JXB Advance Access originally published online on April 8, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1433-1435, June 1, 2004
© 2004 Oxford University Press
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An 
-crystallin gene, ACD31.2 from Arabidopsis is negatively regulated by FPF1 overexpression, floral induction, gibberellins, and long days
Received 17 December 2003; Accepted 18 February 2004
1 Institute of Developmental Biology, University of Cologne, Gyrhofstrasse 17, D-50923 Cologne, Germany
2 University of Liège, Plant Physiology, B22, Sart Tilman, B-4000 Liège, Belgium
* To whom correspondence should be addressed. Fax: +49 221 470 5164. E-mail: John.Chandler{at}uni-koeln.de
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Abstract |
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A gene sequence was isolated from a differential display experiment to find transcripts altered in expression by overexpression of FLORAL PROMOTING FACTOR 1 (FPF1) in Arabidopsis thaliana. The gene, ACD31.2, encodes an
-crystallin domain containing protein with homology to small heat shock proteins. In addition to down-regulation by FPF1 overexpression, the ACD31.2 transcript is also down-regulated by long days, floral induction, and by gibberellin in wild-type plants. Expression is highest in leaves and stems. Key words: Arabidopsis thaliana, FPF1, gibberellin, photoperiod.
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Floral induction in Arabidopsis is a complex trait subject to many environmental and physiological stimuli such as photoperiod, involving the endogenous clock, temperature (vernalization), and gibberellins (GAs) (Mouradov et al., 2002). Major genes involved in some of these pathways have been cloned (Simpson and Dean, 2002) and for some of these, such as CONSTANS, the target genes and signal transduction pathway have been well-characterized (Suarez-Lopez et al., 2001). However, less is understood concerning the gibberellin floral promotion pathway involving the FLORAL PROMOTING FACTOR1 (FPF1) gene (Kania et al., 1997). FPF1 is specifically expressed and highly up-regulated in the apical meristem of Arabidopsis following floral induction. The gene modulates the competence to flower (Melzer et al., 1999) and overexpression results in early flowering and largely phenocopies plants treated with GAs. The response of 35S::FPF1 overexpressing plants to paclobutrazol and GA suggests that FPF1 has a role in GA perception (Kania et al., 1997).
In order to understand which genes are involved in the FPF1 overexpression phenotype, a differential display experiment (GenHunter kit) (Liang and Pardee, 1992) was performed, comparing FPF1 overexpressing plants with the wild type, either before or after floral induction. One transcript which was down-regulated in FPF1 overexpressing plants was sequenced and the cDNA compared with the genomic sequence in the database. The gene, At1g06460 (accession number AAF24817
[GenBank]
consists of seven exons (Fig. 1a) with an ORF of 909 bp and putatively encodes a protein of 302 amino acids which corresponds to the protein ACD32.1, a member of the -crystallin family of proteins in Arabidopsis whose nomenclature was established by Scharf et al. (2001). The C-terminal part of the protein shows homology to small heat shock proteins, with 23 amino acid residues from 92 in accordance with the HSP20 consensus sequence (Fig. 1b).
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The down-regulation of ACD32.1 expression from the differential display experiment (Fig. 1c) was confirmed by a northern blot analysis, using total RNA isolated from the apical region of either Ler or FPF1 overexpressing plants grown in non-inductive short-day conditions or following an inductive treatment of four long days. ACD32.1 transcript expression was not only reduced in the FPF1 overexpressing transgenic plants, but was also reduced in the wild type and transgenics following floral induction (Fig. 1d). The expression of ACD32.1 in various tissues of intact Arabidopsis plants was also monitored by northern hybridization. The probe for all hybridization experiments was a 167 bp ACD32.1 fragment amplified by PCR using the primers 5'-CTTGACTGTGACAG GTAGACG-3' and 5'-AATCCCATCATAAATTCAGCG-3'. The highest transcript levels were found in leaves and stems, with significantly lower transcript amounts in inflorescences and virtually no expression in the root (Fig. 1e) The expression was higher in all tissues when plants were grown in non-inductive short-day conditions compared with inductive long days (Fig. 1e). Analysis of ACD32.1 transcript levels after spraying intact flowering plants with 10–4 M GA3 and harvesting material 2 d later showed that ACD32.1 expresion is negatively regulated by gibberellins in all the tissues analysed (Fig. 1e).
To date, there are few examples of genes whose expression is negatively regulated by GA. The gibberellin biosynthesis genes GA3Ox1 and GA20Ox1, 2 and 3 are subject to negative feedback regulation by GA (GA3Ox1, Cowling et al., 1998; GA20Ox1, 2, and 3, Phillips et al., 1995). The down-regulation of ACD32.1 by daylength may be a direct result of GA levels, since it is known that the concentration of many active GAs and GA20Ox1 expression is higher in long days (Xu et al., 1997). To confirm the negative regulation of ACD32.1 by GA, an independent semi-quantitative RT-PCR experiment was performed after reverse transcription of 3 µg total RNA (Superscript II, Invitrogen) from leaves with or without treatment with 10–4 M GA3 for 2 d. PCR was performed for 20 cycles and the RT-PCR gels were Southern blotted and hybridized with the corresponding gene fragments. As a control, the ribosomal elongation initiation factor 4A (eIF4A) was amplified as representing a constitutive gene, with primers 5'-AAACT CAATGAAGTACTTGAG-3' and 5'-TGACCTTCTCAATTTG CTGAG-3'. The invariant expression of eIF4A showed that gene expression in general was not altered by GA treatment (Fig. 1f). In addition, expression of the GA3ox1 gene, a gene known to be negatively regulated by GA treatment was monitored using primers 5'-TAACCATTCTGTACCAGAACA-3' and 5'-TGTTCGAAG ATACTCTTTCCA-3'. The down-regulation of the GA3ox1 trancript on GA treatment confirmed the efficacy of the treatment and allows the interpretation that the down-regulation of the ACD32.1 transcript observed in this experiment (Fig. 1f) and in Fig. 1e is unequivocally due to the GA treatment.
The conclusion that expression of the ACD32.1 gene is negatively regulated by GA concentration is further supported by experiments with paclobutrazol, an inhibitor specific to the mono-oxygenases involved in the oxidation of ent-kaurene to ent-kaurenoic acid, which reduces active GA biosynthesis (Radermacher, 1989). Rosettes were sprayed with paclobutrazol (37 mg l–1) weekly until the appearance of buds and expression of ACD32.1 was found to be up-regulated (Fig. 1g) as would be predicted by a reduction in active GA concentration.
The family of ACD proteins in Arabidopsis is newly characterized and consists of 25 ORFs (Scharf et al., 2001). Knowledge about the function of these proteins and the relevance of their homology to small heat shock proteins is limited. However, one member of the group, RTM2 has been implicated in resistance to tobacco etch virus in Arabidopsis (Whitham et al., 2000). Two further family members; ACD44.3 and ACD55.2 contain an ARID (AT-rich interaction) domain and are DNA-binding proteins with a helix-turn-helix motif (Tucker et al., 2000). The hypothesis that ACD proteins are involved with DNA binding functions is further supported by ACD114.3 which has multiple -crystallin domains and is a chloroplast nucleoid DNA-binding protein (Nakano et al., 1997).
Since ACD32.1 transcript expression is repressed by floral induction and factors which promote flowering such as gibberellins, long days, and FPF1 overexpression, it can be speculated whether this gene has a role in the repression of flowering.
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Acknowledgements |
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This work was supported by the Swiss Federal Institute of Technology and the Swiss Federal Office of Education and Science as part of an EC-project (GMOF).
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