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. 2003 Aug;132(4):2135-43.
doi: 10.1104/pp.103.023309.

PAUSED encodes the Arabidopsis exportin-t ortholog

Christine A Hunter  1 , Milo J Aukerman, Hui Sun, Maria Fokina, R Scott Poethig
Affiliations

PAUSED encodes the Arabidopsis exportin-t ortholog

Christine A Hunter et al. Plant Physiol. 2003 Aug.

Abstract

Los1p/exportin-t (XPOT) mediates the nuclear export of tRNAs in yeast and mammals. The requirements for this transport pathway are unclear, however, because los1 mutations do not affect yeast growth, and the phenotype of XPOT mutations in mammals is unknown. Here, we show that PAUSED (PSD) is the Arabidopsis ortholog of LOS1/XPOT and is capable of rescuing the tRNA export defect of los1 in Brewer's yeast (Saccharomyces cerevisiae), suggesting that its function has been conserved. Putative null alleles of PSD disrupt the initiation of the shoot apical meristem and delay leaf initiation after germination, the emergence of the radicle and lateral roots, and the transition to flowering. Plants doubly mutant for psd and hasty, the Arabidopsis ortholog of exportin 5, are viable but have a more severe phenotype than either single mutant. These results suggest that PSD plays a role in tRNA export in Arabidopsis, but that at least one-and perhaps several-additional tRNA export pathways also exist. The PSD transcript is broadly expressed during development and is alternatively spliced in the 3'-untranslated region. No temporal or spatial difference in the abundance of different splice forms was observed. We propose that the mutant phenotype of psd reflects defects in developmental events and cell/tissue types that require elevated levels of protein synthesis and are therefore acutely sensitive to a reduction in tRNA export.

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Figures

Figure 1.
Figure 1.
The psd mutation delays leaf production and disrupts inflorescence morphology. Photos of a wild-type Col plant (A) and a psd-6 mutant plant (B) 14 DAP show the reduction in leaf number caused by the delay in leaf initiation. Cotyledons (c) and leaf numbers are indicated. The resulting change in leaf shape and identity can be seen in the outlines of leaves from wild-type (C) and psd-6 (D) plants. Leaves lacking abaxial trichomes are shown in light gray, leaves with abaxial trichomes in dark gray, and leaves lacking adaxial trichomes in black. Bracts are underlined. E, The rate of initiation of LFY:GUS positive leaf primordia in Col and psd-6 shows the initial delay in the psd-6 mutant. Both wild-type and psd-6 plants initiate their first adult leaf primordium (leaf 4.9 in Col and 2.7 in psd-6) at 5 DAP (arrows). In the inflorescence of wild-type plants (F), secondary inflorescences are subtended by bracts, and siliques are distributed in an even pattern. In psd-6 (G) some secondary inflorescences lack bracts (arrow) and the phyllotaxy is disrupted (arrowhead). Bars = 5 cm for A and B and 1 cm for F and G.
Figure 2.
Figure 2.
PSD is required in the SAM, but not the RAM. Confocal images of mature seeds stained with Hoechst show that the SAM of mature seeds (A) is greatly reduced in psd-6 embryos (B). At 3 DAP, wild-type seedlings (C) have a well-ordered SAM with visible leaf primordia, whereas in psd-6 (D), the central region of the SAM is disrupted and leaf primordia are reduced in size. At 3 DAP, a section of a wild-type seedlings (E) shows distinct cell layers in the SAM that are missing in psd-6 seedlings of the same age. This defect appears more severe in the central zone of the SAM, because intact cells in the peripheral zone have begun to initiate leaf primordia. Compared with wild type (G), the roots of psd-6 mutant seedlings (H) are slightly disordered and have an increased amount of starch at 3 DAP, although all cell layers appear to be present. The thickening of the psd-6 mutant root appears to be caused by an enlargement of cells in the cortical layer. Bars = 20 μm.
Figure 3.
Figure 3.
PSD has homology to Los1p/XPOT. A, The structure of the PSD transcript is shown, with the coding region in black and the UTRs in white. The three alternate spliced forms of exons 13 and 14 are diagramed, and polyadenylation sites are shown with arrowheads. The position and nature of the four psd mutations are indicated with arrows. B, A comparison of the Arabidopsis PSD (At), human XPOT (Hs), and fission yeast (Schizosaccharomyces pombe (Sp) and Brewer's yeast (Sc) Los1p proteins. Identical amino acids boxed in black; conservative amino acid changes are shaded gray.
Figure 4.
Figure 4.
PSD is expressed throughout development. A, Northern blot showing the PSD transcript from wild-type and psd-6 mutant plants. A single transcript is detected in wild type and is reduced in size and expression level by the psd-6 inversion. B, Northern blot showing the presence of PSD transcript in the roots (Rt), rosette leaves (Lf), and floral buds (Fl) of mature plants. C, Southern blot of DdeI-digested reverse transcriptase (RT)-PCR products showing the presence of the three alternatively spliced forms of the PSD 3′-UTR in 8-, 15-, and 22-d shoot apices, and in roots, rosette leaves, and floral buds. If present, the 13A/14B form would migrate between the 13B/14A and 13B/14B forms.
Figure 5.
Figure 5.
PSD complements the Brewer's yeast los1 mutant phenotype. Serial dilutions of parent strain X2316-3C, the los1-1 temperature-sensitive mutant strain 201-1-5, or 201-1-5 containing the ADH:PSD construct were grown at permissive (22°C) or restrictive (34°C) temperature. The ADH:PSD construct is able to partially suppress the slow growth phenotype of 201-1-5 on SDC (-ade) plates.
Figure 6.
Figure 6.
The psd-1; hst-1 double mutant plant shows a combination of the psd and hst phenotypes and is greatly reduced in size.
See this image and copyright information in PMC

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