Comparison of the bolting times of wild type and <i>NGAL1</i> over-expression lines.

<p>The average numbers of leaves of wild type and OE lines at bolting were calculated from randomly selected plants of each genotype (n≥28). Bolting times were calculated as days after germination (DAG). Data were presented in the form of mean±standard deviation (s.d.). Comparisons were made between wild type and each of the OE lines. Statistical significance was evaluated by <i>p</i> values generated by Student’s <i>t-</i>test.</p

Phenotypes of <i>abs2-1D</i>.

<p>A. Three-week-old wild type, <i>abs2-1D</i>/+ heterozygous and <i>abs2-1D</i> homozygous plants. Plants were grown at 22°C under continuous illumination of ∼100 µmol·m⁻²·s⁻¹. B. Cotyledons and rosette leaves of three-week-old wild-type, <i>abs2-1D</i>/+ heterozygous and <i>abs2-1D</i> homozygous plants. From left to right are two cotyledons and rosette leaves that were arranged in the order of their initiations. C. Comparison of the fifth rosette leaves of three-week-old wild type, <i>abs2-1D</i>/+ heterozygous and <i>abs2-1D</i> homozygous plants. Leaves were flattened between glass slides before photographing (Bars, 2 mm).</p

Identification of a loss-of-function mutant allele of <i>NGAL1</i>.

<p>A. T-DNA insertion site in Salk_146872 (<i>ngal1-1</i>). Lines represented introns and intergenic regions and boxes represented exons. 5′ and 3′ UTRs were indicated by shaded boxes. Approximate positions of the PCR primers used in B and C were marked with arrows. B. PCR-identification of <i>ngal1-1</i> homozygous mutant. The T-DNA insertion was flanked by two LB sequences. C. Expressions of <i>NGAL1</i> in wild type and <i>ngal1-1</i> mutant. Total RNAs were extracted from flower tissues and RT-PCRs were carried out with indicated primers and cycle numbers. D. Phenotypes of two-week-old wild type and <i>ngal1-1</i> homozygous seedlings. E. Floral tissues of five-week-old wild type and <i>ngal1-1</i> homozygous plants. F. Comparison of root phenotypes of one-week-old wild type and <i>ngal1-1</i> homozygous plants.</p

The cloning of <i>ABS2</i>.

<p>A. <i>abs2-1D</i> was genetically linked with T-DNA. Total leaf DNAs were extracted from 16 progenies of an <i>abs2-1D</i>/+ heterozygous plant. The DNAs were digested with <i>Hind</i>III and restriction fragments were separated with electrophoresis followed by transfer to a nylon membrane. The blot was probed with ³²P labeled <i>BAR</i> gene sequences. Plants that did not show <i>abs2-1D</i> phenotypes were indicated by arrows. B. Confirmation of a single T-DNA insertion in <i>abs2-1D</i>. Genomic DNAs from <i>abs2-1D</i> plants were digested with indicated restriction enzymes. After electrophoresis and transfer to a nylon membrane, the blot was hybridized with ³²P labeled <i>BAR</i> gene sequences. There is one <i>Eco</i>RI site in the probe sequence so two hybridizing bands were observed. C. Cloning of <i>abs2-1D</i>. In the <i>abs2-1D</i> mutant, activation tagging T-DNA was inserted between At2g36080 and At2g36090. Solid lines represent intergenic regions, while white boxes represent genes in the vicinity of the T-DNA insertion. The right border of the T-DNA was facing At2g36080. D. Semi-quantitative RT-PCR analysis of the expression levels of At2g36080 and At2g36090 in wild-type, <i>abs2-1D/+</i> heterozygous and <i>abs2-1D</i> homozygous mutants. <i>Actin2</i> expression was shown as a control. Total cellular RNAs were extracted from the aerial parts of two-week-old seedlings. 1 µg DNase I treated RNA from each sample was used for cDNA synthesis. RT-PCRs were performed with indicated numbers of cycles.</p

<i>NGAL1</i> tissue expression profile and NGAL1 protein localization.

<p>A. Expressions of <i>NGAL1</i> in different tissues of wild type plants were determined by semi-quantitative RT-PCR. Total RNAs were extracted from roots, two-week-old seedlings, rosette leaves, stems, cauline leaves, siliques and flower tissues and semi-quantitative RT-PCRs were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049861#pone-0049861-g002" target="_blank">Figure 2D</a>. <i>Actin2</i> expression was shown as a control. B–E. Tissue expression pattern of <i>NGAL1</i> examined by histo-chemical GUS staining. Illustrated are one-week-old seedling (B), flower (C), silique (D) and two-week-old seedling (E) from transgenic plants expressing <i>P_NGAL1::GUS</i> fusion construct. F. Nuclear localization of <i>NGAL1-GFP</i> fusion protein in Arabidopsis leaf protoplasts. Nuclei of protoplasts were stained by Hoechst 33342. GFP fluorescence and bright field (BF) images of Arabidopsis protoplasts were compared to show the sub-cellular localization of GFP (cytosol and nucleus) and NGAL1-GFP (nucleus).</p

Flower phenotypes of <i>NGAL1</i> OE lines.

<p>A. Floral tissues of wild type and <i>NGAL1</i> OE plants. Note the conspicuous absence of the flower petals in <i>NGAL1</i> OE lines. B–E. Individual flower phenotypes of wild type and <i>NGAL1</i> OE plants. Individual flowers from wild type (B), two <i>NGAL1</i> OE lines, OE-2 (C and E) and OE-3 (D), were shown. Note the filamentous structure found in some flowers from OE lines (pointed by the white arrow head).</p

Phenotypes of <i>NGAL1</i> over-expression lines.

<p>A. Phenotypes of two-week-old wild type and three independent <i>NGAL1</i> over-expression (OE) lines, OE-2, OE-3 and OE-4. B. Cotyledons and rosette leaves detached from two-week-old wild-type, OE-2, OE-3 and OE-4 plants. From left to right were two cotyledons and rosette leaves that were arranged in the order of their initiations. C. Comparison of the fifth rosette leaf of two-week-old wild type, OE-2, OE-3 and OE-4 plants (Bars, 2 mm). D. Semi-quantitative RT-PCR analysis of the expression levels of <i>NGAL1</i> in wild-type, <i>abs2-1D</i>/+ heterozygous, <i>abs2-1D</i> homozygous, OE-2, OE-3 and OE-4 plants. RT-PCRs were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049861#pone-0049861-g002" target="_blank">Figure 2D</a>.</p

The Over-Expression of Two Transcription Factors, ABS5/bHLH30 and ABS7/MYB101, Leads to Upwardly Curly Leaves

<div><p>Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental cues. We are interested in dissecting these regulatory circuits genetically and report here the isolation of two Arabidopsis dominant mutants, <i>abnormal shoot5-1D</i> (<i>abs5-1D</i>) and <i>abs7-1D</i> identified through activation tagging screens. Both <i>abs5-1D</i> and <i>abs7-1D</i> display an intriguing upwardly curly leaf phenotype. Molecular cloning showed that the elevated expression of a bHLH transcription factor ABS5/T5L1/bHLH30 or a MYB transcription factor ABS7/MYB101 is the cause for the abnormal leaf phenotypes found in <i>abs5-1D</i> or <i>abs7-1D</i>, respectively. Protoplast transient expression assays confirmed that both ABS5/T5L1 and ABS7/MYB101 are targeted to the nucleus. Interestingly, the expression domains of auxin response reporter <i>DR5::GUS</i> were abnormal in leaves of <i>abs5-1D</i> and <i>ABS5</i>/<i>T5L1</i> over-expression lines. Moreover, cotyledon venation analysis showed that more areoles and free-ending veins are formed in <i>abs5-1D</i>. We found that the epidermis-specific expressions of <i>ABS5</i>/<i>T5L1</i> or <i>ABS7</i>/<i>MYB101</i> driven by the Arabidopsis <i>Meristem Layer 1</i> promoter (<i>P_AtML1</i>) were sufficient to recapitulate the curly leaf phenotype of <i>abs5-1D</i> or <i>abs7-1D</i>. In addition, <i>P_AtML1::ABS5</i> lines exhibited similar changes in <i>DR5::GUS</i> expression patterns as those found in 35S-driven <i>ABS5</i>/<i>T5L1</i> over-expression lines. Our work demonstrated that enhanced expressions of two transcription factors, ABS5/T5L1 and ABS7/MYB101, are able to alter leaf lamina development and reinforce the notion that leaf epidermis plays critical roles in regulating plant organ morphogenesis.</p></div

Phenotypes of <i>abs5-1D</i>.

<p><b>A</b>. Leaf rosettes of five-week-old wild type and <i>abs5-1D</i> mutant. To have a clear view of the rosette leaves, the inflorescence stems were removed prior to photographing. <b>B–C</b>. Overview of the transverse sections of the eighth rosette leaf from three-week-old wild type (<b>B</b>) and <i>abs5-1D</i> (<b>C</b>). Bars: 500 µm. <b>D–E</b>. Transverse sections of the mid-vein regions of wild type (<b>D</b>) and <i>abs5-1D</i> (<b>E</b>) leaf. Bars: 50 µm.</p

Expression analysis of <i>ABS5</i>/<i>T5L1</i>.

<p><b>A</b>. Semi-quantitative RT-PCR analysis of <i>ABS5</i>/<i>T5L1</i> gene expression in various tissues: roots, two-week-old seedlings, rosette leaves, stems, cauline leaves, siliques and flowers. The expression of <i>Actin2</i> was used as a control. <b>B</b>. Nuclear localization of ABS5-GFP fusion protein in Arabidopsis leaf protoplasts. Wild type leaf protoplasts were transformed with <i>P_35S::GFP</i> or <i>P_35S::ABS5-GFP</i>. A single representative protoplast was shown for each transformation. Nuclei were specifically stained by the fluorescent dye Hoechst33342. Hoechst33342, GFP, chlorophyll autofluorescence signals were monitored by fluorescence microscopy. Bright field (BF) images served as controls for protoplast integrity. Bars: 10 µm.</p