Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

Laser microsurgery: a versatile tool in plant (electro) physiology

Summary In plant cells the cell wall is a formidable obstacle in many physiological studies such as patch-clamp measurements and cell labelling with antibodies. Enzymatic digestion of the cell wall, in order to release a protoplast, has a number of disadvantages; therefore we worked out an alternative method to gain access to the plasma membrane. The wall of specialized cells from three higher plant species and one unicellular alga were perforated using the focussed UV light of a nitrogen laser. In order to enhance the absorption of the UV light by the walls, a dye was used that binds specifically to cell wall components. Extrusion of the protoplast or parts thereof was controlled by a regulated gradual decrease of the osmolarity of the solution surrounding the cells. Cytoplasmic streaming and chloroplast circulation were maintained in the protoplasts, demonstrating their viability after the wall perforation with the laser. Continuous deposition of new cell wall material by the polar tip of pollen tubes after surgical removal of the wall at the tip is another demonstration of the viability of the cells. Formation of high resistance seals between the plasma membrane and a patch pipet was surprisingly difficult. The role of Hechtian strands and continuing synthesis of cell wall material in seal formation is further investigated. Other applications for the surgical laser are: fusion of two cells or vacuoles, analysis of the composition of specific parts of the cell wall, and release of the vacuole from an identified cell type for patchclamp studies.Abbreviations CFW calcofluor white - PM plasma membrane     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Involvement of transglutaminase in the formation of covalent cross-links in the cell wall ofCandida albicans

Activity of the enzyme glutaminyl-peptide-γ-glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N^∈-(γ-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms ofCandida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.     

Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress

The cell walls in the new white roots of jack pine (Pinus banksiana Lamb.) were observed to constrict around the shrinking protoplast of osmotically stressed roots, and pressure was maintained via an apparent adjustment of cell-wall size and elasticity. These elastic alterations of the cell wall permitted the root cells to maintain full turgor despite the loss of most of the water in the tissue. The constriction of the root cell wall around the dehydrating protoplasts to maintain turgor may reflect changes in cell wall structure. We found that these shrinking root cells synthesize and secrete into the intercellular fluid a set of proteins. These proteins become tightly associated (i.e. guanidine HCl- and sodium dodecyl sulfate-insoluble) with the cell wall but can be released from the matrix, after briefly boiling in 0.1% sodium dodecyl sulfate, by the combination of guanidine HCl, CaCl₂ and dithiothreitol. However, these cell-wall proteins became insoluble with time. The proteins could subsequently be destructively extracted from the wall with acid NaClO₂ treatments. After these proteins were incorporated into the cell walls, the roots adopted a new, smaller maximal tissue volume and elastic coefficients returned to normal levels. Received: 8 July 1998 / Accepted: 19 November 1998     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Immunohistochemical and immunocytochemical detection of SchS34 antigen in <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> spores and spore impacted mouse lungs

The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2–3× the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.     

The structure and biochemistry of charophycean cell walls: I. Pectins of Penium margaritaceum

Summary. Plant cell walls are essential for proper growth, development, and interaction with the environment. It is generally accepted that land plants arose from aquatic ancestors which are sister groups to the charophycean algae (i.e., Streptophyta), and study of wall evolution during this transition promises insight into structure–function relationships of wall components. In this paper, we explore wall evolutionary history by studying the incorporation of pectin polymers into cell walls of the model organism Penium margaritaceum, a simple single-cell desmid. This organism produces only a primary wall consisting of three fibrillar or fibrous layers, with the outermost stratum terminating in distinct, calcified projections. Extraction of isolated cell walls with trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid yielded a homogalacturonan (HGA) that was partially methyl esterified and equivalent to that found in land plants. Other pectins common to land plants were not detected, although selected components of some of these polymers were present. Labeling with specific monoclonal antibodies raised against higher-plant HGA epitopes (e.g., JIM5, JIM7, LM7, 2F4, and PAM1) demonstrated that the wall complex and outer layer projections were composed of the HGA which was significantly calcium complexed. JIM5 and JIM7 labeling suggested that highly methyl esterified HGA was secreted into the isthmus zone of dividing cells, the site of active wall secretion. As the HGA was displaced to more polar regions, de-esterification in a non-blockwise fashion occurred. This, in turn, allowed for calcium binding and the formation of the rigid outer wall layer. The patterning of HGA deposition provides interesting insights into the complex process of pectin involvement in the development of the plant cell wall. Correspondence and reprints: Department of Biology, Skidmore College, 815 North Broadway, Saratoga Springs, NY 12866, U.S.A.     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae)

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.Abbreviations CWH complete cell walls isolated from the homogenates - CWM maternal cell walls accumulated in the medium - KC ketocarotenoid - SC secondary carotenoids - SP sporopollenin     

Analysis of Root Growth by Impedance Spectroscopy (EIS)

Electrical impedance spectroscopy (EIS) is investigated as a non-destructive method for monitoring root growth of tomato. This paper aims to (i) review the basic principles of EIS applied to the characterisation of the different parts of the soil–root–stem-electrode continuum, (ii) experiment the validity of the relationship between root weight and root capacitance taking into account the influence of the soil and plant electrodes position, (iii) describe an EIS analysis of the root growth of tomato plants. All experiments were carried out in 50 dm³ containers either in hydroponics at 930 μS for the test of root fresh or dry weight and root capacitance relationships, or in a potting mix (oxisol) for electrode placement tests and EIS estimation of root growth. Electrical measurements of the soil–root–stem-electrode continuum were done with a two-electrode measuring system using unpolarisable Ag–AgCl electrodes. A ‘root cutting’ and a ‘progressively immersed root system’ experiments were carried out in order to validate the relationship between root capacitance and root mass at 1 kHz. The effects of soil electrode and plant electrode placement were also tested, pointing out the sensitivity of the method to the insertion height of the “plant electrode” into the stem. For the root growth experiment, Impedance Spectra (IS) measurements were made just before harvesting the roots for dry weight and length determination. Measurements were made 14, 22, 26 and 39 days after planting, until flowering. The IS of the soil–root–stem-electrode continuum was modelled by a lumped electric circuit consisting of a series resistor R ₀ for the soil and of four parallel resistance (R _i )-capacitance (C _i ) circuits for the other components of the circuit. The model had nine parameters whose values were estimated by Complex Nonlinear Least Squares curve fitting. A stepwise ascendant regression was used to identify the electrical parameters that better correlated with root dry mass or length increment: C ₃ and C ₄ were well correlated with root dry mass with a r ² of 0.975, whereas root length was explained by the combination of 1/R ₃, C ₃, 1/R ₂ and 1/R ₁ with a r ² of 0.986. This work may be considered as a new methodological contribution to the understanding of root electrical properties in the non-destructive diagnosis of root systems.     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

Proline-rich cell wall proteins accumulate in growing regions and phloem tissue in response to water deficit in common bean seedlings

Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119–1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation. Marina Battaglia and Rosa M. Solórzano contributed equally to this work.     

Mechanisms of infection of plants by nitrogen fixing organisms

Heterotrophic nitrogen-fixing microorganisms can enter plants via wounds, root hairs or intact epidermises. All at some stage need the ability to digest primary cell walls and/or middle lamellas. None appears to digest secondary walls. The ability of any organism to infect a particular plant reflects (a) the enzymes produced by the microorganism (and possibly, as part of its reaction, the plant); (b) the exact nature of the primary wall; (c) the distribution of secondary walls. Plants may respond to infection by hypersensitive and other reactions which could be triggered by production of cell wall fragments. Infection threads of secondary wall material may be essential for root hair infection and where cell boundaries are crossed. Entry into host cells other than by infection threads involves a delicate balance between endophyte and host. This may only be achieved in one or a few cells, which may then divide repeatedly to produce a symbiotic structure.     

Directional cell-to-cell communication in theArabidopsis root apical meristem III. Plasmodesmata turnover and apoptosis in meristem and root cap cells during four weeks after germination

Summary Plasmodesmata frequency and distribution in root cap cells ofArabidopsis thaliana root tips were characterized during four weeks after germination to understand the symplasmic control of apoptosis. Apoptotic cells in some of the root apical-meristem cells and in root cap cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reaction and characterized by electron microscopy. Starting at the second week after germination, cells in the outermost layers of the root cap showed typical apoptotic features, including nuclear DNA fragmentation, chromatin condensation, cytoplasmic vacuolation, and organelle destruction. Intercellular connections, indicated by the frequency and number of plasmodesmata per cell length, were significantly reduced in the walls of outer root cap cells. This shows that cells become symplasmically isolated during the apoptosis process. In apoptotic root cap cells, the majority of nonfunctional plasmodesmata were observed to be associated with degenerated endoplasmic reticulum; this state was prior to the detection of any nuclear DNA fragmentation. Other nonfunctional plasmodesmata were sealed by heterogeneous cell wall materials. However, in immature epidermal and cortical cells in 4-week-old arrested roots the endoplasmic reticulum associated with plasmodesmata became disconnected as a result of protoplast condensation and shrinkage. No degenerated endoplasmic reticulum was observed in these cells. These observations suggest that the apoptotic processes in the root body and the root cap are different.     

Fractional changes in phenolic acids composition in root nodules of Arachis hypogaea L.

Phenolic acids are active antimicrobial compounds and root signaling molecules that play important roles in plant defense responses. They are generally present in plants as glycosides or esters. A range of soluble and bound phenolic acids were detected in roots and root nodules of Arachis hypogaea L., among which five were identified by high performance liquid chromatography (HPLC) coupled with UV–Vis diode array detector (DAD), viz., p-coumaric acid (p-com), p-hydroxybenzaldehyde (HBAld), p-hydroxybenzoic acid (HBA), caffeic acid (CA) and protocatechuic acid (PA). Para-coumaric acid was constitutively present in all fractions whereas HBA was present in the soluble form only in young nodules. CA and PA were mostly present in the wall bound fraction. The root nodules contain higher concentration of phenolic acids than non-nodulated roots and presence of peroxidase and polyphenol oxidase indicate the metabolism of phenolic acids in roots and root nodules. These results indicate that phenolic acids (p-com and CA) in bound-glycosidic or ester forms were major components in cell wall fortification which provide protection to the root nodule from pathogen attack.     

Early death at medium acidification and survival after low pH adaptation inCryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.     

Stomata and plasmodesmata

Summary In developing epidermal tissue ofPhaseolus vulgare L. complete plasmodesmatal connections occurred between guard cells and epidermal cells and between sister guard cells of a stoma but they were not seen in fully differentiated tissue. However, incomplete, aborted plasmodesmata were occasionally seen in the common guard/epidermal cell wall, usually connected to the epidermal cell protoplast, in mature tissue. Plasmodesmatal connections between neighbouring epidermal cells were commonly observed in tissue at all stages of development. In all locations, the plasmodesmata were usually unbranched occurring singly or in small pit fields; very rarely branched, incomplete plasmodesmata were also seen in the wall between mature guard and epidermal cells. The significance of these findings were related to stomatal functioning and to the development of plasmodesmata in general.     

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.     

In-situ Raman microprobe studies of plant cell walls: Macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P.

Native-state organization and distribution of cell-wall components in the secondary wall of woody tissue from P. mariana (Black Spruce) have been investigated using polarized Raman microspectroscopy. Evidence for orientation is detected through Raman intensity variations resulting from rotations of the exciting electric vector with respect to cell-wall geometry. Spectral features associated with cellulose and lignin were studied. The changes in cellulose bands indicate that the pyranose rings of the anhydroglucose repeat units are in planes perpendicular to the cross section, while methine C–H bonds are in planes parallel to the cross section. Changes in bands associated with lignin indicate that the aromatic rings of the phenyl-propane units are most often in the plane of the cell-wall surface. However, regions where lignin orientation departs from this pattern also occur. These results represent direct evidence of molecular organization with respect to cellular morphological features in woody tissue, and indicate that cell-wall components are more highly organized than had been recognized. Studies carried out in order to establish the usefulness and sensitivity of the Raman technique to differences of composition within the cell walls provide evidence of variations in the distribution of cellulose and lignin. Such compositional differences were more prominent between the walls of different cells than within a particular cell wall.