Journal of Experimental Botany, Vol. 51, No. 351, pp. 1765-1766,
October 2000
© 2000 Oxford University Press
Gene Note |
Characterization of auxin-induced ARRO-1 expression in the primary root of Malus domestica
Department of Botany, University College Dublin, Belfield, Dublin 4, Ireland
Received 1 June 2000; Accepted 19 July 2000
Abstract
ARRO-1, a novel 2-oxoacid-dependent dioxygenase (2-ODD) is up-regulated during IBA-induced adventitious root formation in stem discs of Malus domestica. Analysis of ARRO-1's expression profile in the primary root of apple seedlings indicates that it is also highly up-regulated in the root in response to both IAA and IBA, but not 2,4-D. Auxin-derived evolution of ethylene can be discounted as the source of ARRO-1 induction as ARRO-1 is not induced in the root following treatment with the ethylene precursor ACC. Constitutive expression in the primary root further suggests that ARRO-1's role may be linked to the regulation of natural auxin levels within plant tissues. Significantly, orthologues of ARRO-1 have been identified in Arabidopsis thaliana by means of DNA database analysis which will enable the further molecular characterization of this class of 2-ODD.
Key words: Apple, Arabidopsis, auxin, dioxygenase, primary root.
ARRO-1 (Adventitious Rooting Related Oxygenase), exhibits prolonged up-regulation during indole-3-butyric acid (IBA)-induced adventitious root formation in apple stem discs (Butler and Gallagher, 1999
). ARRO-1 (AJ225045) encodes a 2-oxoacid-dependent dioxygenase, but to date, shares less than 31% amino acid sequence identity with other dioxygenases such as the 1-amino-cyclopropane-1-carboxylate (ACC) oxidases and gibberellin 3ß-hydroxylases, suggesting it represents a novel dioxygenase class.
ARRO-1 is expressed in stem discs that are induced to form root-initials 48 h after the beginning of an auxin treatment. Consequently, its detection in the root tissues of the intact plant is an appropriate step in determining ARRO-1's specificity to the root development process. In this further characterization of ARRO-1, its expression was examined in the primary root of apple seedlings. Apple seeds (cv. Golden Delicious) were stratified by incubation at 4 °C for 1 week and then soaked for 12 h in water. The outer testae were removed and seeds placed in the light on filter paper soaked with sterile distilled water. Germination occurred after approximately 2 d. The roots of intact, 6-d-old seedlings were immersed either in IBA, 2,4-dichlorophenoxyacetic acid (2,4-D) or IAA (each at 75 µM) for 5 h and then transferred to water. Roots were harvested 24 h after the beginning of this treatment. Both adventitious and prolific lateral root formation occurred in each treatment, however, this was less pronounced in those roots treated with 2,4-D, which also began to develop callus.
Total RNA was extracted from the primary roots (according to De Vries et al., 1988) and used in Northern blot analysis. This analysis clearly shows that ARRO-1 is up-regulated in the primary root in response to the IBA and IAA treatments (Fig. 1A). However, it is unclear whether ARRO-1 is independently expressed in response to both exogenous IAA and IBA or whether its up-regulation by the latter reflects the underlying conversion of IBA to IAA. It has been reported that exogenously supplied IBA is converted to IAA in apple (Van der Krieken et al., 1992). Therefore, it is possible that ARRO-1 is induced by elevated levels of IBA-derived IAA and not directly by IBA. ARRO-1 was not induced by 2,4-D, even though adventitious and lateral root formation occurred following its application to the primary root. 2,4-D may mediate its auxin effect via an alternative pathway from that which is stimulated by IBA and IAA (Ribnicky et al., 1996).
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Seedlings were also placed in GA3 (50 µM) or ACC (300 µM) for 24 h before harvesting for RNA isolation and Northern blot analysis. ARRO-1 is not induced by ACC, which is rapidly metabolized by ACC oxidase to form ethylene (Zarembinski and Theologis, 1994
, and references therein). This further eliminates auxin-induced ethylene as the source of ARRO-1 induction. GA3 also failed to induce ARRO-1 suggesting it is not involved in the numerous pathways of GA metabolism (Hedden, 1997, and references therein). This evidence coupled with the manner in which ARRO-1 is constitutively expressed in the root (Fig. 1A
, lane 1) and up-regulated in response to IBA and IAA suggest that it represents a novel class of 2-ODD which plays a part in the regulation of auxin metabolism following exposure to elevated levels of specific auxins.
DNA database analysis has recently identified an Arabidopsis thaliana genomic sequence (AC007576) that shares significant identity with ARRO-1. Two predicted open reading frames (ORFs) have been identified within this sequence, each encoding a putative polypeptide that, respectively, shares 56% (AAD39298) and 57% (AAD39299) amino acid sequence identity (
74% similarity) with ARRO-1 (Fig. 2). The genes are positioned in tandem on Chromosome I and share 76% amino acid sequence identity with one another. Northern blot analysis shows that both genes are expressed in Arabidopsis (ED Butler and TF Gallagher, unpublished data). The presence of two very similar gene copies within the Arabidopsis genome supports previous observations of a small multigene family in apple (Butler and Gallagher, 1999). Southern blot analysis (Fig. 1B) shows that ARRO-1 also hybridizes strongly with several fragments in the pear (Pyrus communis) and plum (Prunus domestica) genomes suggesting that ARRO-1 is relatively conserved within the Rosaceae, but not in more divergent species such as birch (Betula pendula).
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The expression dynamics of ARRO-1 detailed here, emphasizes the unique circumstances in which this gene is expressed. This study coupled with the identification of the ARRO-1 orthologues in Arabidopsis will aid in the development of strategies to analyse further the expression and roles of ARRO-1 in plant development. These will include in situ hybridization and transgenics, utilizing both over-expression and knock-out strategies.
Notes
1 To whom correspondence should be addressed. Fax: +353 1 7061153. E-mail: eoin.butler{at}ucd.ie 
References
Butler ED, Gallagher TF.1999. Isolation and characterization of a cDNA encoding a novel 2-oxoacid-dependent dioxygenase which is up-regulated during adventitious root formation in apple (Malus domestica ‘Jork 9') stem discs. Journal of Experimental Botany 50, 551–552.
De Vries S, Hoge H, Bisseling T.1988. Isolation of total and polysomal RNA from plant tissues. In: Gelvin SB, Schilperoort RA, Verma DPS, eds. Plant molecular biology manual . Dordrecht: Kluwer Academic Publishers, B6: 1–13.
Hedden P.1997. The oxidases of gibberellin biosynthesis: their function and mechanism. Physiologia Plantarum 101, 709–719.
Ribnicky DM, Ili
N, Cohen JD, Cooke TJ.1996. The effects of exogenous auxins on endogenous indole-3-acetic acid metabolism. Plant Physiology 112, 549–558.[Abstract]
Van der Krieken WM, Breteler H, Visser MHM.1992. Uptake and metabolism of indolebutyric acid during root formation on Malus microcuttings. Acta Botanica Neerlandica 41, 435–442.
Zarembinski TI, Theologis A.1994. Ethylene biosynthesis and action: a case of conservation. Plant Molecular Biology 26, 1579–1597.[Web of Science][Medline]
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