The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root growth and leaf vascular patterning

Abstract

The POLARIS (PLS) gene of Arabidopsis was identified as a promoter trap transgenic line, showing beta-glucuronidase fusion gene expression predominantly in the embryonic and seedling root, with low expression in aerial parts. Cloning of the PLS locus revealed that the promoter trap T-DNA had inserted into a short open reading frame (ORF). Rapid amplification of cDNA ends PCR, RNA gel blot analysis, and RNase protection assays showed that the PLS ORF is located within a short ( approximately 500 nucleotides) auxin-inducible transcript and encodes a predicted polypeptide of 36 amino acid residues. pls mutants exhibit a short-root phenotype and reduced vascularization of leaves. pls roots are hyperresponsive to exogenous cytokinins and show increased expression of the cytokinin-inducible gene ARR5/IBC6 compared with the wild type. pls seedlings also are less responsive to the growth-inhibitory effects of exogenous auxin and show reduced expression of the auxin-inducible gene IAA1 compared with the wild type. The PLS peptide-encoding region of the cDNA partially complements the pls mutation and requires the PLS ORF ATG for activity, demonstrating the functionality of the peptide-encoding ORF. Ectopic expression of the PLS ORF reduces root growth inhibition by exogenous cytokinins and increases leaf vascularization. We propose that PLS is required for correct auxin-cytokinin homeostasis to modulate root growth and leaf vascular patterning.

Figure 1.

GUS Expression in the PLS-GUS Promoter Trap Line AtEM101. (A) Heart-stage embryo, with 5 h of GUS staining, showing GUS activity in the basal region. Magnification ×100. (B) Seven-day-old seedling root tip, with 5 min of GUS staining, showing activity in columella and, weakly, in young vascular cells. Bar = 50 μM. (C) Seven-day-old seedling root tip, with 1 h of GUS staining, showing activity throughout the root tip. Magnification ×7. (D) Aerial parts of a 12-day-old seedling, with 8 h of GUS staining, showing activity in leaf vascular tissues. Magnification ×5. (E) RNA gel blot analysis showing PLS-GUS fusion transcript levels in the pls mutant using 10 μg of RNA from 7-day-old total seedlings (Se), siliques (Si), root tips (Rt), roots with tips removed (Ro), and aerial parts (Ae). The top gels shows a GUS fusion transcript, and the bottom gels shows ethidium bromide–stained 25S rRNA. Note that silique RNA runs more rapidly through gels than that from other tissues.

Figure 2.

pls Mutant Phenotype. (A) pls (top) and wild-type (wt; bottom) seedlings, 14 days after germination, showing the short-root phenotype of the pls mutant. (B) Primary root growth of wild-type C24, pls mutant, and C24/pls heterozygote seedlings. (C) The hypocotyl-root junction of pls (left) and wild-type (right) seedlings, 14 days after germination, showing the increased frequency of anchor roots (arrows) in the pls mutant. (D) Rosette leaves of wild-type (left) and pls mutant (right) seedlings, 12 days after germination, showing the decreased frequency of higher order vascular strands in the pls mutant. (E) Enlargement of leaves shown in (D) showing detail of the reduced vascularization of the pls mutant.

Figure 3.

PLS Encodes a Small Transcript. (A) PLS locus. Closed rectangles indicate transcripts, and arrows indicate ORFs (for GENE X and PLS). The T-DNA insertion site is indicated. (B) The PLS gene sequence shows transcription start site 2 (inverted arrowhead), the GENE X polyadenylation site (boldface), the 9–amino acid uORF (underlined), the 36–amino acid PLS ORF (amino acids shown), and the PLS polyadenylation sites (arrows). The asterisk marks the position of the T-DNA insertion site (in the 25th codon; L).

Figure 4.

PLS Expression. (A) RNA gel blot [5 μg of poly(A)⁺ RNA] showing the ∼500-nucleotide (nt) PLS transcript. (B) RNase protection assays to map the PLS transcript initiation site in wild-type and AtEM101 seedlings using a radiolabeled antisense PLS transcript. Transcript start sites (1 and 2) are shown in the bottom panel (arrows) and in diagrammatic form above. M, size markers (radiolabeled phiX174 Hinf1 DNA); Y−, yeast RNA control; YRNase, RNase-treated yeast RNA control; pls, 50 μg of total RNA from AtEM101 seedlings; Colt, 50 μg of total RNA from wild-type seedlings (Columbia); ColA+, 5 μg of poly(A)⁺ RNA from wild-type seedlings (Columbia). (C) Activity of the 370-bp promoter region (pPLS::GUS expression) in the root tip of a 7-day-old transgenic seedling with 24 h of GUS staining. Magnification ×7. (D) Activity of the 1190-bp promoter region in the root tip of a 7-day-old transgenic seedling with 24 h of GUS staining. Magnification ×5.

Figure 5.

The PLS Gene Promoter is Auxin Inducible. (A) RNA gel blot showing PLS-GUS fusion transcript levels after treatment with auxin (10 μM 1-NAA) or cytokinin (10 μM kinetin). Seedlings were grown for 7 days on hormone-free medium and then transferred to the hormones for the times indicated. The top gel shows the PLS-GUS transcript, and the bottom gel shows ethidium bromide–stained 25S RNA. Relative transcript levels were normalized compared with untreated controls, and values are shown below. Each lane contained 10 μg of total RNA. (B) PLS-GUS fusion transcript levels after treatment of 7-day-old AtEM101 seedlings with 10 μM active auxins (1-NAA and 2,4-D) and inactive analogs (2-NAA and 2,3-D) for 24 h. Each lane contained 20 μg of total RNA. Unt., untreated. (C) Competitor PCR analysis showing the increase in abundance of the native PLS transcript (POLARIS) in untreated 7-day-old wild-type seedlings (unt) and after treatment of 7-day-old wild-type seedlings with 10 μM 1-NAA for 2 and 24 h. The competitor lanes show the abundance of amplicon derived from a cleaved (with ClaI) synthetic PLS cDNA fragment that was identical to wild-type PLS sequence except for an introduced ClaI site and that was introduced into the PCR as an internal standard. The ACTIN3 transcript was amplified as a control, and control amplifications lacking reverse transcriptase are shown (−RT). (D) PLS-GUS fusion transcript levels in transgenic lines containing either the 370-bp (pS) or 1190-bp (pL) promoter fragments linked to gusA after growth on 10 μM 1-NAA for 24 h (+) or unsupplemented medium (−). Each lane contained 20 μg of total RNA.

Figure 6.

Gene Expression in a pls Mutant Background. (A) and (B) RNA gel blot analysis of transcript abundances of ARR5/IBC6 (A) and ARR6/IBC7 (B) genes in wild-type (WT) and pls mutant seedlings 7 days after germination. 25S RNA is shown as a loading control. Each lane contained 10 μg of total RNA. (C) and (D) ARR5/IBC6::GFP gene fusion activities in the root tips of transgenic wild-type (C) and pls mutant (D) seedlings 7 days after germination, viewed with the confocal microscope. Magnification ×100. (E) and (F) RNA gel blot analysis of transcript abundances of IAA1 (E) and IAA2 (F) genes in wild-type and pls mutant seedlings 7 days after germination. 25S RNA is shown as a loading control. Each lane contained 10 μg of total RNA.

Figure 7.

Complementation of pls with the PLS ORF-Containing Partial cDNA. (A) Fourteen-day-old seedlings of the wild type (C24), pls mutant, and complementation line 113C showing restoration of wild-type root architecture to the complemented mutant. (B) Primary root length of pls, the wild type (C24), and eight independent complementation lines (77 to 113C) containing the partial PLS cDNA at 9 days after germination. All lines show significantly longer roots than the pls mutant. (C) RT-PCR analysis of seedlings of the pls mutant (pls) and transgenic lines mPLS ORF 1 to 5 (lanes 1 to 5, respectively). The top gel shows amplification of the 487-bp mPLS ORF cDNA from RNA extracted from each of the five mPLS ORF transgenic lines but not from the pls mutant. The bottom gel shows amplification of the control ACT3 cDNA in pls and each mPLS ORF transgenic line. Control experiments lacking reverse transcriptase showed no amplification products (data not shown). M, molecular mass markers (kD).

Figure 8.

Effects of Overexpressing a PLS ORF-Containing Partial cDNA in Wild-Type Seedlings. (A) RNA gel blot analysis of wild-type (Columbia [Col]) and five independent transgenic overexpressers of the partial PLS cDNA (designated 38 to 70) probed with radiolabeled PLS cDNA. 25S RNA is shown as a loading control. Under the exposure conditions used, the autoradiograph does not reveal the low-abundance PLS transcript in the wild-type sample. Each lane contained 10 μg of total RNA. (B) Primary root length of wild-type (Columbia) and five independent PLS-overexpressing lines grown for 7 days in the presence of 0.1 to 5 μM BA. Note that wild-type and overexpressing seedlings, grown in the absence of exogenous BA but under otherwise identical conditions, exhibited no significant difference in primary root length. (C) Rosette leaves of wild-type (left) and PLS-overexpressing (right) seedlings 12 days after germination showing the increased frequency of higher order vascular strands and lack of trichomes in the overexpressers. Arrows indicate trichomes.