PAUSED, a putative exportin-t, acts pleiotropically in Arabidopsis development but is dispensable for viability

Abstract

Exportin-t was first identified in humans as a protein that mediates the export of tRNAs from the nucleus to the cytoplasm. Mutations in Los1p, the Saccharomyces cerevisiae exportin-t homolog, result in nuclear accumulation of tRNAs. Because no exportin-t mutants have been reported in multicellular organisms, the developmental functions of exportin-t have not been determined. Here, we report the isolation and characterization of two Arabidopsis exportin-t mutants, paused-5 and paused-6. The mutant phenotypes indicate that exportin-t acts pleiotropically in plant development. In particular, paused-5 and paused-6 result in delayed leaf formation during vegetative development. The two paused mutations also cause the transformation of reproductive organs into perianth organs in the hua1-1 hua2-1 background, which is partially defective in reproductive organ identity specification. The floral phenotypes of hua1-1 hua2-1 paused mutants resemble those of mutations in the floral homeotic gene AGAMOUS. Moreover, paused-5 enhances the mutant phenotypes of two floral meristem identity genes, LEAFY and APETALA1. The developmental defects caused by paused mutations confirm the important roles of exportin-t in gene expression in multicellular organisms. In addition, a paused null allele, paused-6, is still viable, suggesting the presence of redundant tRNA export pathway(s) in Arabidopsis.

Figure 1.

psd phenotypes in seedlings. A, A 15-d-old Landsberg erecta (Ler) plant. B, A 15-d-old psd-5 plant with fewer true leaves than wild type at the same stage. C, A 15-d-old psd-5 plant with no true leaves. D, A 15-d-old psd-5 plant with a terminal leaf. E, A 14-d-old Columbia (Col) plant. F and G, 14-d-old psd-6 plants with fewer true leaves than Col. One cotyledon was absent in G. H, A 20-d-old psd-6 plant with a terminal leaf. I, A 20-d-old F₁ plant from a cross between Col and Ler. J, A 20-d-old F₁ plant from a cross between psd-5 and psd-6. Fewer rosette leaves are found than the corresponding wild type (I) at the same stage. K, A 13-d-old psd-5 hua2-1 (Ler) transgenic seedling containing pPZP211-HEN5p3/4 showing normal true leaf formation. L, A 13-d-old psd-5 hua2-1 (Ler) transgenic seedling containing pPZP-35S-green fluorescent protein (GFP) showing the psd-5 phenotype. The magnification in K and L is different from that in A through J.

Figure 2.

Floral phenotypes of psd mutants and interactions with mutations in A, B, and C genes. A, A hua1-1 hua2-1 (Col) flower. B, An early hua1-1 hua2-1 psd-5 flower with petals instead of stamens in the third whorl (arrow). C, A late hua1-1 hua2-1 psd-5 flower with petals in the third whorl and an additional flower in the fourth whorl. D, A hua1-1 hua2-1 psd-6 (Col) flower showing stamen-to-petal transformation in the third whorl. E, Comparison of hua1-1 hua2-1 (1), hua1-1 hua2-1 psd-5 (2), and hua1-1 hua2-1 psd-6 (3 and 4) gynoecia. F, A hua1-1 hua2-1 psd-6 flower with an internal flower in the fourth whorl. G, A psd-5 flower with normal floral organ types. H and I, Abaxial surfaces of the base of a hua1-1 hua2-1 (H) and a hua1-1 hua2-1 psd-5 valve (I). J and K, Abaxial surfaces of the apical portion of an Ler (J) and a psd-5 valve (K). The psd-5 valve has a few cells with typical sepal epicuticular striations (arrow). L, A hua1-1 hua2-1 ag-1 flower. M, A hua1-1 hua2-1 psd-5 ag-1 flower. N, A hua1-1 hua2-1 ap2-2 flower. O, A hua1-1 hua2-1 psd-5 ap2-2 flower. P, A hua1-1 hua2-1 pi-3 flower with filaments (arrow) in the third whorl. Q, A hua1-1 hua2-1 psd-5 pi-3 flower with sepals in the third whorl. R, A hua1-1 hua2-1 ap1-1 flower. S, A hua1-1 hua2-1 psd-5 ap1-1 “flower” with leaf-like organs in a spiral phyllotaxy, and an internal terminal structure with carpel character. Size bars in H through K = 10 μm.

Figure 3.

The PSD gene and protein. A, Positional cloning of PSD. The black lines represent BACs with the BAC names indicated above. The recombination frequencies at the markers (vertical lines) are shown below the BACs as number recombination/number chromosome. The PSD genomic structure is shown with rectangles representing exons and lines representing introns. The psd-5 single nucleotide deletion is marked as a star in exon 9 and the psd-6 T-DNA insertion mutation is represented as an inverted triangle in exon 1. B, A clustalW alignment of PSD protein with exportin-t (human, Homo sapiens) and Los1p (Saccharomyces cerevisiae). The amino acids showing identity and ones showing similarity are outlined, and highlighted in dark and light gray, respectively. The lines below the alignment denote the Ran-binding domain.

Figure 4.

AP1, AG, AP3, and PSD RNA accumulation as determined by in situ hybridization. The brown/purple color represents positive hybridization signals. A, A stage 12 hua1-1 hua2-1 flower without AP1 expression in the inner two whorls. B, A hua1-1 hua2-1 psd-5 flower with ectopic AP1 expression in the inner two whorls (arrows). C, A stage 5–6 hua1-1 hua2-1 flower showing AG RNA in the center. D, A stage 6 hua1-1 hua2-1 psd-5 flower showing AG RNA in the center. E, A stage 12 hua1-1 hua2-1 flower with AP3 expression in the second and third whorls. F, A stage 12 hua1-1 hua2-1 psd-5 flower showing ectopic expression of AP3 on the adaxial side of the ovary (arrow). G, A longitudinal section of an Ler inflorescence hybridized to a PSD sense probe. H through J, Hybridization of Ler (H and I) and ag-1 (J) tissues to a PSD antisense probe. PSD expression can be detected in young stamens and carpels (white arrows) in a stage 10 flower and in young floral meristems (black arrow; H), in the ovules of a stage 12 flower (I) and in the central young floral organs and the meristem (arrow) in the ag-1 flower (J). Size bar = 50 μm.

Figure 5.

Interactions between psd and lfy. A, A lfy-5 inflorescence with flowers. B, A psd-5 lfy-5 primary inflorescence (arrow) without the formation of flowers. C, A magnification of the inflorescence stem in B. Outgrowths that appear undifferentiated (arrow) are found along the stem and the internodes are more compact than those in lfy-5. D, A lfy-6 inflorescence with abnormal flowers lacking petals and stamens. E, A psd-5 lfy-6 inflorescence, which produces filamentous organs at positions normally occupied by flowers and terminates in a structure with stigmatic tissues at the top. F, A psd-5 lfy-6 inflorescence terminating in filamentous organs that sometimes have two-branched trichomes (arrow), suggesting that the organs have leaf character.

Figure 6.

AG RNA and protein accumulation. A, AG RNA accumulation in various genetic backgrounds as determined by RNA filter hybridization (top panel). The same blot was hybridized with UBQ5 for comparison (bottom panel). The numbers shown below indicate the relative abundance of AG mRNA among different genotypes. These numbers were derived from statistical analysis of three independent experiments. Note that although some hybridization signals may appear saturated in this picture, the signal intensity was in the linear range of phosphorimager quantitation. B, AG protein accumulation in different genotypes (arrow, top panel) as determined by western blotting. As a loading and blotting control, phosphoenolpyruvate (PEP) carboxylase (Kandasamy et al., 2002) accumulation is shown in the bottom panel. The psd-6 (Ler) strain was obtained by two backcrosses of psd-6 (Col) to Ler.

Figure 7.

PSD RNA accumulation in total RNAs from different tissues and genotypes (top panel) as determined by RNA filter hybridization. The same blot was hybridized with a UBQ5 probe for comparison (bottom panel). Inflo., Inflorescences.