of the woody species examined were supercooled to around –10°C upon freezing temperatures and were not frozen extracellularly. It seems likely that DTA failed to reveal the low temperature exotherm (LTE), that is produced by breakdown of supercooling in the xylem ray parenchyma cells as a consequence of the overlap between the high temperature exotherm and the LTE in each case. The xylem ray parenchyma cells in these woody species were very sensitive to dehydration, and supercooling had, to some extent, a protective effect against freezing injury. It is suggested that the capacity for supercooling of xylem ray parenchyma cells of tropical and subtropical woody species might be the result of inherent structural characteristics, such as rigid cell walls and compact xylem tissues, rather than the result of positive adaptation to freezing temperatures. The present and previous results together indicate that the responses of xylem ray parenchyma cells in a wide variety of hardwood species to freezing temperatures can be explained as a continuum, the specifics of which depend upon the temperatures of the growing conditions. Received: 24 January 1997 / Accepted: 13 May 1997     

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.     

Seasonal ultrastructural alterations in the plasma membrane produced by slow freezing in cortical tissues of mulberry (Morus bombyciz Koidz. cv. Goroji)

Seasonal alterations in the ultrastructure of the plasma membrane produced by slow freezing were examined in cortical parenchyma cells of mulberry twigs (Morus bombyciz Koidz. cv. Goroji) grown in northern Japan. In freezing-sensitive summer, freezing produced distinct aparticulate domains with accompanying inverted hexagonal_II (H_II) phase transitions in the plasma membrane. In autumn and spring, during cold acclimation and deacclimation, freezing produced aparticulate domains in the plasma membrane without accompanying H_ii phase transitions. In winter, when the twigs were freezing-tolerant, freezing did not produce ultrastructural alterations in the plasma membrane. A significant relationship was recognized between the percentages of cells with aparticulate domains in the plasma membrane, regardless of the presence or absence of H_II phase transitions, and the occurrence of freezing injury throughout all seasons and at all freezing temperatures tested in each season. The aparticulate domains in the plasma membranes were shown to be produced by the close apposition of membranes due to freezing-induced dehydration and deformation of cells. Although the precise mechanisms that cause injury as a result of the formation of aparticulate domains in the plasma membrane remain unclear, our results indicate that the development of cold acclimation paralleled the process whereby cells developed the ability to reduce and finally to prevent the formation of aparticulate domains in the plasma membrane that would otherwise result from freezing-induced cellular dehydration and deformation that brings membranes into close proximity with one another.     

The positional distribution of cell death of ray parenchyma in a conifer, Abies sachalinensis

We monitored the distribution of death of secondary xylem cells in a conifer, Abies sachalinensis. The cell death of tracheids, which are tracheary elements, occurred successively and was related to the distance from cambium. Thus, it resembled programmed cell death. By contrast, the death of long-lived ray parenchyma cells had the following features: (1) ray parenchyma cells remained alive for several years or more; (2) in many cases, no successive cell death occurred even within a given radial cell line of a ray; and (3) the timing of cell death differed among upper and lower radial cell lines and other lines of cells within a ray. These results indicate that the death of long-lived ray parenchyma cells involves a different process from the death of tracheids. The initiation of secondary wall formation and the lignification of ray parenchyma cells in the current year's annual ring were delayed in the upper and lower radial cell lines of a ray. In addition, the density of distribution and orientation of cortical microtubules in such cells were different from those in cells in other radial lines. Ray parenchyma cells in the previous year's annual ring within the upper and lower radial cell lines of a ray contained many starch grains. Our results indicate that positional information is an important factor in the control of the pattern of differentiation and, thus, of the functions of ray parenchyma cells that are derived from the same cambial ray cells.     

Differences in patterns of cell death between ray parenchyma cells and ray tracheids in the conifers <Emphasis Type="Italic">Pinus densiflora</Emphasis> and <Emphasis Type="Italic">Pinus rigida</Emphasis>

Differences in patterns of cell death between ray parenchyma cells and ray tracheids in the conifers Pinus densiflora and Pinus rigida were clarified. Differentiation and cell death of ray tracheids occurred successively and both were related to the distance from the cambium. In this respect, they resembled those of longitudinal tracheids. Thus, the cell death of short-lived ray tracheids could be characterized as time-dependent programmed cell death. In contrast, ray parenchyma cells survived for several years or more, and no successive cell death occurred, even within a single radial line of cells in a ray. Thus, the features of death of the ray parenchyma cells were different from those of ray tracheids. Cell death occurred early in ray parenchyma cells that were in contact with ray tracheids. The initiation of secondary wall thickening occurred earlier in ray parenchyma cells that were in contact with ray tracheids in Pinus densiflora than in others. In addition, localized thickening of secondary walls occurred only in ray parenchyma cells that were in contact with ray tracheids in Pinus rigida. Moreover, no polyphenols were evident in such cells in either species. Therefore, ray parenchyma cells that were in contact with ray tracheids appeared not to play a role in the formation of heartwood extractives. Our observations indicate that short-lived ray tracheids might affect the pattern of differentiation and, thus, the functions of neighboring long-lived ray parenchyma cells in conifers.     

Seasonal changes in the freezing behavior of xylem ray parenchyma cells in four boreal hardwood species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. Copyright 1999 Academic Press.     

Diurnal differences in the supply of glucomannans and xylans to innermost surface of cell walls at various developmental stages from cambium to mature xylem in Cryptomeria japonica

Summary. We investigated the diurnal differences in the innermost surface of tracheid cell walls at various developmental stages from cambium to mature xylem. Cryptomeria japonica saplings were cultivated in a growth chamber with a light cycle set at 14 h of light and 10 h of darkness. Samples were collected from the saplings during both the light and dark periods. The innermost surface of cell walls was immunogold-labeled with anti-glucomannan or anti-xylan antiserum and was observed by field emission scanning electron microscopy. Diurnal differences in the aspect of the innermost surface of cell walls were seen only in S₂-layer-forming tracheids; cellulose microfibrils were clearly evident during the light period, and amorphous material containing glucomannans and xylans was prevalent during the dark period. Cellulose microfibrils were present at the primary-wall formation and S₁-layer-forming stages, and many warts were observed in the mature tracheids, regardless of the time of sampling. The densities of labeled glucomannans on the innermost surface of cell walls in S₁- and S₂-forming tracheids and of labeled xylans in S₂-forming tracheids during the dark period were significantly higher than those during the light period. These results suggest a diurnal periodicity in the supply of cell wall matrix containing hemicellulose to the innermost surface of developing secondary walls. Correspondence and reprints: Laboratory of Bio-material Physics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Present address: Chair of Climate Change Science for Forestry and Water Resources, Graduate School of Science and Technology, Niigata University, Niigata, Japan.     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997     

Histochemistry, ultrastructure and possible significance of dead parenchyma cells with specialized walls in the leaf and rhizome of Sansevieria

Abstract. The internal parenchyma of the leaf and rhizome in 36 species of Sansevieria is made of dead cells and living cells arranged in a regular pattern. Intercellular spaces are lacking. The walls of dead cells consist of an inner amorphous layer positive to the fluorescence test for callose, a middle suberin-like layer and an outer fibrillar layer. In about half of the species examined, the inner layer forms distinctive thickenings. Detached leaves of Sansevieria lose water very slowly, and are able to recover it quickly. The pattern of leaf dehydration appears to be related to leaf morphology, whereas no relation is evident between the pattern of leaf rehydration and leaf morphology. Neither leaf dehydration nor leaf rehydration pattern is affected by the presence of wall thickenings in the dead parenchyma cells. The fresh weight per unit volume of both turgid and droughted leaves is nearly 1, denoting that the dead cells are filled with water and do not undergo substantial cavitation during drought. The data indicate that the dead parenchyma cells of Sansevieria are a specialized water-storing system.     

Determination of the Role of Cold Acclimation-Induced Diverse Changes in Plant Cells from the Viewpoint of Avoidance of Freezing Injury

Received 4 January 1999/ Accepted in revised form 7 April 1999     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Effects of ethylene on the orientation of microtubules and cellulose microfibrils of pea epicotyl cells with polylamellate cell walls

Summary Following a 5 hours ethylene treatment, cortical cells of Pea (Pisum sativum L. var Alaska) epicotyl third internode showed a change in the orientation of both microtubules near the plasma membrane and recently deposited cellulose microfibrils. Control cortical cells had mostly transverse microtubules. The ratio of the average frequency of transverse to longitudinal microtubules was 6.0. After 5 hours of ethylene treatment, cortical cells had mostly longitudinal microtubules, with the ratio of transverse to longitudinal microtubules equal to 0.1. Epidermal cells were more variable than cortical cells with regard to the frequency of longitudinal and transverse microtubules. Observation of cortical cell walls in conventionally stained thin sections revealed that recent deposition of microfibrils had been primarily transverse in almost all of the control cortical cells sampled. In contrast, more than half of the ethylene-treated cortical cells had recent deposition oriented primarily longitudinally. This change in microtubule and microfibril orientation may be early enough to constitute the primary effect of ethylene leading to radial cell expansion.Research supported by NSF grant PCM 78-03244, A1, 2 to PBG and by a Research Corporation grant to WRE.     

Ultrastructure of the intestinal cells of Aspiculuris tetraptera after in vivo treatment of mice with mebendazole and thiabendazole

Ultrastructural changes in the intestinal cells of female Aspiculuris tetraptera following in vivo treatment with mebendazole or thiabendazole are described. Major changes included a marked reduction in the luminal microbial flora and the appearance of large numbers of autophagic vesicles in the cytoplasm of the intestinal cells. Some mebendazole-treated nematodes were more severely affected, with a complete loss of cellular integrity and a collapse of the intestinal lumen. The possible role of the intestinal cell microtubules in inducing these anthelmintic effects is discussed.     

Use of antisera to localize callose,xylan and arabinogalactan in the cell-plate,primary and secondary walls of plant cells

Antibodies to cellobiose, L-arabinopyranose, L-arabinofuranose, D-galactose, oligosaccharides containing 14 xylose, oligosaccharides containing 14 glucose, and oligosaccharides containing 13 glucose have been raised in rabbits. The antisera have been characterized to show the specificity of binding to particular polysaccharides. They have been used for immunocytology using the electron microscope to locate the polymers in dividing and differentiating cells of bean (Phaseolus vulgaris L.) root, bean callus tissue and cells of Zinnia elegans L. in vitro. Arabinogalactans have been shown to be present in the cell-plate and primary walls but not in secondary thickening. Xylan as distinct from xyloglucan was found in the primary walls but not in the cell-plate. It was present in large amounts in the secondary thickening. Callose was found in the cell plate and also in the young growing wall. In the wall it was specifically located at the plasmodesmata. The use of the antibody against L-arabinofuranose enabled a specific organelle to be detected which was membranous and which occurred within the cytoplasm and also within the vacuole of the cells. Membranes carrying polymers containing L-arabinofuranose were also found in layers just under the plasmamembrane.Abbreviations L-Araf L-arabinofuranose - L-Arap L-arabinopyranose - BSA bovine serum albumin - Gal galactose - D-Galp D-galactopyranose - Glc glucose - Xyl xylose     

Epidermal derivatives as xylem elements and transfer cells: a study of the host-parasite interface in two species of Triphysaria (Scrophulariaceae)

Summary Haustoria ofTriphysaria pusilla andT. versicolor subsp.faucibarbata from a natural habitat were analysed by light and electron microscopy. The keel-shaped edge of the secondary haustorium generally splits the epidermis and cortex of the host root parallel to the root axis, and penetrates to the host vascular tissue. Anticlinally elongated epidermal cells of the haustorium constitute most of the host/parasite interface. Some of these epidermal cells are divided by oblique cell walls. Some of their oblique daughter cells as well as some undivided epidermal cells differentiate into xylem elements. Single epidermal cells occasionally intrude into the vascular tissue of the host and individual host cells can be invaded. The surface area of the plasmalemma in parasitic parenchymatous interface cells is increased by the differentiation of wall labyrinths characteristic of transfer cells and by the development of membrane-lined cytoplasmic tubules or flattened sacs which become embedded in the partly lignified interface cell-wall. Mycorrhizal fungal hyphae enter the xylem bridge in some haustoria. Implications of these observations for the function of the haustorium are discussed.     

Regional differences in the distribution of golgi cells in the cerebellar cortex of man and some other mammals

Summary The number of Golgi cells per unit volume was determined in different regions of the cerebellar cortex of man and of ten other mammals. Despite the general belief in the uniform architecture of the cerebellar cortex, regional differences in the distribution of Golgi cells were found. In the inferior parts of the vermis, the number of Golgi cells per unit volume is twice that in the corresponding hemispheres. In addition, there are differences between the anterior and inferior parts of the vermis. These differences are a feature of the cytoarchitecture of the cerebellum in man and all the investigated mammals. The ratio of Purkinje cells to Golgi cells was also determined and found to differ in different species. In man, this ratio is 11.5, while in the monkey and cat it is almost 11.9 and in the rat 13.3. These differences in the ratio of Purkinje cells to Golgi cells are discussed from the point of view of cerebellar evolution.Supported by the Deutsche Forschungsgemeinschaft.     

Significance of the cytoskeleton for cytoplasmic organization and cell organelle dynamics in epithelial cells of fresh-water sponges

Summary The cytoskeleton in epithelial cells ofSpongilla lacustris is constructed of microtubules radiating from the nuclear region and terminating at the cell periphery as well as microfilaments forming a cortical layer beneath the plasma membrane and distinct fibers in the cytoplasmic matrix. Single frame analysis and in vivo labeling of mitochondria with Rh 123, endosomes or lysosomes with TRITC-BSA, endoplasmic reticulum (ER) with DiOC₆ (3) and dictyosomes with C₆-NBD-ceramide points to the microtubular system as a candidate for controlled cytoplasmic organization and active transport of these cell organelles. In epithelial cells with an intact microtubular system, mitochondria and endosomes or lysosomes show a regular shuttle movement between the nucleus and the cell periphery with a velocity of 1.3–1.4 m/s; the ER forms a more or less dynamic two-dimensional network in the entire cytoplasmic matrix, and dictyosomes are arranged in a ring-like manner around the nucleus. In epithelial cells treated with colchicine or colcemid, mitochondria and endosomes or lysosomes gather in the perinuclear region and become immobile; the ER accumulates near the cell center, whereas most dictyosomes distribute randomly over the whole cytoplasm. Transformation of the microfilament system with cytochalasin D has no influence on cell organelle distribution and dynamics but impedes cell locomotion and cell surface activities.Abbreviations BSA bovine serum albumin - C₆-NBD-ceramide 6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)caproyl]sphingosine - DiOC₆(3) 3,3-dihydroxyloxacarbocyanine jodide - DMSO dimethylsulfoxide - EGTA ethylenediaminetetraacetic acid - GTX glycerol-Triton-X-100 - PBS phosphate buffered saline - PEG polyethylene glycol - PIPES 1,4-piperazine-N,N-bis-(2-ethanesulfomc) acid - Rh 123 rhodamine 123 - TRITC tetramethylrhodamine isothiocyanate     

A study of cells present in peripheral lymph of pigs with special reference to a type of cell resembling the langerhans cell

Summary Large mononuclear cells with long, actively moving cytoplasmic veils were observed in lymph coming from the skin. The enzyme histochemistry and ultrastructure of these cells suggested that they are related to epidermal Langerhans cells and interdigitating cells in the lymph node. It has been reported that Langerhans cells and interdigitating cells play a role in contact hypersensitivity by taking up antigen and presenting it to thymus-dependent lymphocytes, and it is likely that the veiled cells in the lymph are also involved.After skin-painting with 1-fluoro-2,4-dinitrobenzene (DNFB), the veiled cells in lymph coming from the site of painting became more active and were observed contacting other cells present in the lymph; many large cellular aggregates were found. Since neutrophilic leucocytes and mononuclear phagocytes were the predominating cell types in this lymph, there was no evidence for a massive recruitment of immunocompetent lymphocytes at the site of painting.Neonatally thymectomized pigs do not develop allergic reactivity to DNFB. It is of interest that the number of veiled cells and their ability to form large cellular aggregates was not affected in these animals. Therefore, it is unlikely that the defect in responsiveness can be attributed to a failure in the function of veiled cells.     

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.