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Gene expression analysis for identifying candidate genes for
controlling oxidative stress in rubber tree
J. Leclercq
1, V. Gébelin
1, M. Chabaud
1, M. Rio
1, G.Oliver
1, H. Chrestin
2and P. Montoro
1*1
UMR DAP, Department BIOS, CIRAD, TA A-96 / 03, Avenue Agropolis, 34 398 Montpellier Cedex 5, France.
2
UMR CLIFA, IRD, University of Mahidol, Rama 6 Road, Bangkok, Thailand
ABSTRACT
Hevea brasiliensis is the main source of natural rubber which is biosynthesized in latex
cells. The high metabolic productivity required for latex regeneration after each tapping can be enhanced by ethylene application, which optimizes the production potential in rubber tree. However, excessive metabolism activation can lead to Tapping Panel Dryness (TPD). Expression analyses of several genes involved in the reactive oxygen species (ROS) scavenging systems have been studied in healthy trees, TPD trees and also in young budded plants. Here, we presented the expression patterns obtained by semi-quantitative and real time RT-PCR of genes involved in ascorbate-glutathione cycle. Three genotypes (PB260, PB217 and RRIM600) with contrasting metabolism have been selected at the immature stage for this study. Their expression patterns have been monitored along the day, and in response to both ethylene stimulation and wounding treatments.
Keyword: Hevea brasiliensis, gene expression, genetic transformation, oxidative burst 1. Introduction
Hevea brasiliensis is the main source of natural rubber which is biosynthesized in latex
cells. Ethephon application can enhance the biosynthesis activity required for latex regeneration after each tapping, optimizing the yield potential of rubber tree. However, a good management of both tapping frequencies and ethephon applications is required for avoiding excessive metabolic activation which can lead to Tapping Panel Dryness (TPD). This physiological disorder is a consequence of an oxidative stress in the latex cells, leading to membrane damages, flocculation of rubber particles and plugging of the latex vessels (Chrestin, 1984, Chrestin 1989). Various systems that keep active oxygen under control exist in plants. Many antioxidant enzymes catalyze redox reactions, many of which rely on electrons supplied by reductants of low molecular weight such as ascorbate and glutathione (Noctor and Foyer, 1998). Many genes involved in detoxifying the reactive oxygen species (ROS) have been isolated in rubber tree (Sookmark, 2005; Leclercq and Montoro, unpublished data). Expression analyses of many genes involved in detoxifying the reactive oxygen species have been studied in healthy trees, TPD trees (Sookmark, unpublished data) and also in young budded plants. In our study, young budded plants from three genotypes (PB 260, PB 217 and RRIM 600) with contrasting metabolism activity and response to
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ethylene stimulation have been selected for studying the expression of seven genes such as
CuZnSOD, MnSOD, catalase, APX1 and 2, GCL cytosolic and chloroplastic isoforms, firstly
by semi-quantitative PCR on PB260 clone and secondly by real-time PCR on PB260, PB217 and RRIM600 clones.
2. Materials and Methods
Plant materials: Young budded plants from three genotypes (PB 260, PB 217 and RRIM 600) coming from Sembawa station (Indonesia) and CIRAD (French Guyana) have been checked for clonal conformity and grown in suitable conditions in Montpellier. Plants with one growth unit were then treated either with 1 µL.L-1 ethylene in hermetical boxes during 4 hours, or wounded on leaves and bark for 15 minutes and 4 hours. Untreated plants were also collected at 8:00 am, 12:00 am and 4:00 pm the same day to measure the impact of photosynthesis. For abiotic stress treatments, one plant has been subjected to drought (9 weeks without water), to light stress (24 hours under 860 µmol.m2s-1 and control plant under 180 µmol.m2s-1), to a cold stress (between 8-15°C for one night) and to a 3 day flooding. RNA extraction from leaves: Total RNAs were extracted as described in Morcillo et al. (2006). cDNA synthesis were performed as described in Leclercq et al. (2008). Expression analyses were done by semi-quantitative RT-PCR (Leclercq et al. 2008) and by real time PCR (LightCycler 480, Roche, Germany) using HbActine as an internal control.
3. Results and discussion
3.1 Expression analysis by semi-quantitative RT-PCR on clone PB260
As shown in Table 1, only CuZnSOD responded to wounding, ethylene, light and drought treatments. In response to wounding and ethylene treatments, the majority of the genes responded positively.
Table 1: Gene expression analysis in PB260 clone by semi-quantitative RT-PCR during the day, in response to
several abiotic stress.
Photosynthesis Wounding Ethylene 4h Light Drought Cold stress Flooding
GCL chl = ++ + = = = - GCL cyto = ++ ++ = = = = CuZnSOD = ++ ++ ++ ++ = = MnSOD + + = = = = = CAT ++ + = + - = = APX1 +++ + + = - + - APX2 = + + = - + -
3.2 Expression analysis by real time RT-PCR on clone PB260, PB217 and RRIM600 In Table 2, are shown the expression patterns obtained by real-time RT-PCR for clone PB260, PB217 and RRIM600. All the genes tested were regulated during the day excepted for GCL cytosolic for which transcripts could not be detected. In response to ethylene and wounding, the results obtained by real time RT-PCR are slightly different from the one obtained by semi-quantitative RT-PCR. Preliminary comparative analysis of the behaviour of three clones showed that PB260 and PB217 had the same behaviour in response to ethylene, different from RRIM600. By contrast, RRIM600 and PB217 had the same behaviour during the day, different from PB260.
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Table2: Gene expression analysis in PB260, PB217 and RRIM 600 clones by real-time RT-PCR during the
day, in response to ethylene and wounding treatments in young budded plant.
Gene Treatment PB260 PB217 RRIM600
Photosynthesis + ++ + Ethylene + + -- CuZnSOD Wounding - -- nd Photosynthesis + ++ ++ Ethylene = - + MnSOD Wounding + + nd Photosynthesis +++ + + Ethylene - - = Catalase Wounding + - nd Photosynthesis + + +++ Ethylene = - = APX1 Wounding + - - -transient nd Photosynthesis +++++ ++++ ++++ Ethylene - - - - - - - - - -- APX2 Wounding - - - -- - - - -- nd
Photosynthesis Not detected Not detected Not detected
Ethylene +++ + +
GCL cytosolic
Wounding - - - - transient nd
3. Conclusions
This comparative expression analysis will be soon completed with 2 more biological repetitions. Hopefully, this approach will allow the identification of molecular markers for early discriminating high yielding clones. New candidate genes involved in oxidative burst tolerance will be also identified and introduced by genetic transformation in rubber tree for functional analysis.
Acknowledgement: This work was supported by the Institut Français pour le Caoutchouc. 4. References
- Chrestin, H., Bangrantz, J., d’Auzac, J., and Jacob, J.L.( 1984) Role of the lutoidic tonoplast in the
senescence and degeneration of the laticifer of Hevea brasiliensis. Zeitschrift-für-Pflanzenphysiologie. 114 (3), 261-268.
- Chrestin, H. (1989) Biochemical aspects of bark dryness induced by overstimulation of rubber trees xith
Ethrel. In: d’Auzac J., Jacob J.L., Chrestin H. editors. Physiology of rubber tree latex. Boca Raton (FL): CRC Press, 1989.
- Leclercq, J., Fliegmann, J., Tellström, V., Niebel, A., Cullimore, J.V., Niehaus, K., Küster, H., Ebel, J. and Mithöfer, A. (2008) Identification of a multigene family encoding putative b-glucan-binding
proteins in Medicago truncatula. J Plant Physiol, 165(7):766-76.
- Morcillo, F., Gagneur, C., Adam, H., Richaud, F., Singh, R., Cheah, S-C., Rival, A., Duval, Y., and Tregear J.W. (2006) Somaclonal variation in micropropagated oil palm. Characterization of two novel
genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin.Tree Physiology, 26:585–594.
- Noctor, G. and Foyer, C. (1998) Ascorbate and glutathione: Keeping active oxygen under control.Annual
Review of Plant Physiology and Plant Molecular Biology 49: 249-279.
- Sookmark, U. (2005) International Workshop on Tapping Panel Dryness, RRII, Kottayam, , Kerala, India,
