Characterization of cDNAs encoding two glycine-rich proteins in
chickpea (
Cicer arietinum
L.): accumulation in response to fungal
infection and other stress factors
Holger Cornels
a, Yuki Ichinose
b, Wolfgang Barz
a,*
aInstitut fu¨r Biochemie und Biotechnologie der Pflanzen,Westfa¨lische Wilhelms-Uni6ersita¨t Mu¨nster,Hindenburgplatz55,
48143Mu¨nster,Germany
bOkayama Uni6ersity,Graduate School of Natural Science and Technology,Tsushima-naka1-1-1,Okayama700,Japan Received 15 September 1999; received in revised form 27 December 1999; accepted 27 December 1999
Abstract
In chickpea plants infected with the pathogenic fungus Ascochyta rabiei [Pass.] Labr. several mRNAs for two glycine-rich proteins (GRPs) were identified by differential cDNA screening. The main part of the deduced amino acid sequences of the 14.6 kD GRP1 and the larger GRP2 consists of glycine-rich repetitive elements essentially as found for GRPs in other plants. Tyrosine residues in conserved positions inside these repetitive motifs suggest an involvement of the GRPs in a polymerization process by oxidative cross-linking, i.e. cell wall fortification. Both GRP transcripts are induced by infection with A. rabiei, showing a maximum of expression 5 days post infection. Wounding of leaves and the stress of water treatment (performed as a control) also seem to induce the accumulation of GRP transcripts. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Chickpea; Cicer arietinum; Fungal infection; Glycine-rich proteins; GRPs; Stress
www.elsevier.com/locate/plantsci
1. Introduction
Glycine-rich proteins (GRPs) are a class of sim-ple-structured proteins, often consisting of repeti-tive amino acid patterns with a high glycine content. They resemble animal keratins or silk fibronins, i.e. proteins that are characterized by both strength and flexibility. GRPs were first de-scribed by Condit and Meagher for petunia in 1986 [1]. Until now, GRPs and/or their corre-sponding genes have been identified in a variety of plants including both monocotyles and dicotyles [2]. Their specific function is still a topic of discus-sion but most of them are thought to be structural cell wall components like HRGPs (hydroxypro-line-rich glycoproteins) or PRPs (pro(hydroxypro-line-rich proteins). Glycine-rich proteins were found mainly in the vascular tissue [3,4]. For some GRPs the
localization in the cell walls of vascular elements has been shown [5]. In addition to developmental regulation GRPs seem to be expressed in response to a variety of abiotic stress factors, including cold [6 – 8], water stress [7,9] and wounding [10]. GRPs also seem to play a role in plant – microbe-interac-tions: Their induction after bacterial [11], viral [12 – 14] and fungal [8] infection has been reported. Here we describe the isolation and characteriza-tion of cDNAs encoding two GRPs which accu-mulate in chickpea (Cicer arietinum L.) plants in response to fungal infection and other stress factors.
2. Materials and methods
2.1. Library construction and selection of
cDNA-clones
In order to identify genes involved in the de-* Corresponding author. Tel.: +49-251-8324790; fax: +
49-251-832837.
E-mail address:[email protected] (W. Barz)
fense mechanisms of chickpea against the infection with the fungus Ascochyta rabiei [Pass.] Labr. (cause of the ‘Ascochyta blight’ disease), two cDNA libraries were constructed from poly(A)RNA extracted from 18-day-old chickpea plants (resistant cultivar ILC 3279) 4 days after infection with spore-supensions of Ascochyta
rabiei cultivar XXI or after treatment of plants
with sterile water, respectively [15]. These libraries were compared by differential dot-blot hybridization with radioactively-labelled probes constructed from chickpea cDNA. Clones that showed significant differences in signal intensity when hybridized to the two cDNA libraries where transfected into E. coli for further investigation and sequencing.
2.2. Growth, infection and wounding of chickpea
plants
To induce germination, chickpea seeds (resis-tant cultivar ILC 3279) were watered for 12 h and kept in darkness for 2 days at high humid-ity. Seedlings then were planted in a standard soil – sand mixture (2:1) and grown for two weeks (12 h light/darkness, 20°C). On the 14th day of growth the plants were either sprayed with a suspension of Asochyta rabiei cultivar XXI spores (106 spores/ml, 500 ml (5×105
spores)/plant) or treated with sterile water (con-trol). After the treatment the plants were kept at high humidity to allow the fungal spores to ger-minate.
Plants were wounded at the same developmen-tal stage when infection and water-treatment were performed (14 day old). All fully developed leaves of the experimental plants were cut two times, avoiding to wound the rachis. Those plants were kept separated from the infected and water-treated plants to avoid unwanted interfer-ence of effects such as ethylene signalling.
2.3. Northern-analysis
Total RNA was isolated from 0.5 g of chick-pea leaves (fresh weight) using the ‘RNAqueous-MIDI™-Kit’ (Ambion, USA). Concentration of RNA was determined by UV absorbance. The RNA was seperated by gelelectrophoresis (20 mg RNA per lane, 1% agarose in MOPS-buffer, 12.5% Formalin (37% formaldehyde)) and
blot-ted to an uncharged nylon-membrane (Qiagen, Germany) by alkaline transfer [16]. The RNA was heat-fixed at 80°C for 30 min.
Visualization of the seperated rRNA by ethidi-umbromide- (gel) and methyleneblue-staining (membrane) were performed as a loading con-trol.
T7-RNA polymerase (‘RNA Transcription Kit’, Stratagene, USA) was used to construct digoxigenin-labelled antisense RNA probes (DIG RNA Labelling Mix, Boehringer, Germany) by in vitro transcription of the GRP-encoding cDNA clones.
Hybridization was performed under highly stringent conditions (68°C, 12 – 18 h, buffer: 5×
SSC, 0.02% SDS, 0.1% laurylsarcosine, 2% Blocking-Reagent (Boehringer, Germany), 50% formamide). Unspecifically bound probe was re-moved by two washes at room temperature (2×
SSC, 0.1% SDS, 15 min) followed by two washes at 68°C (0.1×SSC, 0.1% SDS, 20 min).
Digoxigenin was immuno-localized with ‘Anti-DIG-AP’ and the substrate ‘CDPstar™’ (Boehringer, Germany) according to the manu-facturers instructions. Chemiluminescence result-ing from the processresult-ing of the substrate by the alkaline phosphatase was visualized by exposure of the membranes to X-ray films.
3. Results
By differential hybridization we found over 100 cDNA clones that appeared to be increased or decreased in the level of expression in re-sponse to infection. These clones were named ‘INR’ (infection responding) [15]. By sequencing and comparison to databases 14 clones with sig-nificant homologies to glycine-rich proteins were identified, mostly among those earlier classified as ‘increased expression’ after infection. Eleven of these cDNAs encode for a GRP of 148 amino acids with a glycine content of 36%, among them three full-length clones (INR5, INR20, INR41). The deduced 14.6 kD protein was named GRP1. The three other clones contain an open reading frame for a larger GRP of more than 208 amino acids (GRP2, \20 kD) with an
cAJ012829) are presented in Fig. 1. A theoreti-cal pI of 5.78 was computed for GRP1. Com-puter aided prediction of the secondary structure of both proteins lead to no significant results, although the repetitive amino acid pattern (see below) suggests a regular structure.
Both proteins show the same basic pattern: a N-terminal region with structural similarities to putative cell wall signal peptides of GRPs from other plants [11,12] (37 amino acids in GRP1) is followed by a region of glycine-rich repeats. The similar structure of the two proteins and their repetitive elements is shown in Fig. 1. The pre-dominant motif of the repetitive elements in both proteins is GGGNYGX, where in most cases, X is asparagine or histidine. GRP1 contains ten re-peats of the GGGNYGX element whereas GRP2 has 16 repetitions of this motif resulting in the larger size of GRP2. Besides the main repetitive element both GRP sequences also include some smaller glycine-containing motifs (e.g. GGH,
GXN). Similar repetitive elements are common among GRPs in other plants [4,17,18].
Database comparison of the chickpea GRP se-quences revealed high homologies to a large number of GRPs from other plants (i.e. bean, tomato, tobacco, rice), especially several
Medicago species. The N-terminal region of
GRP1 shows a similarity of over 80% to the N-terminus of the protein MFSTRESS, a GRP
from Medicago falcata L. induced by
environ-mental stress [18] (Fig. 2). Overall similarity of the GRPs from the two members of the Fabacea family is 62%.
In order to verify the inducibility of the GRP mRNAs northern-hybridizations were performed using digoxigenin-labelled RNA-probes con-structed from the GRP cDNA-clones INR20 (representing the GRP1 open reading frame flanked by 19 bp on the 5%-end and 200 bp of non-coding region on the 3%-end) and INR51 (a GRP2 cDNA clone that is 29 bp shorter on the
Fig. 1. Repetitive amino acid structure of the two chickpea GRPs. (A) Deduced amino acid sequence of GRP1; (B) deduced amino acid sequence of GRP2.Bold=main repetitive element (GGGNYGX).
Fig. 3. Results of northern-hybridizations with digoxigenin-labelled probes constructed from INR20 (GRP1) and INR51 (GRP2, 17 bp shorter than INR23 on the 5%-end). Dpi, days post infection/water treatment; 20 mg total RNA/lane; detection of digoxigenin with Anti-DIG-AP and chemiluminescence substrate CDPstar®(Boehringer); control plants were sprayed with sterile
water.
Fig. 4. Results of comparative northern-hybridizations with digoxigenin-labelled probes constructed from INR20 (GRP1) and INR51 (GRP2) using total RNA from untreated, water-treated, wounded and infected plants. dpi=days post infection/other treatment; 20 mg total RNA/lane; detetection of digoxigenin with Anti-DIG-AP and chemiluminescence substrate CDPstar®
(Boehringer).
5%-end but otherwise identical to INR23). To test the specifity of the probes a variety of sequenced and identified chickpea cDNA clones were sepa-rated by gelelectrophoresis along with the GRP cDNAs and blotted to a nylon membrane. Upon hybridization to the probes used only the GRP cDNAs showed signals, whereas no hybridization to any other cDNA could be observed (data not shown). The northern results are shown in Fig. 3. A cross-hybridization to both GRP transcripts occurs, especially when using the larger probe constructed from INR51 for hybridization. Nev-ertheless, a strong and transient induction with a maximum 5 days post infection (dpi) was found for both GRPs. The smaller GRP1 mRNA (transcript size about 0.8 kb) appears to respond faster and accumulates to higher levels than the larger GRP2 transcript (about 1 kb). Such re-sults are qualitatively reproducible, although in different kinetics slight quantitative differences especially in the controls could be observed (as
for probe INR51 in Figs. 3 and 4). Such differ-ences in the level of expression in the controls may result from developmental regulation or other stress factors (see Section 4).
signals in the untreated plants may be due to developmental regulation of the GRPs.
4. Discussion
Regarding the amino acid sequences of the two GRPs, it is noteworthy that each main repetitive motif in both proteins contains the amino acid tyrosine in a conserved position. The phenolic rings of this acid are the putative essential sites for oxidative cross-linking of structural cell wall proteins like HRGPs (hydroxyproline-rich glyco-proteins) or GRPs during incorporation into the complex network of phenolic substances of the cell wall [3], maybe as part of a cell wall fortification process. In chickpea cell cultures, the insolubiliza-tion of two cell-wall proteins (a putative HRGP and a proline-rich protein (PRP)) after elicitor-in-duced oxidative burst has been demonstrated [19]. Because of the comparatively late maximum of expression (5 dpi) it is not very likely that the GRPs in chickpea are involved in the direct de-fense against infection with A. rabiei. This and the inducibility by wounding suggest that they may rather play a role in a more general stress-response system, for example as part of a cell wall repair mechanism (as suggested for bean and soybean in reference [5]). It also appears possible that they are responsible for the more effective sealing of vascu-lar elements during the late phases of A. rabiei
growth in the apoplastic space [20].
Gene activation as shown for GRP1 and GRP2 in the control plants sprayed with water (Figs. 3 and 4), which especially in case of GRP2 (probe INR51) led to an induction nearly as high as triggered by infection or wounding, most likely results from multiple stress factors which are difficult to demonstrate in detail. However, the effects of such ‘manipulation stress’ must be taken in consideration in related studies.
Acknowledgements
Financial support by Deutsche Forschungsge-meinschaft and Fonds der chemischen Industrie (to W. Barz) is gratefully acknowledged. Y. Ichi-nose was a recipient of an Alexander-von-Hum-boldt fellowship and supported by Japan Society for Promotion of Science.
References
[1] C.M. Condit, R.B. Meagher, A gene encoding a novel glycine-rich structural protein of petunia, Nature 323 (1986) 178 – 181.
[2] S.H. Cheng, B. Keller, C.M. Condit, Common occurence of homologues of petunia glycine-rich protein-1 among plants, Plant Mol. Biol. 31 (1996) 163 – 168.
[3] G.I. Cassab, Plant cell wall proteins, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 281 – 309.
[4] A.M. Showalter, Structure and function of plant cell wall proteins, Plant Cell 5 (1993) 9 – 23.
[5] U. Ryser, M. Schorderet, G.F. Zhao, D. Studer, K. Ruel, G. Hauf, B. Keller, Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein, Plant J. 12 (1) (1997) 97 – 111.
[6] D.P. Horvath, P.A. Olson, Cloning and characterization of cold-regulated glycine-rich RNA-binding protein genes from leafy spurge (Euphorbia esula L.) and com-parison to heterologous genomic clones, Plant Mol. Biol. 38 (1999) 531 – 538.
[7] S. Laberge, Y. Castonguay, L.P. Vezina, New cold- and drought-regulated gene from Medicago sati6a, Plant
Physiol. 101 (1993) 1411 – 1412.
[8] A. Molina, M. Mena, P. Carbonero, F. Garcı´a-Olmedo, Differential expression of pathogresponsive genes en-coding two types of glycine-rich proteins in barley, Plant Mol. Biol. 33 (1997) 803 – 810.
[9] J. Go´mez, D. Sanchez-Martinez, V. Stiefel, J. Rigau, P. Puigdome`nech, M. Page`s, A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich protein, Nature 334 (1988) 262 – 264. [10] E. Yasuda, H. Ebinuma, H. Wabiko, A novel
glycine-rich/hydrophobic 16 kDa polypeptide gene from to-bacco: similarity to proline-rich protein genes and its wound-inducible and developmentally regulated expres-sion, Plant Mol. Biol. 33 (1997) 667 – 678.
[11] K. Pawlowski, P. Twigg, S. Dobritsa, C. Guan, B.C. Mullin, A nodule-specific gene family fromAlnus gluti
-nosa encodes glycine- and histidine-rich proteins ex-pressed in the early stages of actinorhizal nodule development, Mol. Plant-Microbe Interact. 10 (5) (1997) 656 – 664.
[12] R.X. Fang, Z. Pang, D.M. Gao, K.Q. Mang, N.H. Chua, cDNA sequences of a virus-inducible, glycine-rich protein gene from rice, Plant Mol. Biol. 17 (1991) 1235 – 1257.
[13] S.M.S. Naqvi, K.S. Park, S.Y. Yi, H.W. Lee, S.H. Bok, D. Choi, A glycine-rich RNA-binding protein gene is differentially expressed during acute hypersensitive re-sponse following Tobacco Mosaic Virus infection in tobacco, Plant Mol. Biol. 37 (1998) 571 – 576.
[14] J.A. Van Kan, B.J.C. Cornelissen, J.F. Bol, A virus-in-ducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene, Mol. Plant-Microbe Interact. 1 (1988) 107 – 112.
expressed in Ascochyta rabiei-inoculated chick-pea plants and elicited cell cultures as detected by differential cDNA-hybridization, Z. Naturforsch., in press.
[16] P. Chomzynski, One-hour downward alkaline capillary transfer for blotting of DNA and RNA, Anal. Biochem. 201 (1) (1992) 134 – 139.
[17] M. Jose`, P. Puigdome`nech, Structure and expression of genes coding for structural proteins of the plant cell wall, New Phytol. 125 (1993) 259 – 282.
[18] M. Luo, L. Lin, R.D. Hill, S.S. Mohapatra, Primary
structure of an environmental stress and abscisic acid-inducible alfalfa protein, Plant Mol. Biol. 17 (1991) 1267 – 1269.
[19] O. Otte, W. Barz, The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubiliza-tion of two cell wall structural proteins, Planta 200 (2) (1996) 238 – 246.
[20] G. Ko¨hler, C. Linkert, W. Barz, Infection Studies of
Cicer arietinum(L.) with GUS-(E.colib-glucuronidase) transformed Ascochyta rabiei Strains, J. Phytopathol. 143 (1995) 589 – 595.
