Journal of Experimental Botany, Vol. 51, No. 348, pp. 1189-1200,
July 2000
© 2000 Oxford University Press
Original papers |
Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins
1 Laboratoire de Physiologie Cellulaire et Morphogenèse Végétales, USTL/INRA. Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cedex, France
2 Laboratorium voor Genetica. Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium
Received 10 September 1999; Accepted 2 March 2000
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Abstract |
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In Cichorium hybrid clone ‘474’ (C. intybus L., var. sativumxC. endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8-fold compared with those in the medium of a non-embryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa ß-1,3- glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin-like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin-like protein present in the conditioned media of the embryogenic ‘474’ hybrid and of a non-embryogenic variety, a 2–8-fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis-related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.
Key words: Cichorium, leaves, somatic embryogenesis, extracellular proteins, ß-1,3-glucanases, chitinases, osmotin-like proteins, microsequencing, immunoblots.
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Introduction |
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Nearly three decades ago, Pathogenesis-Related (PR) proteins were shown to be present in the leaves of Samsun NN and Xanthi tobacco plants during the hypersensitive reaction against tobacco mosaic virus (TMV) (Gianinazzi et al., 1970
; van Loon and van Kammen, 1970). Many proteins with similar properties have since been isolated from tobacco and other plant species, both dicots and monocots (van Loon, 1985; Carr and Klessig, 1989; Linthorst, 1991). The major tobacco PR proteins have been classified into five majors groups: PR1–PR5 (Bol, 1990; Linthorst, 1991) based on their serological properties and later on sequence data,. These proteins were found to be induced not only upon virus infection (Pfitzner and Goodman, 1987; Lawton et al., 1992 Tornero et al., 1994), but also upon pathogenesis caused by fungi (Tahiri-Alaoui et al., 1990; Niderman et al., 1995) or bacteria (Huang et al., 1994). In fact, it became apparent that PR protein accumulation occurred under different stress conditions, whether caused by wounding (Grosset et al., 1990; Warner et al., 1992), plant hormones, such as ethylene (Meller et al., 1993; Sessa et al., 1995), salicylic acid (Chen et al., 1993; Shah and Klessig, 1996), heavy metals (Awade et al., 1991; Didierjean et al., 1996), heat (Eckey-Kaltenbach et al., 1997), cold (Hon et al., 1995) or UV light (Brederode et al., 1991).
So-called PR proteins were also shown to accumulate in healthy plants (Regalado and Ricardo, 1996; Gruner and Pfitzner, 1994), in particular, in an organ- and tissue-specific manner during development programmes such as germination (Casacuberta et al., 1992), senescence (Hanfrey et al., 1996), and flowering (Lotan et al., 1989; Ori et al., 1990; Cote et al., 1991; van Eldik et al., 1996). Therefore, besides having a function in the defence system, PR proteins have functions related to the development of the plant. Both ß-1,3-glucanases and chitinases have also been found to be associated with plant development (Neale et al., 1990; Ori et al., 1990; Cote et al., 1991; van Eldik et al., 1996). In carrot, chitinases may be involved in the generation of signal molecules that stimulate somatic embryogenesis (de Jong et al., 1992; Kragh et al., 1996).
Previously it has been shown that during somatic embryogenesis in Cichorium, the protein patterns in the explant tissue and in the medium changes and that some of these changes are associated with the induction and initiation of somatic embryogenesis. The proteins involved in these changes were named Somatic Embryogenesis Related (SER) proteins (Hilbert et al., 1992; Boyer et al., 1993; Helleboid et al., 1995). Major proteins, SER 37.7 kDa, were isolated from the medium and were identified as extracellular ß-1,3-glucanases (Helleboid et al., 1998) that may be involved in the degradation of the callose wall surrounding embryogenic cells and small embryos.
In this paper, two more major proteins present in the medium of embryo culture of the Cichorium hybrid ‘474’ were identified by microsequencing as chitinases and osmotin-like proteins. The identity of chitinase was confirmed by the demonstration of chitinase activity, whereas the putative osmotin-like protein was recognized by antibodies against tobacco osmotin-like proteins. ß-1,3- glucanases, chitinase and osmotin-like proteins were shown to accumulate in the medium of embryo cultures to a much higher level when compared to their level in the medium of cultures of a non-embryogenic Cichorium variety.
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Materials and methods |
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Somatic embryogenesis culture conditions
Plantlets of chicory hybrid clone ‘474’ (Cichorium intybus L., var. sativumxC. endivia L., var. latifolia) were grown in vitro as described previously (Helleboid et al., 1995
). Seeds of the non-embryogenic Cichorium intybus var. witloof cv. ‘Flash’ were sterilized, germinated and cultured in vitro. Leaf fragments from 6-week-old plantlets were cultured for 4 d at 35 °C in darkness in 50 ml of an agitated liquid basal medium containing sucrose (60 mM) and glycerol (330 mM) to induce somatic embryogenesis. The presence of glycerol allowed the activation of the mesophyll cells for embryogenic competence, but impaired cell divisions (Robatche-Claive et al., 1992). Synchronized cell divisions only occurred after the transfer of the 4-d-induced leaf-fragments to glycerol-free medium (expression condition).
Conditioned medium protein extraction
Embryo culture media were harvested and passed through a Faltenfilter MN 7131/4 (Osi) and through a Millipore (Bedford) 0.22 µm filter, dialysed for 72 h against distilled water, and lyophilized. Proteins were extracted on 50 ml lyophilized-medium as described by Helleboid et al. (Helleboid et al., 1998) and proteins were resuspended in SDS sample buffer (20% glycerol, 100 mM DTT, 2% SDS, and 62.5 mM TRIS-HCl, pH 6.8) or in lysis buffer (Damerval et al., 1986). Samples were stored at -70 °C until analysis.
Protein polyacrylamide gel electrophoresis (PAGE)
SDS-PAGE were performed on 1 mm thick slab gels with a 12.5% acrylamide separating gel and a 4% acrylamide stacking gel (Laemmli, 1970). Gels were run at 200 V constant voltage in a Mini-Protean II electrophoresis cell (Bio-Rad). Native electrophoresis was realized as SDS-PAGE, except that SDS was absent. 2D-PAGE were realized as described by Boyer et al. (Boyer et al., 1993), except that ampholytes (4%) consisted of 90% ampholytes pH 3–10 and 10% ampholytes pH 5–7 (Bio-Rad). Proteins were stained with silver nitrate as described previously (Blum et al., 1987). The prestained markers containing phosphorylase B (142.9 kDa), serum albumin (97.2 kDa), ovalbumin (50 kDa), carbonic anhydrase (35.1 kDa), trypsin inhibitor (29.7 kDa), and lysozyme (21.9 kDa) were supplied by Bio-Rad.
In vivo labelling of conditioned medium protein
Leaf fragments were cultured as described for somatic embryogenesis and 35S-methionine was added 24 h before the end of each culture step (3 d of induction and 3 d or 7 d after the transfer of 4 d induced leaf fragments on expression medium). Culture media were harvested and protein was isolated as before. Proteins were separated by SDS-PAGE, the gel dried and exposed to ß-max film (Amersham).
Protein microsequencing
After SDS-PAGE, the gel was equilibrated in buffer consisted of 50 mM boric acid, 0.1% SDS, set to pH 8. Proteins were transferred overnight at 35V constant voltage onto a polyvinylidene difluoride membrane (PVDF: Bio-Rad) in a Mini-Protean II cell. After transfer, this membrane was rinsed and stained for 5 min in a solution containing 0.1% Naphthol Blue Black (w/v), 45% methanol (v/v) and 7% (v/v) acetic acid. Thereafter the membrane was rinsed with distilled water and dried in air. Bound proteins were digested in situ according to a modified Bauw et al. (Bauw et al., 1989) method. The band of interest, excised from the PVDF membrane, was firstly immersed in 500 µl of a 0.2% polyvinylpyrrolidone (PVP; 40 kDa) solution in methanol and stored at 4 °C. Electroblotted protein was digested at 37 °C for at least 4 h by adding 1 ml of 1 M CaCl2 and 1 ml of 1 mg ml-1 porcine trypsine solution. Resulting tryptic peptides were collected and membrane pieces were washed once with 100 µl of 80% (v/v) formic acid and twice with 100 µl of distilled water. All combined solutions were submitted to a reverse-phase HPLC using a HP 1050 Ti HPLC (Hewlett Packard), containing a reverse-phase C4 column (0.46x25 cm; Vydac Separations Group) equilibrated with 0.1% trifluoroacetic acid in water. Peptides were eluted on a Waters-Millipore HPLC apparatus with a linearly increasing gradient of acetonitrile (from 0% to 70%), with UV absorbance detection at 214 nm and peptide fractions were dried in a Speed Vac (Savant) concentrator and stored at -20 °C prior to sequence analysis. Automated peptide sequencing was performed using a 473A Applied Biosystems sequencer (Perkin Elmer) equipped with an on-line phenylthiohydantion amino acid-derivative analyser. Amino acid sequence comparisons were realized using the SWISS-PROT data bank.
Immunoblot assays and lectin labelling
After electrophoresis, electro-transfer was realized according to Helleboid et al. (Helleboid et al., 1998). Resulting membranes blocked overnight in TBS (25 mM TRIS-HCl, pH 7.4; 0.5 M NaCl) containing 2% PVP at room temperature, were exposed to primary serum (1 : 2000 dilution) in TBST (25 mM TRIS-HCl, pH 7.4; 0.5 M NaCl; 0.1% Triton X100) containing 1% PVP for 2 h. After three washes of 10 min in TBST, membranes were incubated for 2 h with alkaline phosphatase-conjugated-goat-anti-rabbit antibodies. Finally, after washes with TBST, alkaline phosphatase signal was developed using 0.03% nitroblue tetrazolium and 0.015% 5-bromo-chloro-3-indoyl phosphate in a solution 10 mM NaCHCO3 and 1 mM MgCl2 at pH 9.8. Alkaline-phosphatase lectin labellings were performed as described for immunoblots assays except that primary serum was replaced by alkaline phosphatase-conjugated-lectins (Amersham) and the second step with secondary antibodies was omitted.
32 kDa protein purification and chitinase enzyme assay
Conditioned medium of 11-d-old embryos cultures was collected, dialysed, concentrated, and loaded on an Econo-Pac cartridge (Bio-Rad). After elution of unbounded protein, a 0.1–1 M NaCl gradient was applied to the column and all the collected fractions were tested for the presence of the 32 kDa protein band after protein denaturing extraction. To test chitinase activity, proteins were firstly separated on a native polyacrylamide gel and this gel was overlayed by a glycol chitin gel according to de Jong et al. (de Jong et al., 1992). Chitinase activity was visible as a non-fluorescent dark band.
Quantification of protein and enzyme accumulation
For each experiment to compare protein bands or PR proteins accumulation, a quantification of detected labelling or specific signal was realized by scanning and comparing the signal intensity using a Sun Sparc work station.
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Results |
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Protein accumulation in conditioned embryo culture medium
Analysis by SDS-PAGE of the embryo-culture expression medium conditioned by leaf fragments of Cichorium clone ‘474’ after their transfer from the induction medium revealed the presence of three major bands of 25, 32 and 38 kDa. Total protein and the three major proteins were highly accumulated (respectively 4-, 5- and 2.5-fold) in the embryogenic hybrid culture medium (EHCM) compared to a non-embryogenic variety culture medium (NEVCM) (Fig. 1
). After staining with the periodic acid–Schiff reagent, positive signals were obtained for all three protein bands, suggesting that the 38 kDa, 32 kDa and 25 kDa proteins are glycoproteins (not shown, but see Helleboid et al., 1998).
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As these proteins were highly accumulated in the embryogenic hybrid culture medium, the time-course analysis of the accumulation of the 25, 32 and 38 kDa proteins has been compared. The proteins patterns extracted from equal amounts of conditioned media collected at different time points during the embryo culture were analysed. As shown in Fig. 2
, the major polypeptide bands appeared in the induction medium on day 3, whereas the 32 kDa band could be detected as early as day 1. The staining intensities of the bands in the protein fractions of 3-day-old induction medium have been compared with those of 3-d-old expression medium (day 4+3; note that at day 4 the leaf fragments were transferred to expression medium). It could be concluded that the accumulation of total protein, particularly the three described proteins, in the medium is subsequently higher during the expression step of somatic embryogenesis compared to the induction step.
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Using in vivo [35S]-methionine labelling experiments during 24 h of three culture steps (day 3, day 4+1 and day 4+7), the de novo synthesis of these three proteins was confirmed. The sequential accumulation of 38 kDa protein was 6- and 12-fold higher at the day 4+3 and day 4+7 stages compared to the day 3 stage. At the same time, 32 kDa protein accumulation was, respectively, 4- and 6-fold higher at the day 4+3 and day 4+7 stages compared to the day 3 stage (Fig. 3
). These results indicated that somatic embryogenesis in Cichorium ‘474’ leaf tissue is accompanied by the accumulation in the culture medium of proteins, especially the three major 38, 32 and 25 kDa bands. Moreover, their higher accumulation rate during the expression step, especially relevant for 38 kDa and 32 kDa protein bands was positively correlated with the expression step of somatic embryogenesis and so could be implicated in somatic embryos development. In such a way, it was decided to determine their identity. In a previous study, the 38 kDa band was identified initially as acidic ß-1,3-glucanases by microsequencing, serological relationship to other plant ß-1,3-glucanases and demonstrating its glucanase activity (Helleboid et al., 1998). To identify the 32 kDa and 25 kDa proteins a similar strategy was used.
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Identification of the 32 kDa protein band
Upon microsequencing, the amino acid sequences from two tryptic peptides of the 32 kDa protein band were unambiguously identified (Fig. 4A). The identified sequences, covering 23 amino acids, shared 82% identity with plant chitinases from tobacco (Lawton et al., 1992) and Arabidopsis (Samac et al., 1990). These results suggested that the protein (s) present in the 32 kDa protein band might be plant chitinase (s).
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In order to confirm the identity of the 32 kDa band as chitinase (s), Western blots were probed with polyclonal antibodies raised against chitinases from tobacco (Legrand et al., 1987
) and carrot (de Jong et al., 1995). However, neither of the antibodies seemed to recognize the 32 kDa protein band (data not shown). In a further attempt to identify if the 32 kDa protein band had chitinase activity, proteins in conditioned embryo-culture expression medium were fractionated on an Econo-PAQ cartridge column, and an aliquot of each fraction was analysed by SDS-PAGE. The 32 kDa protein band was found to be present as a single major protein in one fraction (Fig. 5A). Subsequently, the purified 32 kDa protein fraction was submitted to native PAGE and after electrophoresis the gel was overlayed with a glycol chitin gel to detect chitinase activity (de Jong et al., 1992). As shown on Fig. 5B, two dark bands, being the result of glycol chitin degradation, indicated the presence in the purified 32 kDa protein fraction of at least two proteins able to degrade the artificial chitinase substrate glycol chitin. Nevertheless, it was possible that this two protein band contained more than two protein cores, because enzymatic assays were realized on a monodimensionnal PAGE separation. For this reason, the 32 kDa purified band was submitted to a 2D-PAGE. This revealed the presence of three or four acidic proteins with slightly different isoelectric points (Fig. 5C). Unfortunately, it was not possible to test the chitinase activity of each of these proteins, because (i) chitinase activity needs a larger amount of protein, and (ii) 2D-PAGE denatures protein and could discard their potential chitinase activity.
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Taken together, the results obtained after microsequencing and the demonstration of the chitinase activity in the purified 32 kDa protein fraction indicate that at least two proteins of 32 kDa present in the medium of embryo cultures are chitinases.
Identification of the 25 kDa protein band
For the 25 kDa protein band, three tryptic peptides were sequenced (Fig. 4B
), and the sequences, 43 amino acids in total, shared 60% identity with tobacco osmotin-like (thaumatin) like proteins (Cornelissen et al., 1986; Takeda et al., 1991; Kumar and Spencer, 1992; Nelson et al., 1992), suggesting that the protein (s) in the 25 kDa protein band may be osmotin-like protein (s). An antiserum raised against tobacco osmotin-like protein (Stinzi et al., 1991) was used to test the putative identity of the 25 kDa protein (s). Upon immunoblotting, the antibodies recognized the 25 kDa protein band in conditioned embryo-culture expression medium (Fig. 6A). Unfortunately, no signals were obtained for the 25 kDa protein (s) after 2D-PAGE (data not shown), preventing a more detailed analysis of the protein (s) present in the 25 kDa protein band. Despite the lack of knowledge concerning the number of proteins present in the 25 kDa protein band, the results obtained after microsequencing and immunoblotting indicate the presence of at least one osmotin-like protein in the medium of Cichorium embryo cultures.
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As indicated above, the 25 kDa protein band reacted with DBA, a lectin that specifically binds to N-acetyl-galactosamine. To investigate this further, the lectin-affinity blotting was performed with a partly (gel-) purified 25 kDa protein fraction (Fig. 6B
). As shown in Fig. 6D, the DBA-AP conjugate bound to the 25 kDa protein on a Western blot, whereas in the presence of N-acetyl-galactosamine, this binding was prevented (Fig. 6E). It thus could be concluded that the glycoprotein (s) in the 25 kDa protein band contain N-acetyl-galactosamine in their carbohydrate moiety. Whether the osmotin-like protein (s) is (are) glycosylated could not be determined, because data concerning the number of proteins in the 25 kDa protein band were lacking.
Comparisons of 38, 32 and 25 kDa protein accumulation in embryogenic and non-embryogenic culture media
Previous results (Helleboid et al., 1998) and the results from this study suggested that the accumulation of proteins and particularly the 38 kDa, 32 kDa, and 25 kDa proteins in the culture medium during Cichorium ‘474’ somatic embryogenesis are, at least in part, caused by increased levels of ß-1,3-glucanases, chitinases and osmotin-like protein (s). As for the 32 kDa and 25 kDa protein bands, it was not possible unambiguously to identify each of the proteins present in the bands, however, and therefore this needed to be verified. Furthermore, ß-1,3-glucanases, chitinases and osmotin-like protein (s) are generally classified as pathogenesis or stress-related proteins in other plants (Linthorst, 1991). Since tissue culture conditions may be considered as a stress, particularly those applied to realize somatic embryogenesis in Cichorium, the accumulation of chitinases and osmotin-like proteins may be caused by the culture conditions per se and are not specifically involved in somatic embryogenesis. This was tested by comparison of the proteins accumulating in the culture medium of the Cichorium embryogenic hybrid with those in a non-embryogenic variety of Cichorium after applying embryo-culture conditions.
Proteins extracted from equal amounts of conditioned culture media (3-d-old embryo-culture induction medium, and 1-, 3-, and 7-d-old embryo culture expression medium) were used specifically to investigate the accumulation of the 38 kDa ß-1,3-glucanases, the 32 kDa chitinases and the 25 kDa osmotin-like proteins (s). To detect the 38 kDa ß-1,3-glucanases protein band, the specific antiserum P38-SH raised against these proteins (Helleboid et al., 1998) was used. As shown in Fig. 7A and B, immunoblots revealed an increase in the level of the 38 kDa ß-1,3-glucanases in the medium of both the embryogenic hybrid and the non-embryogenic variety of Cichorium. However, the level of the 38 kDa ß-1,3-glucanases accumulation was, respectively, 4- and 5-fold higher at day 4+3 and day 4+7 of embryogenic hybrid ‘474’ expression step compared to the same steps of the non-embryogenic variety. Moreover, while this level was, respectively, 5-, 10- and 12-fold higher during the expression step compared to the induction step of the embryogenic hybrid ‘474’, the increase was only up to 1.6-fold higher in the same conditions for the non-embryogenic variety. Nevertheless, ß-1,3-glucanases accumulation was 2-fold higher at day 3 of the induction step in NEVCM compared to EHCM (Fig. 7C).
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Since no significant signal was obtained with antibodies raised against carrot and tobacco chitinases, the accumulation of the 32 kDa chitinase band in culture media of the embryogenic hybrid and the non-embryogenic variety was monitored by the demonstration of chitinase activity in glycol chitin overlay gels. Results of these assays revealed an increase in chitinase activity, displayed by two dark bands, during both cultures (Fig. 8B
, C), similar to the 32 kDa purified protein band (Fig. 8A). Moreover, while both chitinase bands were found to have similarly accumulated during the induction step, their accumulation was 2-fold (day 4+1 and day 4+3) and 3-fold (day 4+7) higher for the embryogenic ‘474’ hybrid compared to the non-embryogenic variety during the expression step (Fig. 8D).
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For the 25 kDa protein band, proteins extracted from 3-d-old and 7-d-old embryo-culture expression media conditioned by the ‘474’ hybrid and the non-embryogenic variety, were analysed by immunoblotting using the antiserum raised against tobacco osmotin-like protein (Stinzi et al., 1991
). In the media of both cultures a 25 kDa protein band was detectable, but again the level to which protein (s) accumulated was 2-fold higher for the embryogenic ‘474’ hybrid (Fig. 9A, C) than for the non-embryogenic variety (Fig. 9B, C) during the expression step.
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These results indicate that direct somatic embryogenesis in Cichorium ‘474’ is accompanied by an increase in the level of 38 kDa ß-1,3-glucanases, 32 kDa chitinases and 25 kDa osmotin-like proteins in culture media. In view of their lower levels in media from cultures of a non-embryogenic Cichorium variety, only part of this increase can be explained by the culture conditions per se, suggesting that the accumulation of these three proteins in the expression medium could be correlated with the process of somatic embryogenesis.
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Discussion |
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Previously, 38 kDa proteins strongly accumulated in culture medium during the expression step of Cichorium ‘474’ hybrid somatic embryogenesis were identified as ß-1,3-glucanases (Helleboid et al., 1998
). In this study, two other protein bands strongly accumulated in culture medium of ‘474’ embryogenic hybrid have been identified as chitinases and osmotin-like-proteins. Therefore, three major extracellular SER proteins in Cichorium have been identified as proteins known to accumulate during pathogenesis in other plants. A higher accumulation of proteins and, particularly the three protein bands, was observed in the expression medium during Cichorium hybrid somatic embryogenesis when compared to the non-embryogenic variety cultured under similar conditions. In culture medium of the non-embryogenic variety they remained at a low level or slowly increased during the entire culture period.
PR proteins were originally identified as additional bands present in native or SDS-PAGE in TMV-infected leaves. Initial characterization revealed them to be low molecular weight compounds that can be extracted at low pH and appear rather resistant to various plant and commercially available proteases. In the absence of any known functions, the tobacco PR proteins were grouped, PR1 to PR5, solely on the basis of their electrophoretic mobility and immunological cross-reactivity. PR proteins, extracted from leaves intercellular fluids of tobacco (Jamet and Fritig, 1986), were found to be acidic and appear to be well adapted to function in this environment. Pathogenesis-related (PR) proteins have been defined as proteins coded for by the host plant, but induced by various pathogens (van Loon, 1990) as well as under stress situations such as wounding (Warner et al., 1992), hormones such as ethylene (Sessa et al., 1995), heat (Eckey-Kaltenbach et al., 1997) and also in developmental processes (Lotan et al., 1989). However, the high accumulation of PR proteins in the culture medium during Cichorium somatic embryogenesis is not likely to be the result of stressful conditions such as heat, hormone and osmoticum, in view of their very low accumulation levels when using a non-embryogenic variety in the same culture conditions. For the three types of PR protein, a similar accumulation rate can be observed. Indeed, these three proteins were barely accumulated during the induction step in both embryogenic and non-embryogenic genotypes. Further, accumulation rate rapidly increased in the embryogenic hybrid culture medium (until 8-fold higher after the transfer an expression medium compared to the induction step), whereas it only slowly increased (up to 2-fold at 7 d after the transfer compared to the induction step) over all the culture. For ß-1,3-glucanases, the rate is even higher for the embryogenic genotype during the induction step. This observation could be explained by a differential specific activation pattern for the different genotypes.
Besides their participation in the plant defence, ß-1,3-glucanases are likely to have functions in normal developmental processes. During pollen formation in tobacco, tetrads were shown to be surrounded by a callose wall (Worrall et al., 1992). Degradation of this callose wall was necessary to release fertile pollen into the anther locule (Worrall et al., 1992) and is probably performed by an anther-specific ß-1,3-glucanase (Bucciaglia and Smith, 1994), suggesting a specific role for ß-1,3- glucanases during pollen formation. During Cichorium somatic embryogenesis, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. The callosic deposition disappeared as embryos grew (Dubois et al, 1991). For this reason, in previous work it was hypothesized that the culture medium accumulated ß-1,3-glucanases (Helleboid et al., 1998) that may be responsible for the degradation of the callose in the cell wall surrounding the embryogenic cells, very similar as in pollen development (Bucciaglia and Smith, 1994). Other work on ß-1,3-glucanases and callose localization supports this hypothesis. Recently, cloning of ß-1,3-glucanases cDNAs expressed during Cichorium somatic embryogenesis and expression analysis support this hypothesis by demonstrating exclusive expression of a ß-1,3-glucanase CG1 clone during somatic embryogenesis process (unpublished results).
Moreover, this finding revealed the presence of at least two chitinase isoforms in embryogenic and non-embryogenic culture medium. These observations were similar to those of Kragh et al. (Kragh et al., 1996) who also observed the presence of different chitinases (EP3) isoforms in carrot embryogenic medium with apparently no correlation between the embryogenic capacity of a given cell line and the EP3 isoenzymes produced. In Cichorium, a rate of up to 8-fold in the culture medium accumulation of chitinase is demonstrated between the embryogenic and the non-embryogenic lines. The chitinase biochemical function resides in the ability of some plant chitinases partially to degrade fungal cell walls (Broekaert et al., 1988). However, several of the isolated plant chitinases do not possess antifungal activity in vitro (Woloshuk et al., 1991). Chitinases genes were also shown to be expressed in the absence of pathogens (de Jong et al., 1992; Kragh et al., 1991; Shinshi et al., 1987). Chitinases were also found in transmitting tissues of tomato flowers (Harikrishna et al., 1991). Therefore, it appears reasonable to assume that plant-produced chitinases may have other functions apart from their role in the defence mechanism. During carrot somatic embryogenesis, a secreted protein, that is responsible for the observed rescue of ts11 embryos arrested at the globular stage, was identified as an EP3 chitinase (de Jong et al., 1992). Morever, since the effect of chitinases was mimicked by Rhizobium-produced Nod factors, it was proposed that the chitinases are involved in the generation of signal molecules essential for embryogenesis in ts11 (de Jong et al., 1993). Further experiments revealed that these chitinases are not only necessary for ts11 temperature-sensitive mutant somatic embryogenesis rescue, but also seems to have a ‘nursing’ function as revealed by comparisons with zygotic embryogenesis (van Hengel et al., 1998). Sacco de Vries research team addressed the question of what the role of endochitinase is during somatic embryogenesis. They have shown that certain arabinogalactan proteins (AGPs) contain endochitinases cleavage sites. In addition, both EP3 endochitinases and AGPs that are present in immature carrot seeds, or are secreted in the medium of suspension-cultured cells can promote the formation of protoplast-derived somatic embryos (van Hengel, 1998). In Cichorium, large amounts AGPs were rercently detected in embryogenic culture medium. Moreover, as previously described for the three PR proteins, this accumulation is higher in EHCM than in NEVCM (Chapman, unpublished results). This observation suggests a possible involvement of the 32 kDa chitinases identified in this study in the cleavage of specific AGPs as previously described by van Hengel (1998) in carrot somatic embryogenesis.
These results suggest that both ß-1,3-glucanases and chitinases could have implications in the somatic embryogenesis process. This observation was also in accordance with Dong and Dunstan (Dong and Dunstan, 1997), who also reported the expression of ß-1,3-glucanases and chitinases cDNAs during the spruce somatic embryogenesis process.
Osmotin-like proteins are generally classed in the PR5 group. The first members were discovered in tobacco as TMV-induced protein (Cornelissen et al., 1986), and since these proteins showed high structural homologies with a sweet-tasting protein isolated from fruits of Thaumatococcus daniellii, they were therefore referred to as thaumatin-like (TL) proteins. Osmotin is a TL protein associated with osmotic adaptation and was associated with high NaCl concentrations and was water-stress induced (Singh et al., 1987, 1989). These proteins have antifungal activity against several fungi in vitro and induce the rupture of fungal hyphae at the growing tips (Vigers et al., 1991; Huynh et al., 1992). TL-proteins have been cloned from many plant species, including wheat (Rebmann et al., 1991), oats (Vigers et al., 1991), maize (Roberts and Selitrennikoff, 1990), tobacco (King et al., 1988) or potato (Pierpoint et al., 1990). Not all TL-proteins were found to be induced by pathogen infection or abiotic stresses, as a TL-protein was also found in maize seed as zeamatin where it seems to have antifungal properties (Richardson et al., 1987). Non-pathogenic and non-stress-inducible TL proteins was also found during flower formation (Neale et al., 1990) and in healthy Lupinus albus organs (Regalado and Ricardo, 1996). Nevertheless, in contrast to ß-1,3-glucanases and chitinases, no reports to date have been produced that suggest the implication of such proteins during the somatic embryogenesis process.
Numerous papers concerning the induction of PR proteins upon various stress conditions have appeared and there is no doubt that the Cichorium hybrid ‘474’ embryogenic culture conditions (see Materials and methods) generates some chemical and physical stresses such as cytokinins, wounding, heat (35 °C) and darkness. Moreover, if the PR protein accumulation in the culture medium was due to stressful culture conditions, it cannot explain the relative low level of these proteins in culture medium of the non-embryogenic variety as compared to that of the hybrid ‘474’. For these reasons, the conclusion is that PR proteins may be involved in somatic embryogenesis. As discussed previously, the 38 kDa Cichorium ß-1,3-glucanases could be involved in the degradation of callose, which was localized in walls surrounding the embryogenic cells (Dubois et al., 1991) and chitinase could have similar roles to the EP3 chitinases described by de Vries team during carrot somatic embryogenesis. On the other hand, there is actually no evidence for the implication of osmotin-like proteins in Cichorium somatic embryogenesis. Nevertheless, papers have already speculated on their possible development implication.
To conclude, during Cichorium somatic embryogenesis process, a high accumulation of proteins has been demonstrated, three of which are PR proteins. It will be interesting to separate the regulation pathways of the induction of PR proteins upon stress or development, but this way seems to be complex since limited information is available to date in order to analyse the complex regulatory network involving distinct or partially overlapping pathways.
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Acknowledgments |
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We thank Bernard Fritig (IBMP, Strasbourg, France) for a generous gift of antisera raised against tobacco chitinases and osmotin-like proteins. Many thanks to Sacco de Vries (University of Wageningen, The Netherlands) for helpful discussion and for providing antiserum raised against carrot chitinases and glycol-chitin reagent. Special thanks to Jean-Pierre de Cottignies (UMR 111, Laboratoire de Chimie Biologique, Université de Lille1) for the P38-SH antiserum production. This research was supported by a grant ‘Contrat Plan-Etat-Région' to the Laboratoire de Physiologie Cellulaire et Morphogenèse Végétales, by a post-doctoral fellowship of the Conseil Régional Nord-Pas de Calais to Theo Hendriks and by a Doctoral Fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche to Stéphane Helleboid.
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3 To whom correspondence should be addressed. Fax: +33 3 20337244. E-mail: hilbert{at}univ\|[hyphen]\|lille1.fr
4 Present address: Institut Pasteur de Lille, Unité de recherche sur les Lipoprotéines et l'Athérosclérose, U 325 INSERM, 1 rue du Professeur Calmette, BP 245, F-59019 Lille Cedex, France.
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Abbreviations |
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PR, pathogenesis-related; PVDF, polyvinylidene difluoride; PVP, polyvinylpyrrolidone; EHCM, embryogenic hybrid ‘474’ culture medium; NEVCM, non-embryogenic variety culture medium; TMV, tobacco mosaic virus; TL, thaumatin-like..
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