A New Allele of the SPIKE1 Locus Reveals Distinct Regulation of Trichome and Pavement Cell Development and Plant Growth

The single-celled trichomes of Arabidopsis thaliana have long served as an elegant model for elucidating the mechanisms of cell differentiation and morphogenesis due to their unique growth patterns. To identify new components in the genetic network that governs trichome development, we carried out exhaustive screens for additional Arabidopsis mutants with altered trichome morphology. Here, we report one mutant, aberrantly branched trichome1-1 (abt1-1), with a reduced trichome branching phenotype. After positional cloning, a point mutation in the SPIKE1 (SPK1) gene was identified in abt1-1. Further genetic complementation experiments confirmed that abt1-1 is a new allele of SPK1, so abt1-1 was renamed as spk1-7 according to the literatures. spk1-7 and two other spk1 mutant alleles, covering a spectrum of phenotypic severity, highlighted the distinct responses of developmental programs to different SPK1 mutations. Although null spk1 mutants are lethal and show defects in plant stature, trichome and epidermal pavement cell development, only trichome branching is affected in spk1-7. Surprisingly, we found that SPK1 is involved in the positioning of nuclei in the trichome cells. Lastly, through double mutant analysis, we found the coordinated regulation of trichome branching between SPK1 and two other trichome branching regulators, ANGUSTIFOLIA (AN) and ZWICHEL (ZWI). SPK1 might serve for the precise positioning of trichome nuclei, while AN and ZWI contribute to the formation of branch points through governing the cMTs dynamics. In summary, this study presented a fully viable new mutant allele of SPK1 and shed new light on the regulation of trichome branching and other developmental processes by SPK1

Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:β-glucuronidase reporter expression was down-regulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley (Hordeum vulgare) could act as a repressor on an intron sequence in the KNOX gene Hooded/Barley Knotted3 (Bkn3) and that AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis (Arabidopsis thaliana) could repress KNOTTED-LIKE FROM ARABIDOPSIS THALIANA2 (KNAT2) promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with down-regulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNA interference silencing of OsGRF3, OsGRF4, and OsGRF5 resulted in dwarfism, delayed growth and inflorescence formation, and up-regulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants

Enhanced Electric Conductivity Of Polymer-Derived Sicn Ceramics By Microwave Post-Treatment

The effect of microwave treatment on the electric conductivity and structure of a polymer-derived SiCN ceramic is studied. It is found that the conductivity of the microwave-treated sample is about 40 times higher than that of the conventional heat-treated one at the same temperature and dwell time conventionally. The X-ray diffraction patterns show that both samples are amorphous without obvious crystallization. Raman analysis reveals that the microwave-treated sample exhibited a narrower full width at half maximum and upper-shift of G peak. X-ray photoelectron spectroscopy spectra show that there is a significant sp3-to-sp2 transition of free carbon in the microwave-treated sample. These results suggest that the microwave-treatment can induce a distinct structure evolution of the free carbon, which contributes to the remarkable enhancement of the conductivity of the sample

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

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

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

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

<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