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. 2016 Jan 5:16:2.
doi: 10.1186/s12870-015-0695-y.

Reduced expression of AtNUP62 nucleoporin gene affects auxin response in Arabidopsis

Affiliations

Reduced expression of AtNUP62 nucleoporin gene affects auxin response in Arabidopsis

Martin Boeglin et al. BMC Plant Biol. .

Abstract

Background: The plant nuclear pore complex has strongly attracted the attention of the scientific community during the past few years, in particular because of its involvement in hormonal and pathogen/symbiotic signalling. In Arabidopsis thaliana, more than 30 nucleoporins have been identified, but only a few of them have been characterized. Among these, AtNUP160, AtNUP96, AtNUP58, and AtTPR have been reported to modulate auxin signalling, since corresponding mutants are suppressors of the auxin resistance conferred by the axr1 (auxin-resistant) mutation. The present work is focused on AtNUP62, which is essential for embryo and plant development. This protein is one of the three nucleoporins (with AtNUP54 and AtNUP58) of the central channel of the nuclear pore complex.

Results: AtNUP62 promoter activity was detected in many organs, and particularly in the embryo sac, young germinating seedlings and at the adult stage in stipules of cauline leaves. The atnup62-1 mutant, harbouring a T-DNA insertion in intron 5, was identified as a knock-down mutant. It displayed developmental phenotypes that suggested defects in auxin transport or responsiveness. Atnup62 mutant plantlets were found to be hypersensitive to auxin, at the cotyledon and root levels. The phenotype of the AtNUP62-GFP overexpressing line further supported the existence of a link between AtNUP62 and auxin signalling. Furthermore, the atnup62 mutation led to an increase in the activity of the DR5 auxin-responsive promoter, and suppressed the auxin-resistant root growth and leaf serration phenotypes of the axr1 mutant.

Conclusion: AtNUP62 appears to be a major negative regulator of auxin signalling. Auxin hypersensitivity of the atnup62 mutant, reminding that of atnup58 (and not observed with other nucleoporin mutants), is in agreement with the reported interaction between AtNUP62 and AtNUP58 proteins, and suggests closely related functions. The effect of AtNUP62 on auxin signalling likely occurs in relation to scaffold proteins of the nuclear pore complex (AtNUP160, AtNUP96 and AtTPR).

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Figures

Fig. 1
Fig. 1
AtNUP62 promoter activity and protein distribution. Tissue-specific activity of the AtNUP62 promoter was investigated by histochemical analysis of GUS staining (blue color) in transgenic plants expressing GUS under control of AtNUP62 promoter region. a Stipules at the basis of cauline leaves (arrows). b Enlarged view of stipules at the basis of a cauline leaf. c margin of a cauline leaf. d Inflorescence. e Root tip. f Cotyledon tip. The arrows indicates the localisation of GUS staining. g Young silique and h developing seed from this silique. i Young germinating seedling. j and k, Subcellular localization of AtNUP62::GFP fusion protein (confocal microscopy). The AtNUP62::GFP construct was expressed under control of the cauliflower mosaic virus 35S promoter, and the same construct was used for plant and protoplast transformation. j Root tip of a transgenic 35S::AtNUP62-GFP 10-day old Arabidopsis plant. k Confocal microscopy analysis of AtNUP62::GFP signals in a transiently transformed Arabidopsis cultured cell protoplast
Fig. 2
Fig. 2
Analysis of the atnup62 mutant. a Upper panel: Structure of the AtNUP62 gene and position of the T-DNA insertion in the SALK_037337 (atnup62-1) mutant. Exons are represented by rectangles (9 exons) and introns by black lines. 5‘ and 3‘-UTR are thick red lines. The protein is composed of 739 amino acids. The ORF regions encoding the FG repeat, serine-rich domain (approximately 480 amino acids) and nsp1-C-like domain (amino acids 565 to 647) are figured in pale green and yellow, respectively. The T-DNA insertion site is indicated (LB and RB: left and right borders) with the sequence of the 15 bases just upstream of the insertion site, coming from our own sequencing of the junction region. The two pairs of primers (a and b) used for PCR are indicated. Lower panel: RT-PCR amplification of RNAs prepared from inflorescences of wild-type Col-0 (Col) and atnup62-1 mutant (nup). Left lanes (amplification of cDNAs): after reverse transcription, PCR amplification was carried out using two couples of primers, a or b, for AtNUP62, or a couple of primers targeting the EF1-α gene. Right lanes (labelled Genomic DNA): amplifications of wild-type and mutant genomic DNA with primers B. Ladder: 1 kb plus from Promega, arrow at 1 kb. All amplified DNA fragments are present in the gel at a position consistent with in silico prediction. b Q-PCR analysis of AtNUP62 expression in different genotypes. Results are expressed as fold changes compared to wild type. Values are means and standard errors of two biological samples (three technical replicates per sample). c Phenotype of atnup62-1 mutant and complemented plants, at the adult stage (upper panels, zooms on flowers on top right) and on in vitro germinations on MS/2 medium (lower panels, 6 day-old seedlings). Lower panels, from left to right: wild type, plantlets with 3 cotyledons and 1 cotyledon from the atnup62 mutant, plantlet with abnormal cotyledons from the 35S::AtNUP62-GFP line, close to a plant (on the right) displaying a normal appearance. Arrows indicate abnormal plantlets. Cotyledons of complemented plants are similar to those of wild-type plants
Fig. 3
Fig. 3
Auxin hypersensitivity of the different AtNUP62 genotypes. a Growth on vertical plates (9 day-old plants) on different concentrations of 2,4-D. b Primary root growth measurements on plantlets grown on agar MS/2 medium containing different concentrations of 2,4-D (two experiments). Data are means ± SE. c Bright field images (left panels) and corresponding fluorescence images (right panels) in wild-type control plant (placed on the left) and a 35S::AtNUP62-GFP plantlet, on MS/2 and 2,4-D-containing medium. Seedlings of comparable root size have been chosen. d GUS activity under the control of DR5 auxin-responsive promoter in wild-type and atnup62 mutant genetic background. Left, 11 day-old plantlets, right, 2.5 day-old seedlings
Fig. 4
Fig. 4
Effect of the atnup62-1 mutation on the axr1 mutant phenotype. a Plants grown in the greenhouse. The lower panels show the disappearance of the serrated margins of axr1 plants in the atnup62 axr1 double mutant. b In vitro-grown plantlets on MS/2 medium supplemented with different concentrations of 2,4-D. c Restoration of auxin sensitivity of primary root growth by the atnup62-1 mutation in the axr1 mutant. Data are means ± SE. d Q-PCR analysis of AtNUP62 gene expression in axr1 and the double atnup62 axr1 mutant. Data are means ± SE of two biological samples (three technical replicates per sample)

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