JXB Advance Access originally published online on June 18, 2004
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Journal of Experimental Botany, Vol. 55, No. 402, pp. 1597-1599, July 2004
Journal of Experimental Botany, Vol. 55, No. 402, © Society for Experimental Biology 2004; all rights reserved
GENE NOTES |
A Pinus radiata AAA-ATPase, the expression of which increases with tree ageing
1Laboratorio Fisiología Vegetal, Departamento BOS, Facultad de Biología, Universidad de Oviedo, C/ Catedrático Rodrigo Uría s/n. E-33071, Oviedo, Spain
2Instituto de Biotecnología de Asturias (asociado al CSIC), E-33071, Oviedo, Spain
3Centro Nacional de Investigaciones Oncológicas, Madrid, Spain
4Laboratorio Genética Molecular de Plantas, Centro Nacional de E-28049 Madrid, Spain
* To whom correspondence should be addressed at: Universidad de Oviedo. Fax: +34 98 510 48 67. E-mail: mjcanal{at}correo.uniovi.es
Received 7 April 2004; Accepted 14 April 2004
Abstract
Age-dependent changes in gene expression profiles were studied in vegetative Pinus radiata buds by means of differential display. Among several candidate cDNAs, a 327 bp fragment that shows high homology with an Arabidopsis thaliana 20S proteasome ATPase designated RPT5a was found. Northern hybridization confirmed that the accumulation of this transcript increases with tree ageing, suggesting a possible role of this AAA-ATPase gene in development-related specific proteolysis.
Key words: Ageing, ATPase, expression, Pinus radiata, proteasome, proteolysis, RPT5a
Ageing is a key process in the life cycle of many forestry species, mainly because of its impact on selected plant production. This is the case in radiata pine (Pinus radiata D. Don), which is one of the most important species in terms of annual wood production. Radiata pine plantations produce timber much faster than natural forests, with an estimated annual roundwood production of 370 million m3, which represents almost 25% of the world's production. Most tree characteristics are only expressed definitively after a phase-change, but ageing always implies the loss of some morphogenic potentials, such as rooting ability. That is why breeding programmes are hampered by having to select adult material for later vegetative propagation whereas clonal multiplication is a juvenile-specific ability. It is therefore necessary to improve rejuvenation techniques that allow the transient recovery of rooting potential in adult materials, and also the development of molecular markers of ageing, which would be useful as indicators of morphogenic ability. Some indicators, based on epigenetic parameters such as DNA methylation, have been reported (Fraga et al., 2002
), but little is known about the genetic basis of tree development related to ageing.
One of the characteristic events associated with ageing and senescence is protein degradation. While most unspecific proteolysis is carried out by the cytosolic and vacuolar proteases (Callis, 1995), proteasomes are mainly involved in the degradation of specific proteins, especially messenger proteins and transcription factors such as auxin and cytokinin signalling proteins (Ramos et al., 2001; Smalle et al., 2002), constituting a key point in gene expression modulation. The proteolytic substrates for proteasome degradation need to be ubiquitinated, with the exception of ornithine decarboxylase (Murakami et al., 2000). The tubular 20S proteasome core has several catalytic activities, including threonine protease and peptidyl glutamyl hydrolase (Orlowski and Wilk, 2000), and it can associate with up to two regulatory 19S subunits. These regulatory complexes include six ATPases and at least 11 non-ATPase subunits, that contribute to the unfolding and exposure of substrates to the catalytic nucleus. One of these AAA-ATPase proteins is reported in the present study as an age-dependent expression candidate in Pinus radiata that could then be proposed as a potential marker of ageing.
The ageing-dependent variations in radiata pine (Pinus radiata D. Don) gene expression profiles were studied using a differential display method (Delta Differential Display Kit, Clontech), comparing changes in mRNA levels in the buds of juvenile and adult material. RNA was isolated by LiCl precipitation (Dong and Dunstan, 1996). Vegetative buds located in non-flowering basal branches (epicormic shoots) that developed from the main stem the year before (ES) were collected and considered as juvenile material. The relative juvenility (in terms of rooting ability, cell division rate, and in vitro responses) of epicormic and basal shoots has been widely reported in forestry species.
In vitro tissue culture lines established from basal shoots maintained a noticeably higher rooting capacity (over 90%) than those established from apical branches of the same adult trees that showed rooting percentages under 10%. Moreover, the physiological differences between basal and apical materials in terms of IAA and polyamine levels are remarkable, being lower in the proliferating lines established from juvenile-like basal shoot materials (Ballester et al., 1999). Pinus radiata epicormic shoot buds were compared with two kinds of adult buds: vegetative buds located in non-flowering branches at the mid-region of the main stem (A1) and vegetative buds located in flowering verticils (A2). Both types of adult branches had developed normally from the main stem several years ago. Plant material was collected in February, and the analyses were restricted to the apical 1.5 cm of buds with a similar size and developmental state.
Among several putative differential cDNA fragments, a 327 bp cDNA, designed as Prß288, was obtained from the amplification with the following combination: forward primer 5'-ATTAACCCTCACTAAATCGGTCATAG-3' and reverse primer 5'-CATTATGCTGAGTGATATCTTTTTTTTTGC-3' using advantage KlenTaq Polymerase Mix (50x)TM.
The purified PCR fragment was cloned into pGEM-T Easy vector (Promega) and then automatically sequenced using T7 and SP6 RNA polymerase promoters. The translated blast of the cDNA sequence revealed a 95% amino acid identity with the carboxyl end of the Arabidopsis thaliana 19S proteasome RPT5a ATPase. This high homology is shared with the protease regulatory subunit 6a homologue of a number of Angiosperms (Fig. 1). The complete cDNA sequence is now under determination by 5'- and 3'-RACE.
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The expression of Prß288 in different buds was monitored by northern hybridization. A single-strand [
-32P]CTP-labelled riboprobe was synthesized using T7 and SP6 transcription starting sites on Prß288-pGEM®-T Easy construction. The highest mRNA accumulation was observed in mature buds (A1 and A2 buds), specially in adult vegetative buds of non-flowering branches. By contrast, juvenile-like buds from the epicormic shoots showed much lower expression levels (Fig. 2). The partial recovery of some shoot apex cellular and physiological juvenile-like characteristics (increases in rates of growth and cell division, RNA and protein synthesis) has been widely reported (Lyndon, 1998). The higher Prß288 accumulation in A1 buds than in A2 buds could be consistent with these phase-change related changes.
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Although there is previous evidence of variation in the expression and activity of some proteasome components associated with stress and starvation responses, senescence, and cellular differentiation (Baharami and Gray, 1999
; Basset et al., 2002; Ingvardsen and Veierskov, 2001), this is the first report of a proteasome-related component that is preferentially expressed in adult material. These results are consistent with other reports of proteasomes being implicated in developmental progression (Girod et al., 1999). These ageing-related gene-expression changes are being now validated in different-aged selected P. radiata offspring.
Some proteasome elements have been reported to be implicated in the modulation of auxin and cytokinin signalling pathways (Ramos et al., 2001; Smalle et al., 2002) through the degradation of Aux/IAA and RPN12 proteins, respectively. The results reported in this paper could therefore represent the first suggestion of a possible implication of proteasomes in the modulation of growth and ageing in woody species. Future experiments are centred on the study of specific DNA methylation–demethylation of the promoter and first exon as a possible regulation mechanism of these ageing-related gene-expression levels. According to the authors' experience, ageing-dependent variations in DNA methylation patterns only affect meristematic tissues (Fraga et al., 2002). That is why it is suspected that Prß288 expression variations could be restricted to the meristematic regions from which needles develop inside the buds, thus modulating meristem competence and growing capacity.
Acknowledgements
This work has been supported by the MCT-AGL2000-2126 Project. L Borja Diego was financed by MECD (FPU fellowship) and M Berdasco by MCYT (FPI contract). L Borja Diego would like to thank all the staff at Carmen Castresana's workgroup for advice and encouragement, as well as A Berdasco and J Menéndez for providing plant material.
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