Plant Cell Reports
Plant Cell Reports (1993) 12:268-272
© Springer-Verlag1993
Differential expression of two peanut peroxidase eDNA clones in peanut plants and cells in suspension culture in response to stress Colette
Breda I, Dominique Buffard i, Robert B. van Huystee 2, and Robert Esnault i
1 Institut des Sciences V~g~tales, C.N.R.S., 91198 Gif-sur-Yvette Cedex, France 2 Department of Plant Sciences, University of Western Ontario, London, Ontario N6A 5B7, Canada (R.B.v.H.) Received October 29, 1992/Revised version received January 5, 1993 - Communicated by F. Constabel
ABSTRACT. Peanut (Arachis h y p o g e a L.) peroxidase gene expression was analyzed by measuring the accumulation of trancripts in cultured cells and various plant parts (leaf, stem, root) and upon their treatment with ethylene or wounding, respectively. Two transcripts (prxPNC1 and prxPNC2) corresponding to two peroxidase genes are expressed at higher levels in cultured cells as compared to various plant organs. Analysis of total poly(A) ÷ RNA with an oligonucleotide probe corresponding to a highly conserved region of peroxidase genes showed the expression of three peroxidase related sequences (1,000, 1,400 or 2,600 bp) in stem or leaf but barely detectable in roots. The prxPNC2 transcript transiently expressed at high levels in response to ethylene treatment of cells or wounding of leaves. This suggests that the corresponding gene(s) are expressed in response to stress.
INTRODUCTION Peroxidases (E.C.1.11.1.7.) are ubiquitous in plants and animals and oxidize a vast array of compounds at the expense of H202. Most higher plants exhibit a large number of peroxidase isozymes. The expression, of some of these peroxidase isozymes, is tissue specific, developmentally regulated or in response to external stimuli; thus peroxidase isozymes serve as convenient biochemical markers in genetic, physiological and pathological studies (Greppin et al. 1986). Nucleotide sequences for genes encoding peroxidases or their corresponding cDNA have been cloned from several plant species (Lagrimini et al. 1987, Roberts et al. 1988, Fujiyama et al. 1988, Buffard et al. 1990). Some of these cloned peroxiCorrespondence to: R. Esnault
dase genes correlate to physiological functions in plant cells. Association of an anionic peroxidase gene with cell wall synthesis in tobacco (Lagrimini and Rothstein 1987) and of another anionic peroxidase gene with suberization in tomato and potato (Roberts et al. 1988) have been reported. Peroxidase transcripts accumulated in response to ethylene treatment in cucumber (Morgens et al. 1990) or to pathogen infection in barley (Rebman et al. 1991). However a direct correlation of peroxidase gene at the molecular level to plant growth and development has not been shown so far. Cultured plant cells respond rapidly to environmental stimuli or elicitors (Templeton and Lamb 1988). Consequently they are a simple experimental system for signal perception and modulation of transcription of specific genes. Cultured plant cells synthesize peroxidases and some of these peroxidases are secreted into the medium (Mader and Walter 1986, van Huystee and Chibbar 1987). The extra-cellular peroxidase activity directly correlated to growth kinetics and cell dry weight in cultured cells from a few plant species (Chibbar et aL 1984, Shetty et al. 1990, Zheng and van Huystee 1991 ). Peanut cells in culture secrete two (anionic and cationic) peroxidase isozymes that are readily purified and studied (van Huystee 1987). The cationic isozyme is the major extracellular isozyme in cultured peanut cells. In vivo and in vitro studies revealed that the synthesis of cationic peroxidase is 2% of total protein synthesis in the cultured cells (Stephan and van Huystee 1981, Chibbar and van Huystee 1983). This is a ten-fold higher level than its synthesis in peanut leaves (Chibbar and van Huystee 1983). Gene amplification in cultured cells may be responsible for the elevated peroxidase synthesis. However, no change in the ploidy level was observed in cultured peanut cells (Inoue and van
269
Huystee 1984). The cloning of two peroxidase cDNA from cultured peanut cells (Buffard et al. 1990) allows us to study peroxidase gene expression in plant cell growth and development. We analyzed the expression of peroxidase genes (prxPNC1 and prxPNC2) in cultured peanut cells and various parts of the peanut plant under normal conditions or treated by ethylene (cells) or wounding (leaves). In this report we show the differential expression of genes encoding peroxidase under these conditions,
MATERIAL AND METHODS Plant Cells and Etephon Treatment. Peanut cells (Arachis hypogaea L) were cultured as described (Buffard et al. 1990).
Four days old cells were treated at 1 or 10 mg 14 ethephon in 0.01M phosphate/0.02M citrate buffer pH 3. Control cells were treated with the buffer alone. Ceils for analysis were collected by fdtration over a sterile 3MM Whatman filter paper and frozen in liquid nitrogen. Whole plant Materials and Wounding. Peanut seeds were germinated on sterile filter papers and plants were grown at 24/17°C at 16h day/Sh night cycles in trays with vermiculite watered with Hoagland solution. Thirty days old plants were harvested and roots, stems and leaves were separated. Leaves of 30 days old plants were gently abraded with sandpaper and harvested at 1.5, 4, 7 and 17 h alter wounding. Preparation and quantitative determination of poly(A)+ RNA. The total RNA from cultured cells was extracted as described (Buffard et al. 1990). The RNA from leaf and stem was extracted by the protocol of Hall et al. (1978) and those from roots by the the guanidium thiocyanate/hot phenol method (Maniatis et al. 1982). The poly(A) ÷ RNA was isolated from total RNA with an oligo(dT) column (Buffard et al. 1990). The precise amounts of poly(A) ÷ RNA was determined by the poly(A):poly(U) hybridization as described (Tessier and Esnault 1980). mRNA Analysis. For dot blot hybridization experiments, the poly(A) ÷ mRNA was denatured in 50% formamide, 6% formaldehyde at 65°C for 5 min. Appropriate dilutions prepared in 10x SSC were applied on a wet (10x SSC) Hybond membrane (Amersham) in a Biorad Bio-Dot apparatus. For Northern analysis, 0.25 to 1 #g of poly(A) ÷ RNA from each sample was denatured with formaldehyde and was eleetrophoresed on a 1.2% agarose gel under denaturing conditions. The separated transcripts were transferred by capillarity onto a Hybond (Amersham) membrane. Hybridizations were conducted by using radioactively labelled oligonueleotides or eDNA or biotynylated eDNA probes. Oligonucleotides were end-labelled with T4 Polynucleotide Kinase and 3,[a2p]ATP; labelled eDNA probes were obtained using the "NEN Random Primer Plus Extension Labelling System" and t~[32P]dCTP. The "Tropix Random Labelling Kit" was used for biotinylation of eDNA; detection of the hybridized molecules was made by using the "Southern-Light Plus Kit" from "Tropix" and AMPPD R as substrate for chemiluminescence. Amongst these cDNAs two correspond to the peanut clones (Buffard et al. 1990) and the third to a chaleone synthase clone isolated from alfalfa leaves. The final wash conditions were: 0.2x SSC, 0.1% SDS at 65 ° C for hybridizations with the peroxidase cDNAs and 0.1x SSC, 0.1% SDS at 45°C with the CHS probe. Hybridi-
zation conditions for oligonucleotides were as described (Buffard et al. 1990). The anti-sense oligonueleotide used in this work was :
3' AC(C/A)-AG(G/A)-AC(G/A)-CG(T/A)-CT(G/A)-AT5' (equivalent to : VSCDAI).
RESULTS Peroxidase expression: cell v s whole plant. Northern analysis employing two types of radioactively labelled probes was used to evaluate the expression of the peroxidase genes. The prxPNC1 and prxPNC2 cDNA probes were used in high stringency conditions to avoid cross-hybridization between them and other peroxidase transcripts. The second was an antisense oligonucleotide, corresponding to the structural conserved region detected in previous analysis (Buffard et al. 1990), that will recognize all the peroxidase genes expressed in the cell or plant. Results with cDNA probes (data not shown) indicated that several-fold differences exist in the corresponding transcript levels in cultured cells as compared to intact plants. To allow for comparison of gene expression, 3-fold higher amount of poly(A) + mRNA from organs was used. The prxPNCl and prxPNC2 do not cross-hybridize and are expressed strongly in cells but are barely detectable in the plant organs (Fig. 1A). The results obtained with the antisense oligonucleotide (Fig. 1 B) confirm this conclusion. Data from Fig. 1 suggest that, in the cultured cells, the two eDNA clones prxPNC1 and prxPNC2 expressed at much higher level than any other, which are undetectable in our experimental conditions. In the whole plant the pattern of peroxidase gene expression is quantitatively as well as qualitatively different. The highest expression of peroxidase genes was observed in the stem, followed by leaf and no peroxidase transcripts were detected in the roots (Fig. 1B). Three putative peroxidase transcripts were observed on the Northern blot with RNA from stem tissue were at 1,000, 1,400 and 2,600 bp, the 1,400 bp transcript being the most heavily labelled (Fig. 1B lane S). Similarly, three transcripts were observed for leaves; however, the 1,000 bp transcript was the highly labelled (Fig. 1B, lane L). Influence of external stimuli. Poly(A) + RNA, isolated from ethephon treated cells at 0, 1, 3 and 6 h after treatment, was used for analysing the expression of peroxidase genes, using the dot blot procedure. The two radioactively labelled peroxidase eDNA prxPNC1 and prxPNC2 as well and a heteroIogous chalcone synthase cDNA (from alfalfa, unpublished) probes were used for the expression of corresponding transcripts. The chalcone synthase is a
270
C
A[
prxPNCI
R
S
IB
L
prxPNC2 ,12.6
C
R
S
L
C
R
S
L
Plant Cell Reports (1993) 12:268-272
© Springer-Verlag1993
Differential expression of two peanut peroxidase eDNA clones in peanut plants and cells in suspension culture in response to stress Colette
Breda I, Dominique Buffard i, Robert B. van Huystee 2, and Robert Esnault i
1 Institut des Sciences V~g~tales, C.N.R.S., 91198 Gif-sur-Yvette Cedex, France 2 Department of Plant Sciences, University of Western Ontario, London, Ontario N6A 5B7, Canada (R.B.v.H.) Received October 29, 1992/Revised version received January 5, 1993 - Communicated by F. Constabel
ABSTRACT. Peanut (Arachis h y p o g e a L.) peroxidase gene expression was analyzed by measuring the accumulation of trancripts in cultured cells and various plant parts (leaf, stem, root) and upon their treatment with ethylene or wounding, respectively. Two transcripts (prxPNC1 and prxPNC2) corresponding to two peroxidase genes are expressed at higher levels in cultured cells as compared to various plant organs. Analysis of total poly(A) ÷ RNA with an oligonucleotide probe corresponding to a highly conserved region of peroxidase genes showed the expression of three peroxidase related sequences (1,000, 1,400 or 2,600 bp) in stem or leaf but barely detectable in roots. The prxPNC2 transcript transiently expressed at high levels in response to ethylene treatment of cells or wounding of leaves. This suggests that the corresponding gene(s) are expressed in response to stress.
INTRODUCTION Peroxidases (E.C.1.11.1.7.) are ubiquitous in plants and animals and oxidize a vast array of compounds at the expense of H202. Most higher plants exhibit a large number of peroxidase isozymes. The expression, of some of these peroxidase isozymes, is tissue specific, developmentally regulated or in response to external stimuli; thus peroxidase isozymes serve as convenient biochemical markers in genetic, physiological and pathological studies (Greppin et al. 1986). Nucleotide sequences for genes encoding peroxidases or their corresponding cDNA have been cloned from several plant species (Lagrimini et al. 1987, Roberts et al. 1988, Fujiyama et al. 1988, Buffard et al. 1990). Some of these cloned peroxiCorrespondence to: R. Esnault
dase genes correlate to physiological functions in plant cells. Association of an anionic peroxidase gene with cell wall synthesis in tobacco (Lagrimini and Rothstein 1987) and of another anionic peroxidase gene with suberization in tomato and potato (Roberts et al. 1988) have been reported. Peroxidase transcripts accumulated in response to ethylene treatment in cucumber (Morgens et al. 1990) or to pathogen infection in barley (Rebman et al. 1991). However a direct correlation of peroxidase gene at the molecular level to plant growth and development has not been shown so far. Cultured plant cells respond rapidly to environmental stimuli or elicitors (Templeton and Lamb 1988). Consequently they are a simple experimental system for signal perception and modulation of transcription of specific genes. Cultured plant cells synthesize peroxidases and some of these peroxidases are secreted into the medium (Mader and Walter 1986, van Huystee and Chibbar 1987). The extra-cellular peroxidase activity directly correlated to growth kinetics and cell dry weight in cultured cells from a few plant species (Chibbar et aL 1984, Shetty et al. 1990, Zheng and van Huystee 1991 ). Peanut cells in culture secrete two (anionic and cationic) peroxidase isozymes that are readily purified and studied (van Huystee 1987). The cationic isozyme is the major extracellular isozyme in cultured peanut cells. In vivo and in vitro studies revealed that the synthesis of cationic peroxidase is 2% of total protein synthesis in the cultured cells (Stephan and van Huystee 1981, Chibbar and van Huystee 1983). This is a ten-fold higher level than its synthesis in peanut leaves (Chibbar and van Huystee 1983). Gene amplification in cultured cells may be responsible for the elevated peroxidase synthesis. However, no change in the ploidy level was observed in cultured peanut cells (Inoue and van
269
Huystee 1984). The cloning of two peroxidase cDNA from cultured peanut cells (Buffard et al. 1990) allows us to study peroxidase gene expression in plant cell growth and development. We analyzed the expression of peroxidase genes (prxPNC1 and prxPNC2) in cultured peanut cells and various parts of the peanut plant under normal conditions or treated by ethylene (cells) or wounding (leaves). In this report we show the differential expression of genes encoding peroxidase under these conditions,
MATERIAL AND METHODS Plant Cells and Etephon Treatment. Peanut cells (Arachis hypogaea L) were cultured as described (Buffard et al. 1990).
Four days old cells were treated at 1 or 10 mg 14 ethephon in 0.01M phosphate/0.02M citrate buffer pH 3. Control cells were treated with the buffer alone. Ceils for analysis were collected by fdtration over a sterile 3MM Whatman filter paper and frozen in liquid nitrogen. Whole plant Materials and Wounding. Peanut seeds were germinated on sterile filter papers and plants were grown at 24/17°C at 16h day/Sh night cycles in trays with vermiculite watered with Hoagland solution. Thirty days old plants were harvested and roots, stems and leaves were separated. Leaves of 30 days old plants were gently abraded with sandpaper and harvested at 1.5, 4, 7 and 17 h alter wounding. Preparation and quantitative determination of poly(A)+ RNA. The total RNA from cultured cells was extracted as described (Buffard et al. 1990). The RNA from leaf and stem was extracted by the protocol of Hall et al. (1978) and those from roots by the the guanidium thiocyanate/hot phenol method (Maniatis et al. 1982). The poly(A) ÷ RNA was isolated from total RNA with an oligo(dT) column (Buffard et al. 1990). The precise amounts of poly(A) ÷ RNA was determined by the poly(A):poly(U) hybridization as described (Tessier and Esnault 1980). mRNA Analysis. For dot blot hybridization experiments, the poly(A) ÷ mRNA was denatured in 50% formamide, 6% formaldehyde at 65°C for 5 min. Appropriate dilutions prepared in 10x SSC were applied on a wet (10x SSC) Hybond membrane (Amersham) in a Biorad Bio-Dot apparatus. For Northern analysis, 0.25 to 1 #g of poly(A) ÷ RNA from each sample was denatured with formaldehyde and was eleetrophoresed on a 1.2% agarose gel under denaturing conditions. The separated transcripts were transferred by capillarity onto a Hybond (Amersham) membrane. Hybridizations were conducted by using radioactively labelled oligonueleotides or eDNA or biotynylated eDNA probes. Oligonucleotides were end-labelled with T4 Polynucleotide Kinase and 3,[a2p]ATP; labelled eDNA probes were obtained using the "NEN Random Primer Plus Extension Labelling System" and t~[32P]dCTP. The "Tropix Random Labelling Kit" was used for biotinylation of eDNA; detection of the hybridized molecules was made by using the "Southern-Light Plus Kit" from "Tropix" and AMPPD R as substrate for chemiluminescence. Amongst these cDNAs two correspond to the peanut clones (Buffard et al. 1990) and the third to a chaleone synthase clone isolated from alfalfa leaves. The final wash conditions were: 0.2x SSC, 0.1% SDS at 65 ° C for hybridizations with the peroxidase cDNAs and 0.1x SSC, 0.1% SDS at 45°C with the CHS probe. Hybridi-
zation conditions for oligonucleotides were as described (Buffard et al. 1990). The anti-sense oligonueleotide used in this work was :
3' AC(C/A)-AG(G/A)-AC(G/A)-CG(T/A)-CT(G/A)-AT5' (equivalent to : VSCDAI).
RESULTS Peroxidase expression: cell v s whole plant. Northern analysis employing two types of radioactively labelled probes was used to evaluate the expression of the peroxidase genes. The prxPNC1 and prxPNC2 cDNA probes were used in high stringency conditions to avoid cross-hybridization between them and other peroxidase transcripts. The second was an antisense oligonucleotide, corresponding to the structural conserved region detected in previous analysis (Buffard et al. 1990), that will recognize all the peroxidase genes expressed in the cell or plant. Results with cDNA probes (data not shown) indicated that several-fold differences exist in the corresponding transcript levels in cultured cells as compared to intact plants. To allow for comparison of gene expression, 3-fold higher amount of poly(A) + mRNA from organs was used. The prxPNCl and prxPNC2 do not cross-hybridize and are expressed strongly in cells but are barely detectable in the plant organs (Fig. 1A). The results obtained with the antisense oligonucleotide (Fig. 1 B) confirm this conclusion. Data from Fig. 1 suggest that, in the cultured cells, the two eDNA clones prxPNC1 and prxPNC2 expressed at much higher level than any other, which are undetectable in our experimental conditions. In the whole plant the pattern of peroxidase gene expression is quantitatively as well as qualitatively different. The highest expression of peroxidase genes was observed in the stem, followed by leaf and no peroxidase transcripts were detected in the roots (Fig. 1B). Three putative peroxidase transcripts were observed on the Northern blot with RNA from stem tissue were at 1,000, 1,400 and 2,600 bp, the 1,400 bp transcript being the most heavily labelled (Fig. 1B lane S). Similarly, three transcripts were observed for leaves; however, the 1,000 bp transcript was the highly labelled (Fig. 1B, lane L). Influence of external stimuli. Poly(A) + RNA, isolated from ethephon treated cells at 0, 1, 3 and 6 h after treatment, was used for analysing the expression of peroxidase genes, using the dot blot procedure. The two radioactively labelled peroxidase eDNA prxPNC1 and prxPNC2 as well and a heteroIogous chalcone synthase cDNA (from alfalfa, unpublished) probes were used for the expression of corresponding transcripts. The chalcone synthase is a
270
C
A[
prxPNCI
R
S
IB
L
prxPNC2 ,12.6
C
R
S
L
C
R
S
L
