Cell wall regeneration by protoplasts of Chlamydomonas

Robinson, David G.; Schlösser, U. G.

doi:10.1007/bf00387749

Cited by 37 publications

(20 citation statements)

References 31 publications

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“…At the end of this incubation, the cells were washed free of GLE (time 0) and began to synchronously assemble a new V-wall. As indicated by the control curve in this panel, a detergent-resistant V-wall is normally completed within 2 h (Robinson and Schlö sser, 1978;Waffenschmidt et al, 1993). When vegetative protoplasts were resuspended in media with increasing concentrations of putrescine, a proportional decrease in the number of cells completing the insolubilization program was observed.…”

Section: Inhibition Of Cell Wall Insolubilization With Primary Aminesmentioning

confidence: 84%

A Transglutaminase Immunologically Related to Tissue Transglutaminase Catalyzes Cross-Linking of Cell Wall Proteins inChlamydomonas reinhardtii

Waffenschmidt¹,

Kusch

Woessner

1999

Plant Physiology

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The addition of primary amines to the growth medium of the unicellular green alga Chlamydomonas reinhardtii disrupts cell wall assembly in both vegetative and zygotic cells. Primary amines are competitive inhibitors of the protein-cross-linking activity of transglutaminases. Two independent assays for transglutaminase confirmed a burst of extracellular activity during the early stages of cell wall formation in both vegetative cells and zygotes. When noninhibiting levels of a radioactive primary amine ( 14 C-putrescine) were added to the growth medium, both cell types were labeled in a reaction catalyzed by extracellular transglutaminase. The radioactive label was found specifically in the cell wall proteins of both cell types, and acid hydrolysis of the labeled material released unmodified 14 C-putrescine. Western blots of the proteins secreted at the times of maximal transglutaminase activity in both cell types revealed a single highly cross-reactive 72-kD band when screened with antibodies to guinea pig tissue transglutaminase. Furthermore, the proteins immunoprecipitated by this antiserum in vivo exhibited transglutaminase activity. We propose that this transglutaminase is responsible for an early cell wall protein cross-linking event that temporally precedes the oxidative cross-linking mediated by extracellular peroxidases.

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Section: Inhibition Of Cell Wall Insolubilization With Primary Aminesmentioning

confidence: 84%

A Transglutaminase Immunologically Related to Tissue Transglutaminase Catalyzes Cross-Linking of Cell Wall Proteins inChlamydomonas reinhardtii

Waffenschmidt¹,

Kusch

Woessner

1999

Plant Physiology

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“…Also, resectioning and transfer of shoots at regular intervals result in repeated rejuvenation conserving a relatively stable developmental stage of the protoplast source. Furthermore, when using in vitro plants, surface sterilization and actinomycin, which is often used to prevent microbial growth, are omitted; this is important because actinomycin has been reported to cause structural abberations such as inflations of nuclear membrane, folding of ER, and changes in mitochondrial and chloroplastic shape [19]. Therefore, use of axenic in vitro cultured plant material is highly recommended as donor material for isolation of grapevine leaf protoplasts.…”

Section: Discussionmentioning

confidence: 99%

“…Among the factors which could create problems during isolation and culture of grapevine protoplasts are the variation of donor tissues, the viral infection of donor plants which alters the composition of cell walls [26], the use of actinomycin during culture which causes structural abberations in the cells [19]. Some of these difficulties could be partially overcome by using axenic shoots from in vitro grown plants, tested for virus-freedom.…”

Section: Introductionmentioning

confidence: 99%

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Theodoropoulos¹,

Roubelakis‐Angelakis

1990

Plant Cell Tiss Organ Cult

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Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50-100mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml-~ Cellulase R-10 and M acerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25 × 106 viable protoplasts per g fresh weight and their size ranged from 12 to 44 ~tm. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10 × 105 protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [~4C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN 3, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.

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“…RESULTS g-Lysin Regulation of mRNAs Encoding Proline-Rich Polypeptides. Chlamydomonas cells stripped of their cell walls with g-lysin rapidly regenerate another matrix over 3-4 hr (13,14). To examine the expression of genes encoding proline-rich polypeptides during cell wall regeneration, total RNA was isolated at various times after wall removal and was translated in vitro with [35Sjmethionine or [3Hlproline.…”

Section: Methodsmentioning

confidence: 99%

Cell wall regeneration in Chlamydomonas: accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins.

Adair

Apt

1990

Proc. Natl. Acad. Sci. U.S.A.

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The unicellular alga Chkamydomonas reinharii is surrounded by a cell wall composed entirely of hydroxyproline-rich glycoproteins (HRGPs). When the walls of vegetative cells are removed with the enzyme gamete autolysin (g-lysin), they regenerate a matrix within 3-4 hr. In vitro translation of mRNAs isolated from g-lysin-treated cells showed sinificant increases and decreases in abu of several mRNAs encoding prline-rich polypeptides. Because the population of up-regulated mRNAs is likely to include species encoding cell wall components, expression of genes for two outer wall HRGPs (GP1 and GP2) was analyzed during wall regeneration by using cDNAs isolated from a C. reinharii Agtll library. Transcripts encoding GP1 and GP2 were elevated severalfold within the first hour of regeneration, suggesting that upregulation of HRGP mRNAs is a primary response to cell wall removal by g-lysin. Cell wall regeneration in Chimydomonas provides an accessible system to study HRGP gene expression during matrix development.Chlamydomonas reinhardtii, a unicellular alga, has a multilayered cell wall constructed from hydroxyproline-rich glycoproteins (HRGPs), lacking the abundant carbohydrate polymers of higher plants (1,2). Each wall layer contains a distinct set of components (3-5), which form two major domains. The inner (framework) domain maintains the overall shape of the matrix and resists extraction by chaotropic agents, perchlorate, and sarkosyl/urea solutions (6-8). By contrast, the outer domain is easily solubilized and is capable of in vitro assembly (5-7, 9-11). These properties have been exploited for the purification of the major outer wall HRGPs (GP1, GP2, and GP3), their biochemical and morphological characterization (5), and in vitro assembly of individual sublayers from purified components (11, 12).While studies of in vitro assembly are contributing much to our understanding of HRGP interactions, information on HRGP gene expression during cell wall development is lacking. In this study, abundance of mRNAs encoding proline-rich polypeptides is examined during cell wall regeneration induced by the wall-degrading enzyme gamete autolysin (g-lysin). During the sexual cycle of C. reinhardtii, g-lysin acts to degrade gamete cell walls as a prelude to cell fusion and zygosis. Vegetative cells, which also serve as substrate for g-lysin, respond to wall removal by assembling another matrix within 3-4 hr (13, 14). Here we show that cell wall regeneration is accompanied by significant changes in levels of translatable mRNAs for proline-rich polypeptides; several transcripts increase as an early response to wall removal, while others decline. Using GP1 and GP2 cDNAs isolated from a vegetative Chlamydomonas Agtll library, we demonstrate that levels of mRNAs encoding both outer wall HRGPs increase severalfold during the first hour of regeneration. These observations suggest that changes in gene expression are likely to play an important role in Chlamydomonas cell wall regeneration, which is a useful experimental system to st...

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Cell wall regeneration by protoplasts of Chlamydomonas

Cited by 37 publications

References 31 publications

A Transglutaminase Immunologically Related to Tissue Transglutaminase Catalyzes Cross-Linking of Cell Wall Proteins inChlamydomonas reinhardtii

A Transglutaminase Immunologically Related to Tissue Transglutaminase Catalyzes Cross-Linking of Cell Wall Proteins inChlamydomonas reinhardtii

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Cell wall regeneration in Chlamydomonas: accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins.

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