Journal of Experimental Botany, Vol. 52, No. 358, pp. 1143-1144,
May 1, 2001
© 2001 Oxford University Press
Gene Note |
Drought differentially affects expression of a PR-10 protein, in needles of maritime pine (Pinus pinaster Ait.) seedlings
Equipe de Génétique et Amélioration des Arbres Forestiers, INRA Pierroton, BP 45, F-33610 Cestas, France
Received 2 January 2001; Accepted 4 February 2001
Abstract
A full length cDNA encoding a PR-10 protein was isolated from maritime pine drought-stressed seedlings. The predicted protein contained 150 amino acids, has a molecular mass of 16.7 kDa and an isoelectric point of 5.28. The transcript level of PR-10 displayed a transient accumulation in needles of drought-stressed plants, and was not detectable in root and stem tissues.
Key words: Drought, Pinus pinaster, PR-10.
The pathogenesis related (PR) protein gene family, has been described as responding to biotic and abiotic stresses (e.g. cold, ozone, salt, or heavy metal), and to different hormones such as ethylene, cytokinin or salicilic acid (Pääkkönen et al., 1998
; Kitajima and Sato, 1999). The intracellular PR-10 subset was recently found in conifers as cold stress-regulated (Ekramoddoullah et al., 2000). The metabolic function of PR-10 was characterized with recombinant protein of Lupinus albus, as presenting a ribonuclease activity against several RNA preparations (Bantignies et al., 2000).
A cDNA fragment (528 bp) with homology to PR-10 was identified in a differential screen (cDNA-AFLP; Bachem et al., 1996) for genes of which the mRNA level was altered upon drought stress (C Dubos, unpublished results). 3'-end rapid amplification of cDNA (3' RACE system for rapid amplification of cDNA ends kit, Life technologies-Gibco BRL) using total drought-stressed needles RNA and a gene-specific 5' primer, was used to generate the cDNA fragment corresponding to the 3' end of this gene. The 5'-end sequence was obtained by PCR, using an oriented drought-stressed needle cDNA library (Uni-Zap® XR vector, Statagene) and the universal T7 primer, in combination with a gene-specific 3' primer. Both 5' and 3' sequences matched to the cDNA-AFLP fragment, and made it possible to obtained a full length cDNA (designated as Pp.ap11, accession AJ291767) of 857 bp.
Pp.ap11 has a putative open reading frame of 450 bp encoding a 150 amino acids protein. The protein presented a predicted molecular mass of 16.7 kDa and an isoelectric point of 5.28. Database sequence analysis using BlastX showed that Pp.ap11 was similar to the root cold-induced PR-10 protein of Picea glauca (AAF12812), with 75% and 86% of amino acids identity and similarity, respectively. Multiple alignment of amino acid sequences showed that Pp.ap11 shared the Bet v 1 conserved domain with other conifer PR-10 and ribonuclease class-2 (Gajhede et al., 1996). Bet v 1 contains a P-loop domain GxGGxG involved in the binding of phosphate group of RNA, that is more conserved in conifers (GDGGVG motif, Fig. 1) compared to the other PR-10 or ribonuclease class-2 proteins. The protein sequence of Pp.ap11 and the Picea glauca PR-10 (AAF12810) were shorter in their N-terminal compared to the other PR-10.
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The expression of the maritime pine PR-10 protein was examinated in hydroponicaly drought-stressed seedlings. After sterilization, Pinus pinaster seeds were placed on moistened filter paper for germination. Seedlings were then transfered to an aerated nutrient solution (Nguyen and Lamant, 1989
). After 3 weeks of growth, seedlings were placed in a drought-stressed medium (-0.45 MPa) using polyethylene glycol (PEG 3350) as osmoticum. The expression of Pp.ap11 was analysed on three tissues (needle, stem and root). While the transcript of this gene was detected in needle, no hybridization signal was found in root and stem, indicating that its over-expression was tissue specific (Fig. 2A). The absence of expression in root was also verified using RT-PCR, a more sensitive method.
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To characterize the maritime pine PR-10 further, the time-dependent expression of Pp.ap11 was analysed on needles of five groups of seedlings: one used as a control was raised in growing solution without PEG (-0.08 MPa) and the other were raised in four drought-stressed conditions obtained by increasing level of PEG (-0.15, -0.30, -0.45, and -0.60 MPa). A time-course expression analysis was performed considering five points: just prior stress application and 1, 10, 21, and 45 d after the treatment started (Fig. 2B
). Another group of unstressed plants was raised in the ground, and was used as a second control for each sampling date. Total RNA was used for RT-PCR experiments. Pp.ap11 was similarly expressed in the two control treatments showing that the hydroponic solution did not affect gene expression. It was gradually over-expressed in needles of all stressed conditions from 1–21 d and returned to a low level at 45 d. In addition to this transient over-expression, the transcript level was dose-stress dependent, increasing from -0.15 to -0.60 MPa. The over-expression of ribonuclease gene in salt-stressed barley leaves has been reported (Muramoto et al., 1999), and these authors suggest a remobilization of nucleotides or phosphate for the benefit of fresh organs. Interestingly, Pp.ap11 was always present at a low level in the controls, indicating a minimum expression level for this gene. Under drought stress, the expression of Pp.ap11 was enhanced, similarly to that described in barley (Muramoto et al., 1999), which could suggest the involvement of the maritime pine PR-10 ribonuclase in the transcript turnover.
Acknowledgments
CD is supported by fellowship from Ministère de la Recherche et de la Technologie. We acknowledge funding from the European Union (INCO-IC18-CT97-0200), Région Aquitaine and INRA (ECOGENE No. 92). We thank Raùl Herrera for helpful comments.
Notes
1 To whom correspondence should be addressed. Fax:+33557979088. E-mail: plomion{at}pierroton.inra.fr 
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