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

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>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

<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

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 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

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

Differentially expressed unigenes with significantly enriched pathways.

<p>^f the number of differentially expressed unigenes (DEGs)</p><p>^g the number of all unigenes</p><p>Differentially expressed unigenes with significantly enriched pathways.</p

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

<div><p>Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.</p></div

Comparison of stamens and pistils of untreated (A to E) and chemical hybridization agent (CHA)-SQ-1-treated wheat plants (F to J).

<p>(A and F) the tetrad stage. (B and G) the early-uninucleate stage. (C and H) the later-uninucleate stage. (D and I) the binucleate stage. (E and J) the trinucleate stage. (E and J, top right) the 2% I₂-KI (top right) staining pollen grains. Scale bars are 0.5 mm in A to J and are 50 μm in the top right of E and J.</p