text:
These authors contributed equally to this work.
Original Article
MULTIPASS, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways
Article first published online: 26 AUG 2013
DOI: 10.1111/tpj.12286
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd
The Plant Journal
Volume 76, Issue 2, pages 258–273, October 2013
Additional Information
How to Cite
Schmidt, R., Schippers, J. H. M., Mieulet, D., Obata, T., Fernie, A. R., Guiderdoni, E. and Mueller-Roeber, B. (2013), MULTIPASS, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways. The Plant Journal, 76: 258–273. doi: 10.1111/tpj.12286
Publication History
- Issue published online: 5 OCT 2013
- Article first published online: 26 AUG 2013
- Accepted manuscript online: 15 JUL 2013 09:58AM EST
- Manuscript Accepted: 10 JUL 2013
- Manuscript Revised: 7 JUL 2013
- Manuscript Received: 12 MAY 2013
Funded by
- ERA-NET Plant Genomics program. Grant Numbers: 0313993A, ANR-06-ERAPG-005-01, Federal Ministry of Education and Research in Germany
- Agence Nationale de la Recherche in France
- RicE Functional Genomics
- Agropolis Foundation
- development;
- expansin;
- transcription;
- Oryza sativa ;
- hormone;
- abiotic stress
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Growth regulation is an important aspect of plant adaptation during environmental perturbations. Here, the role of MULTIPASS (OsMPS), an R2R3-type MYB transcription factor of rice, was explored. OsMPS is induced by salt stress and expressed in vegetative and reproductive tissues. Over-expression of OsMPS reduces growth under non-stress conditions, while knockdown plants display increased biomass. OsMPS expression is induced by abscisic acid and cytokinin, but is repressed by auxin, gibberellin and brassinolide. Growth retardation caused by OsMPS over-expression is partially restored by auxin application. Expression profiling revealed that OsMPS negatively regulates the expression of EXPANSIN (EXP) and cell-wall biosynthesis as well as phytohormone signaling genes. Furthermore, the expression of OsMPS-dependent genes is regulated by auxin, cytokinin and abscisic acid. Moreover, we show that OsMPS is a direct upstream regulator of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3, OsEXPB6 and the endoglucanase genes OsGLU5 and OsGLU14. The multiple responses of OsMPS and its target genes to various hormones suggest an integrative function of OsMPS in the cross-talk between phytohormones and the environment to regulate adaptive growth.
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Plant hormones shape plants by modulating growth in response to endogenous and environmental cues (Wolters and Jürgens, 2009). Adaptation to environmental conditions occurs largely through hormone homeostasis (Achard et al., 2006; Carabelli et al., 2007; Park et al., 2007). Major developmental growth regulators include auxin, brassinolide (BL), cytokinin (CK) and gibberellic acid (GA), whereas abscisic acid (ABA) and ethylene are often implicated in stress responses (Wolters and Jürgens, 2009). Under unfavorable conditions, plants produce stress hormones to promote survival. These hormones are essential for modulating the redistribution of resources from growth to plant protection or defense (Wang et al., 2007; Wilkinson et al., 2012).
Auxin and GA are positive plant growth regulators that may be counteracted by stress hormones such as ABA (Park et al., 2007; Yaish et al., 2010). ABA synthesis is induced by salt stress in rice (Oryza sativa) (Moons et al., 1995), with the level and duration correlating with salt tolerance. In Arabidopsis thaliana, over-expression of a GH3 enzyme gene involved in auxin inactivation results in reduced levels of free auxin and increased stress tolerance (Park et al., 2007). During abiotic stress, expression of various GH3 genes is modulated by ABA. A connection between auxin and ABA has also been implied for the rice transcription factor (TF) ABI5-like1 (ABL1), which controls stress-responsive genes through ABA-responsive elements in their promoters. The abl1 mutant shows reduced ABA responsiveness but hypersensitivity to auxin (Yang et al., 2011). Another example of cross-talk is provided by the TF OsAP2–39, which is a positive regulator of ABA biosynthesis but also induces expression of ELONGATED UPPERMOST INTERNODE (EUI), encoding a GA-deactivating enzyme (Yaish et al., 2010). ABA also affects the expression of CK biosynthesis and breakdown genes in rice roots (Tsai et al., 2012). The interactions between phytohormones and their regulation at the genetic level are still poorly understood in rice, but have become a major focus in current crop breeding strategies (Wilkinson et al., 2012).
The adaptive growth response at the genetic level is executed by stress-responsive TFs. After the initial growth cessation upon salinity stress, the development of the root system is adapted to reach out for novel water sources. Expansins (EXPs) act as positive regulators of growth, and their expression is induced in elongating tissues (Cho and Kende, 1997; Lee and Kende, 2001). Based on phylogenetic analysis, EXPs are grouped into two related classes: α- and β–EXPs (Cosgrove, 2000). Grasses such as rice contain a far greater number of β–EXP genes than dicots such as Arabidopsis (Lee et al., 2001). Inducible over-expression of OsEXPA4 stimulates plant growth by increasing cell size, while knockdown diminishes plant growth (Choi et al., 2003). OsEXPB3 was shown to be localized to the primary cell wall of the vascular system (Lee and Choi, 2005). Phytohormones are implicated in the regulation of EXP gene expression, as cis-elements specific to GA, auxin, ABA and ethylene are commonly found in the promoters of most EXP genes (Lee et al., 2001). Not surprisingly, the expression of EXP genes is down-regulated during growth under saline conditions (Walia et al., 2005). However, the down-regulation of cell wall-modifying genes is transient. During drought stress, the plant modulates root growth by activating EXPs and xyloglucan:xyloglucosyl transferases in a spatio-temporal manner (Harb et al., 2010; Sasidharan et al., 2011). Thus, modulation of cell-wall extensibility during abiotic stress is an important aspect of the adaptive growth response.
Here we studied the molecular mechanism by which the R2R3-type MYB TF encoded by MULTIPASS (OsMPS) controls adaptive growth. Expression of OsMPS is repressed by the growth-promoting hormones auxin, GA and BL, and growth defects of OsMPS over-expression plants are partially rescued by auxin application. Furthermore, we showed that OsMPS is a direct regulator of genes encoding expansins, and propose that it functions as a hub for multiple hormones to control plant growth.
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
OsMPS is a salt-responsive R2R3-type MYB transcription factor gene from rice
OsMPS (Os02g40530) was identified in a screen for TF genes in rice roots (cv. Nipponbare) responding to short-term salt stress. OsMPS expression is induced after 30 min and 1 h of salt stress (Figure 1a). Up-regulation of OsMPS was also observed in leaves after salt stress (Figure S1). The OsMPS locus spans three exons that form an open reading frame of 870 nucleotides (Figure 1d,e). OsMPS is classified as an R2R3-type MYB TF belonging to clade C14 (Zhang et al., 2012a). This clade includes AtMYB71, AtMYB79 and AtMYB121 from Arabidopsis and two uncharacterized rice MYB TFs (Os04g42950 and Os03g04900). Using the web tool Phytozome (Goodstein et al., 2012), homologous proteins from Sorghum bicolor, Zea mays and Setaria italica were identified and shown to share high sequence identity with OsMPS at the DNA-binding domain (Figure 1f). OsMPS contains a putative nuclear localization signal immediately downstream of the R2R3 domain, and transient expression of the OsMPS coding sequence fused at its 3′ end to the CFP-encoding gene in rice protoplasts revealed a fluorescence signal in the nucleus (Figure 1c).
OsMPS is expressed in vegetative and reproductive tissues
The expression profile of OsMPS was analyzed using the PlaNet Browser (Mutwil et al., 2011). OsMPS is expressed in most tissues, including shoots, roots, anthers and seeds, but not in endosperm, stigma, ovary or the embryo (Figure S2). To examine the tissue-specific expression of OsMPS in planta, a 2 kb sequence directly upstream of the transcriptional start site was cloned and used to drive GUS expression. OsMPS expression was detected in almost all tissues examined (Figure 2). In reproductive tissues, GUS activity was present in the anthers and vascular tissue of the spikelet (Figure 2a,b). Furthermore, OsMPS is expressed in the vascular tissues of the coleoptile and young leaves (Figure 2c,d). GUS activity was detected in the main root and root hairs, but was absent from the growing zone (Figure 2e–g). GUS activity spreads throughout the leaf at the onset of senescence and is wound-inducible (Figure 2h,i). In agreement with the expression data for OsMPS, the roots showed increased GUS activity after 2 h of salt stress (Figure 1b).
OsMPS affects plant growth
To characterize the function of OsMPS in rice, over-expression (OE) and knockdown (KD) lines were generated. For each construct, at least 15 independent lines were established. Based on the expression level of OsMPS, several non-segregating lines (T3 generation) were selected for characterization, including OE1–2, OE2–5, OE6–2 and OE6–5 for over-expression, and KD4–5, KD6–3 and KD9–2 for knockdown (Figure S3).
Over-expression of OsMPS resulted in decreased plant stature at the seedling, vegetative and heading stage (Figure 3a–c). Shoots and roots of OE1–2, OE2–5 and OE6–2 seedlings were significantly shorter than those of empty-vector (EV) seedlings at both 4 and 7 days after sowing (Figure 3d,g), whereas KD6–3 and KD9-2 seedlings had longer roots at 4 or 7 days after sowing, respectively. Upon salt stress, both KD and OE lines showed a shorter shoot and/or root length compared to EV seedlings at 4 and/or 7 days after sowing (Figure 3e,h). As OsMPS over-expression plants were severely reduced in size under control conditions, we determined the relative growth reduction upon salt stress. Interestingly, relative shoot and/or root growth of OE1–2, OE2–5 and OE6–2 seedlings was significantly less affected by salt stress than in EV seedlings (Figure 3f,i). In contrast, KD lines showed a more pronounced relative reduction in shoot and/or root length under salt stress than the EV line.
At the age of 4 weeks, OE plants showed a reduced root size, while KD plants showed an enlarged root structure (Figure 3b). The fresh weight (FW) and dry weight (DW) of OE6–2 shoots and roots were reduced compared with EV plants, while KD4–5 and KD6–3 showed an increased weight for the root and/or shoot (Figure 3j,m). We examined the effect of OsMPS on biomass accumulation under salt stress using 4-week-old plants. Roots and/or shoots of both OE6–2 and KD4–5 displayed a reduced weight compared to the EV control line after 1 week of salt stress (Figure 3k,n). Similarly, KD6–3 plants showed reduced root FW after salt stress, but had a higher shoot FW than the EV line. However, the relative reduction in DW of roots and shoots observed in lines KD4–5 and KD6–3 was stronger than in stressed EV plants (Figure 3l,o). In contrast, the relative root biomass of stressed OE-6–2 plants was less reduced than in EV plants.
As OsMPS affects plant stature throughout its life cycle, we determined epidermal cell sizes of the 2nd leaf of the main tiller of 6-week-old plants. Cells of KD4–5 leaves at the tip and middle region were significantly larger and longer than those of EV leaves (Figure 4). In contrast, over-expression of OsMPS resulted in decreased cell size and length.
OsMPS mis-expression causes metabolic changes related to growth
Metabolism underlies plant development during both optimal and adverse environmental conditions (Stitt et al., 2010; Ribeiro et al., 2012). To determine the effect of OsMPS expression on metabolite levels, a GC–MS-based analysis of primary metabolites in roots and leaves of 4-week-old plants was performed.
With respect to amino acids, OE6–2 roots accumulated significantly higher levels of Pro, Gly, Ile, Lys, Phe, Thr, Tyr and Val than the EV line under control conditions (Table S1). Of note, Phe and Tyr are precursors for the production of phenylpropanoids and the subsequent generation of lignin (Gray et al., 2012). Interestingly, Asp was significantly decreased in OE6–2 roots but increased in KD4–5, while the levels of γ-aminobutyric acid (GABA) were down-regulated in both lines compared to EV roots (Table S1). After 24 h of salt stress, the levels of Ala, Asp, GABA, Glu, Gln, Lys and Thr significantly increased in EV roots compared to control conditions. In the KD4–5 and OE6–2 lines, Asp, GABA, Glu, and Gln increased compared to their levels under control conditions; however, Ala, Lys and Thr did not significantly change in the KD4–5 and OE6–2 lines after salt stress. Furthermore, the levels of Gly, Ile, Pro and Val, which are higher in OE6–2 roots under control conditions, decreased significantly upon salt stress, although not below EV levels (Table S1).
Similar to roots, most amino acids accumulated under control conditions in OE6–2 leaves (Table S2). In EV leaves, only Gly was decreased upon salt stress, whereas, in KD4–5 leaves, lower levels of Ala, GABA, Gly, His, Ile, Pro and Val were observed compared to non-stress conditions. Furthermore, up-regulation of Asp, Ser and Thr was found in OE6–2 leaves.
Roots of OE6–2 plants exhibited higher levels of glucose and trehalose under control conditions, whereas roots of both KD4–5 and OE6–2 had lower levels of raffinose and galactinol. Upon salt stress, raffinose decreased in all lines tested, while galactinol was lower in EV and KD4–5 roots after salt stress as compared to their respective controls. Furthermore, leaves of OE6–2 showed higher levels of most measured sugars compared to EV (Table S2). In leaves, a significant decrease in glucose, fructose, galactose and xylose for KD4–5 and OE6–2 was found as compared to EV plants, but the levels of these metabolites in OE6–2 did not decrease below those of the EV line, whereas this did occur for KD4–5.
Interestingly, in roots and/or shoots of OE6–2 plants, a low level of the tricarboxylic acid cycle intermediates 2–oxoglutarate, malate and fumarate was observed. Tricarboxylic acid cycle intermediates are directly linked to plant development as their abundance is a potential growth signal (van der Merwe et al., 2010; Finkemeier et al., 2013). Upon salt stress, a general increase in tricarboxylic acid cycle intermediates in the roots of all lines examined was observed. In leaves, only the levels of malate in KD4–5 and EV lines decreased significantly upon salt stress compared to their controls.
OsMPS affects heading date and panicle-related traits
OsMPS is expressed in anthers and spikelets (Figure 2a,b), suggesting a role in reproductive development. OE6–2 plants showed a delay in heading and flowering by 21 days, and a reduced number of panicles per plant compared to the EV line (Figure 5a–c). Down-regulation of OsMPS caused early heading and flowering (Figure 5a,b) but KD4–5 plants developed fewer panicles per plant (Figure 5c). On the other hand, altered OsMPS expression did not affect panicle length or the number of spikelets per panicle (Figure 5d,e) but caused a reduction in the number of grains per panicle and plant (Figure 5f,g). Furthermore, grains of OE1–2, OE6–2 and OE6–5 lines were significantly reduced in length and weight compared to EV grains, while those of KD4–5 and KD9-2 showed an increase in length (Figure 5i–k). In combination with the reduced panicle number, OE6–2 plants had a grain yield of 9% and KD4–5 plants had a grain yield of 72% relative to EV plants (Figure 5h).
OsMPS expression is regulated by plant hormones
The involvement of plant hormones in the regulation of OsMPS expression was analyzed in silico and in vivo (Figure 6). The OsMPS promoter used for GUS analysis contains three ABA-responsive elements (ACGT[G/T]), one ARF-specific auxin-response element (AuxRE; TGTCTC) and one GA-responsive element (GARE; TAACAGA) (Figure 6a). ABA induced expression of OsMPS after 2 and 6 h of treatment, whereas auxin (naphthalene-1–acetic acid, NAA) and GA3 transiently repressed OsMPS expression in roots (Figure 6b). CK (6–benzylaminopurine) induced OsMPS expression at 2 and 6 h of treatment, and epi-brassinolide (epi-BL) transiently repressed it. After 6 h of treatment with auxin, GA or epi-BL, the expression of OsMPS was not significantly different from control treatments. These observations indicate that OsMPS acts downstream of multiple hormonal pathways.
Auxin promotes growth by stimulating cell elongation (Zhang et al., 2008), although at high levels, it may inhibit growth. As OsMPS is repressed by auxin, we tested whether growth retardation of OsMPS over-expression seedlings may be rescued by auxin application. Treatment with NAA (0.1 μm; 7 days) stimulated shoot and root growth of OsMPS OE lines and partially restored plant size (Figure 6c,d). EV seedlings appeared unaffected by the minimal level of exogenous NAA used. To assess the involvement of OsMPS in the response to ABA, seedlings were grown for 7 days on MS medium containing 5 μm ABA (Figure 6e). The shoot length of KD4–5 plants was moderately reduced, whereas it was significantly increased in the case of OE6–2 plants relative to EV seedlings.
OsMPS modulates the expression of hormone and cell wall-related genes
An initial microarray analysis on roots of OE6–2, KD4–5 and EV plants was performed to identify genes regulated by OsMPS. This resulted in the identification of 247 genes showing a ≥two-fold down-regulation in OE plants and a ≥two-fold up-regulation in KD plants compared to EV. GO enrichment analysis using PLAZA (Proost et al., 2009) revealed that, among others, the biological processes 'sexual reproduction' and 'cell wall organization/biogenesis' were significantly enriched (Table S3). Furthermore, 460 genes were ≥two-fold up-regulated in OE roots but ≥two-fold repressed in KD roots. GO enrichment analysis on these genes revealed a role in metabolic processes (Table S4). Based on the functional annotation, a subset of genes was selected for quantitative RT–PCR analysis to confirm the microarray data and obtain insight into the function of OsMPS (Table S5). Auxin-related genes, including indole-3–acetic acid inducible protein (OsIAA) genes (i.e. OsIAA4, OsIAA12 and OsIAA20) and auxin response factor (ARF) genes (i.e. ARF10 and ARF19) were found to be repressed in OE6–2 but induced in KD roots (Figure 7a). Furthermore, ARF4 and ARF21 were repressed in OE6–2, and OsIAA11 and OsIAA26 were induced in KD4–5. Moreover, BR biosynthesis and signaling genes were found to be down-regulated upon over-expression of OsMPS (Figure 7a). These genes include OsBRI1, OsBAK1, OsBZR1, OsLIC, OsIBH1 and OsBSL2 (Bai et al., 2007; Wang et al., 2008; Zhang et al., 2009, 2012b), and an EXORDIUM ortholog (Os02g5200; Schröder et al., 2009). In KD lines, only the BR biosynthesis gene OsDWARF11 (Tanabe et al., 2005) showed increased expression. The expression of ACO1 and ACO3, catalyzing the oxidation of 1–aminocyclopropane-1–carboxylic acid to ethylene (Iwamoto et al., 2011), was up-regulated in roots of KD4–5, while expression of OsABA8ox2, encoding an ABA-inactivating enzyme (Zhu et al., 2009), was repressed in OsMPS over-expression roots.
Cell expansion requires cell-wall relaxation, which is achieved by the action of EXPs, endoglucanases (GLUs) and yieldins (Hayashi et al., 1984; McQueen-Mason et al., 1992; Okamoto and Okamoto, 1995). Seven EXP genes were found to be differentially expressed (Figure 7b). OsEXPB3 and OsEXPB6 were strongly induced in KD4–5 roots, but repressed in OE6–2 roots. In addition, OsEXPA4, OsEXPA8 and OsEXPA22 were down-regulated in OE6–2, indicating that OsMPS negatively regulates EXP expression. Furthermore, OsEXPB2 was significantly induced in KD4–5, whereas OsEXPA14 was repressed in both the OE and KD lines. Additionally, OsGLU5 and OsGLU14, which are expressed during lateral root development (Yoshida and Komae, 2006), were induced in KD4–5 roots and/or decreased in OE6–2 (Figure 7c). Reactive oxygen species contribute to cell growth by loosening the cell-wall matrix (Passardi et al., 2004). Enzymes responsible for the production of reactive oxygen species include cell wall-located class III peroxidases (PRXs). Eight PRX genes were found to be significantly down-regulated in OE6–2, while four PRX genes were induced in KD4–5 (Figure 7d). Taken together, the expression analysis supports a role for OsMPS in controlling cell wall-related genes.
Co-expression network analysis reveals a potential role of OsMPS in the regulation of cell-wall remodeling
In order to evaluate whether the genes regulated by OsMPS have an intrinsic association with each other, an extensive co-expression analysis was undertaken using the RiceFREND and ATTED–II web servers (Obayashi et al., 2011; Sato et al., 2013). EXPB genes (OsEXPB2, OsEXPB3 and OsEXPB6) were used as guide genes for both tools. Although RiceFREND is restricted to displaying small gene networks (100 genes), the analysis demonstrated that OsEXPB genes are highly co-regulated with other cell wall-related genes (Figure S4 and Methods S1). Furthermore, expression of the selected EXPB genes was intimately connected with expression of other EXP, PRX, xyloglucan:xyloglucosyl transferase and auxin-related genes, and several ethylene biosynthesis genes (Tables S6 and S7). The obtained co-expressed genes were cross-referenced with the OsMPS microarray data. Many genes co-expressed with OsEXPB2, OsEXPB3 and OsEXPB6 were also differentially expressed in OE6–2 and/or KD4–5. These genes include the EXP genes OsEXPA4 and OsEXPA8, the cell-wall genes OsGLU14 and OsCESA3, the PRX genes OsPRX45, OsPRX46, OsPRX65, OsPRX95, OsPRX108, Os02g14170 and Os08g42030, and the auxin-related genes OsIAA12, OsIAA14 and OsIAA23. As the co-expression associations overlap with OsMPS-dependent genes identified by the microarray studies, the results support the notion that OsMPS functions as a regulator of these genes. Indeed, mining of expression data revealed that OsMPS induction during cold, drought and salt stress co-occurs with down-regulation of genes co-expressed with β–class EXP genes (Figure S4).
The effect of OsMPS on gene expression may be mimicked by auxin, CK and ABA applications
As OsMPS expression is modulated by several hormones, the response of its potential target genes towards auxin, BL, GA, CK and ABA was tested (Figure 7). Down-regulation of OsMPS affected the expression of auxin-related genes in a similar manner as auxin treatment. Five OsIAA genes, ARF10, ARF19 and to some extent two indole-3–acetic acid-amido synthetase genes involved in auxin inactivation (OsGH3.3 and OsGH3.13) were up-regulated after auxin treatment (Figure 7a). GA application also induced several OsIAA genes. Repression of auxin-related genes as in OsMPS over-expression plants was observed in the wild-type after CK treatment. Application of CK reduced the expression of OsGH3.3, OsGH3.13, OsIAA4, OsIAA11, ARF10 and ARF21. Similarly, ABA treatment caused down-regulation of OsGH3.13, OsIAA11 and OsIAA12, while OsIAA20 was induced (Figure 7a). The down-regulation of BR-related genes in OsMPS over-expression plants was only weakly reproduced by hormone treatments.
In OsMPS knockdown plants, several EXP genes were up-regulated, of which OsEXPA4, OsEXPB3 and OsEXPB6 were auxin-inducible. In contrast, OsEXPB2 and OsEXPA14 were repressed after GA treatment, while epi-BL application did not show an effect. Repression of EXP as observed by OsMPS over-expression was also observed after ABA or CK treatment. Both hormones down-regulated the expression of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3 and OsEXPB6, while OsEXPA14 expression was exclusively repressed by CK. In contrast, OsEXPA22 was found to be induced by ABA and CK (Figure 7b). Several cell wall-related genes, including OsGLU5, OsGLU14 and a cellulase-like protein-encoding gene (CSLA1) were auxin-inducible (Figure 7c). The down-regulation of cell wall-related genes in OsMPS over-expression plants was reproduced by CK and ABA treatment. CK treatment resulted in down-regulation of OsGLU5, OsGLU14, CSLA1 and OsCESA3. ABA treatment repressed OsGLU5 and OsGLU14 and weakly down-regulated OsCESA3 (Figure 7c).
Increased expression of OsVIVIPAROUS1 (OsVP1) and CSLD5, as in OE6–2, was observed after CK or GA treatment of wild-type roots (Figure 7c,e). Down-regulation of PRX gene expression by OsMPS over-expression was also observed after treatment with CK or ABA (Figure 7d). Both hormones repressed OsPRX95, OsPRX104 and Os02g14170. In addition, OsPRX45, OsPRX77, OsPRX108 and Os06g48020 were down-regulated only by CK application. While over-expression of OsMPS repressed OsPRX46 and OsPRX52, ABA and CK induced the expression of these genes. Epi-BL treatment affected the expression of OsPRX45, OsPRX95 and OsPRX108 (Figure 7d). Thus, the effect of OsMPS on gene expression overlaps with the response to ABA, CK and auxin.
OsMPS binds to the promoter of expansin and endoglucanase genes
The Arabidopsis OsMPS homolog AtMYB71 was shown to bind a 'TAACTG' DNA sequence (Wang et al., 2002). Therefore, promoter sequences (1.5 kb) of genes differentially expressed in OsMPS transgenic plants were screened for the presence of the AtMYB71 binding site (Figures 8 and S5). Two α–class EXP genes, OsEXPA4 and OsEXPA8, and three β–class EXP genes, OsEXPB2, OsEXPB3 and OsEXPB6, contain the cis-element. Furthermore, several genes involved in cell-wall biosynthesis/modification, e.g. OsGLU5 and OsGLU14, possess the AtMYB71 binding site. We tested whether OsMPS interacts with the 'TAACTG' motif by an electrophoretic mobility shift assay (EMSA) using probes that span the binding site (Figure 8a,b). Incubation of OsMPS with OsEXPA4 promoter probes caused a band shift, which was diminished by addition of unlabeled probe (Figure 8c). Additionally, we observed that OsMPS binds to DNA probes containing the 'TAACTG' motif from the promoters of OsEXPB3, OsEXPB6, OsGLU5 and OsGLU14. No retention was observed when probes were incubated with OsMPS-free protein extract, indicating the specificity of the reaction.
To determine whether OsMPS regulates its proposed target genes in vivo, a transient chromatin immunoprecipitation assay coupled to quantitative PCR (ChIP-qPCR) was performed on wild-type rice protoplasts transformed with the 35S:OsMPS-CFP construct (Figure 8d). OsMPS binds to the promoter of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3 and OsEXP6. Furthermore, enrichment for OsGLU5 and OsGLU14 promoter fragments spanning the AtMYB71-specific binding site was observed, indicating that OsMPS directly modulates the expression of genes involved in cell-wall modulation. In contrast, no enrichment was detected for three negative controls covering upstream sequences from OsEXPA4 (−1.7 kb), OsGLU5 (−2.4 kb) and OsGLU14 (−2.1 kb) that lack the cis-element (Figure 8d).
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Plant hormones allow plants to adapt to changing environments by mediating growth, development and nutrient distribution (Peleg and Blumwald, 2011). Although hormone response pathways have been elucidated in detail, only little is known with respect to molecular components that regulate cross-talk (Depuydt and Hardtke, 2011). Here, we characterized OsMPS and revealed that it may form a common hub for cross-talk between auxin and ABA/CK to control adaptive growth.
TFs play a central role in the regulation of plant responses towards adverse growth conditions (Wolters and Jürgens, 2009). R2R3-MYB proteins are well known for their role in abiotic stress tolerance and development (Park et al., 2010; El-Kereamy et al., 2012; Makkena et al., 2012; Yang et al., 2012). In the absence of stress, knockdown of OsMPS increased biomass accumulation, while OsMPS over-expression impaired growth. However, down-regulation of OsMPS negatively affected biomass accumulation under salt stress, while over-expression resulted in a less severe relative biomass reduction (Figure 3). Metabolic profiling revealed that OsMPS over-expression results in accumulation of amino acids and decreased levels of tricarboxylic acid cycle intermediates, which may indicate a lower rate of protein synthesis and metabolism, correlating with the smaller plant size observed (Stitt et al., 2010; Ribeiro et al., 2012).
OsMPS is transiently induced during the initial phase of salinity stress (Figure 1a), in agreement with the occurrence of growth cessation as the first response towards stress (Chaves et al., 2009). Similar to mis-expression of OsMYB4 (Park et al., 2010), over-expression or knockdown of OsMPS had costly trade-offs regarding panicle development and grain yield. Although their panicles had a similar number of spikelets, a significant decrease in grain number was observed for both lines (Figure 5e,f). A potential role for OsMPS in seed setting is supported by the presence of GUS activity in anthers of reporter lines (Figure 2a). Previously, it was demonstrated that root-specific over-expression of OsNAC10 improves grain yield in rice, while its ectopic expression negatively affects grain filling (Jeong et al., 2010). Therefore, it would be of interest to generate cell type- or tissue-specific OsMPS over-expression lines.
OsMPS expression is modulated by multiple hormones. Auxin, BL and GA, which are known to promote cell elongation (Depuydt and Hardtke, 2011), transiently decrease the expression of OsMPS. Consistently, OsMPS knockdown plants showed increased size and length of leaf epidermal cells (Figure 4). On the other hand, OsMPS is induced by CK and ABA (Figure 6b), and over-expression reduced leaf cell size (Figure 4). The promoter of OsMPS contains ABA-responsive, AuxRE and GARE regulatory elements, suggesting a direct link with upstream TFs controlled by these hormones. The ABA-responsive element is a known recognition site for AREB/ABF TFs including OsABF2 and ABL1, which are important positive regulators of ABA signaling and abiotic stress responses in rice (Zou et al., 2008; Yang et al., 2011).
OsMPS negatively regulates the expression of several ARF and IAA genes (Table S5 and Figure 7a). Interestingly, the growth inhibition of OsMPS over-expression lines was partially rescued by low concentrations of exogenous auxin (Figure 6c–e). In agreement, over-expression of OsIAA11, which is induced in roots of OsMPS knockdown plants, causes an increased crown root length and thickness (Zhu et al., 2012). Furthermore, the ARF1 target gene CROWN ROOTLESS1 (CRL1) is essential for crown root growth (Inukai et al., 2005). Hence, the observed effect of OsMPS on root growth may be through differential expression of auxin-related genes. CK is known to have negative effects on de novo auxin-induced root formation (Kitomi et al., 2011). Interestingly, the expression pattern of AUX/IAA genes in OsMPS over-expression plants is similar to that of wild-type seedlings treated with CK (Figure 7a). In rice, the crown rootless5 (crl5) mutant is impaired in root growth. CRL5 encodes an AP2/ERF TF that is induced by auxin and activates the expression of OsRR1, a negative regulator of CK signaling. Over-expression of either CRL5 or OsRR1 reduces CK sensitivity and increases root formation (Kitomi et al., 2011). Thus, an altered balance between CK and auxin may be responsible for the observed effect of OsMPS on root growth.
A prerequisite for cell elongation is cell-wall loosening to allow for morphological change (Depuydt and Hardtke, 2011). Auxin-induced acidification of the cell wall activates EXPs, which loosen the non-covalent bonds between polysaccharides allowing turgor-driven growth (Rayle and Cleland, 1992; Nakayama et al., 2012). Seven EXP genes were found to be repressed by OsMPS. Moreover, OsMPS binds in vivo to the promoters of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3 and OsEXPB6 (Figure 8d). OsEXPA4 has been reported to enhance plant growth when over-expressed (Choi et al., 2003). Interestingly, all EXP genes directly regulated by OsMPS are repressed by ABA (Figure 7b) and short-term salt stress (Figure S4). We propose that down-regulation of EXP genes during the initial phase of salt stress is mediated by ABA through induction of OsMPS. In addition, OsMPS directly controls the expression of OsGLU5 and OsGLU14 (Figure 8). These GLUs hydrolyze the two major hemicelluloses in type II cell walls (Yoshida and Komae, 2006). OsGLU5 (OsCEL9A) and its paralog OsGLU14 (OsCEL9F) have been previously reported to be auxin-inducible genes that are expressed during lateral root formation (Yoshida et al., 2006). Thus, OsGLU5 and OsGLU14 are positive regulators of growth whose expression is restricted during abiotic stress.
In summary, this study shows that OsMPS is induced by salt stress and the phytohormones ABA and CK (Figure 9). In contrast, hormones promoting growth down-regulate OsMPS. OsMPS acts as a negative regulator of auxin-related genes and cell wall-remodeling genes. The expression level of OsMPS negatively correlates with root biomass accumulation. As root growth is controlled by the balance between CK and auxin, OsMPS may function as a novel mediator between these two hormonal pathways. Taken together, the results show that OsMPS is required for the adaptive growth response in rice during adverse environmental conditions.
Experimental procedures
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Plant material and growth conditions
Analysis was performed using non-segregating transgenic lines (T3 generation) established in the rice Nipponbare background. Soil-grown plants were cultured in phytotrons as described by Degenkolbe et al. (2009). Growth on MS medium or in hydroponic culture was performed as described by Schmidt et al. (2012). Plants were grown at 26/22°C and 75/70% relative humidity (day/night) with a day length of 12 h and a light intensity of 700 μmol m−2 sec−1.
Constructs and rice transformation
The artificial microRNA specific to OsMPS was designed as described by Warthmann et al. (2008) and cloned into the pC5300 OE vector as described previously (Schmidt et al., 2012). For over-expression, the OsMPS coding sequence was amplified by PCR and recombined into pC5300. For the OsMPS:GUS construct, a 2 kb promoter sequence upstream of the transcriptional start site was cloned into pMDC162 (Curtis and Grossniklaus, 2003) using a two-step cloning strategy involving pENTR/D–TOPO (Invitrogen, www.invitrogen.com). Constructs were transformed into rice calli (cv. Nipponbare) via Agrobacterium tumefaciens strain EHA105 as described by Sallaud et al. (2003). To generate 35S:OsMPS-CFP, the coding sequence of OsMPS was cloned into the pGHPGWC vector (Zhong et al., 2008). Protoplast isolation and transformation were performed as described by Zhang et al. (2011). Fluorescence imaging was performed using a confocal laser scanning microscope (SP5; Leica Microsystems, www.leica-microsystems.com). Primers used for cloning are listed in Table S8.
RNA extraction and expression analysis
RNA isolation, cDNA synthesis and expression profiling using quantitative RT–PCR were performed as described previously (Schmidt et al., 2012). Rice ACTIN (Os03g50885) was used as an internal control (Figueiredo et al., 2012). Oligonucleotide sequences used for expression profiling of OsMPS and OsMPS-dependent genes were designed using QuantPrime (Arvidsson et al., 2008) and are listed in Table S9. For microarray analysis, RNA extracted from roots of 4-week-old hydroponically grown plants was hybridized to the Affymetrix 57k Rice Genome GeneChip (Affymetrix, www.affymetrix.com). To identify differentially expressed genes, the log2 transformed signal ratio of each gene was calculated, and a log2 (ratio) ≥1.0 or ≤0.5 was used as the cut-off. All probe sets matching transposable elements were removed from the dataset. Probes were considered for further analysis irrespective of detection call (P: present; M: marginal; A: absent).
Salt and hormone treatments
Dehulled seeds were surface-sterilized as described by Schmidt et al. (2012) and placed on vertical square plates with MS medium containing 100 mm NaCl, 1 μm ABA or 0.1 μm NAA. For estimation of biomass reduction under salt stress (7 days, 100 mm NaCl), plants were grown hydroponically and analyzed as described previously (Schmidt et al., 2013). For expression analysis, 2-week-old hydroponically grown wild-type seedlings were treated with 10 μm NAA, 50 μm GA3, 10 μm epi-BL, 10 μm 6–benzylaminopurine or 50 μm ABA for 2 or 6 h. Roots were harvested and analyzed in triplicate.
Detection of GUS activity
OsMPS:GUS plants (T2 generation) were stained overnight in GUS solution as described by Jefferson (1987).
Cell size measurements
Measurements of leaf blade epidermal cell size and length were performed as described by Barrôco et al. (2006) using CellP software (Olympus, http://www.olympus-europa.com) after imaging with an Olympus BX51 microscope (Olympus) using differential interference contrast optics.
Metabolite profiling analysis
GC–MS analysis was performed on roots and leaves of 4-week-old hydroponically grown plants. Extraction and derivatization of metabolites from tissues for GC-MS analysis were performed exactly as described by Lisec et al. (2006). Peak detection, retention time alignment and library matching were performed using TagFinder 4.0 (Luedemann et al., 2012). Metabolites were quantified based on the peak intensity of a selective mass. The amount of metabolites was analyzed as the relative metabolite level calculated by normalization of the peak intensity to that of the internal standard ribitol and the fresh weight.
Electrophoretic mobility shift assay and ChIP-qPCR
Electrophoretic mobility shift assays were performed using in vitro expressed OsMPS protein as described by Schmidt et al. (2013). To this end, the OsMPS coding sequence was cloned into the pF3A WG Flexi vector (Promega, www.promega.com) and translated using TNT® SP6 High Yield Wheat Germ Mastermix containing 1 μl FluoroTec Green Lys (Promega). EMSAs were performed using Cy5-labeled probes (Table S10) using a LightShift® chemiluminescent assay kit (Pierce, www.piercenet.com) according to the manufacturer's instructions. Separation of protein–DNA complexes was performed on a 5% native polyacrylamide gel. For detection of the Cy5 signal, a Typhoon scanner (GE Healthcare, www.gehealthcare.com) was used.
A transient ChIP-qPCR assay with 35S:OsMPS-CFP was performed as described by Du et al. (2009) using rice protoplasts (Zhang et al., 2011), whereby, after fixation of the cells with formaldehyde, a EpiQuick kit (Epigentek, www.epigentek.com) was used as described previously (Lai et al., 2012). For calculation of enrichment, the obtained CT values were normalized against the input CT, and ΔΔCT [CT(IgG) − CT(anti-GFP)] was calculated. Oligonucleotides were designed using Primer3 (Rozen and Skaletsky, 2000), and are listed in Table S11.
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
This work was in part supported by grants from the ERA-NET Plant Genomics program (TRIESTER, numbers 0313993A and ANR-06-ERAPG-005-01) through the Federal Ministry of Education and Research in Germany and the Agence Nationale de la Recherche in France, and the RicE Functional Genomics (REFUGE) platform, Montpellier, France, funded by the Agropolis Foundation. R.S. thanks the FAZIT Stiftung for a PhD fellowship. We thank Eugenia Maximova (Max Planck Institute of Molecular Plant Physiology, Germany) for microscopic work and Christopher Herbst (University of Potsdam, Germany) for technical assistance.
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
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Supporting Information
- Top of page
- Summary
- Introduction
- Results
- Discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
|Figure S1. OsMPS is induced by salt stress in leaves.
|Figure S2. Expression of OsMPS during vegetative and reproductive growth.
|Figure S3. Expression levels of OsMPS in transgenic lines.
|Figure S4. EXPB co-expression networks and putative target gene expression during abiotic stress.
|Figure S5. Putative direct OsMPS target genes containing the MYB-specific cis-element 'TAACTG' in their promoters.
|Word document
|Methods S1. GO enrichment and co-expression analysis of OsMPS-dependent genes and blast search for OsMPS homologs.
|Table S1. Effect of OsMPS on the metabolite profile of roots.
|Table S2. Effect of OsMPS on the metabolite profile of leaves.
|Table S3. GO enrichment for genes down-regulated by OsMPS.
|Table S4. GO enrichment for genes up-regulated by OsMPS.
|Table S5. Expression of genes regulated by OsMPS and hormone treatments.
|Table S6. Co-expressed genes obtained using the RiceFREND tool.
|Table S7. Co-expression networks obtained using the ATTED–II server.
|Table S8. Sequences of oligonucleotides used for cloning constructs.
|Table S9. Sequences of oligonucleotides used for expression profiling of OsMPS-regulated genes.
|Table S10. Probes used for EMSA.
|Table S11. Probes used for ChIP-qPCR.
|Word document
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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Margaret Ambriscoe (1910 - 2008)
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Comment: Re:Cars!? (Score 1) 176
That's a deep answer. This is the funniest article of the day for me as a flying robot/drone developer.
While the germans are flying drones all over the place, selling them and have a regulatory framework, they are complaining they cannot build/sell autonomous cars... and calling out other countries, mainly the US as beating them in that game.
But in the US, it's the completely reverse... or bizarro situation. While Google and Uber are building autonomous cars, and getting them approved for use, drones on the other hand are DOA with no framework insight... and drone companies are complaining countries like Germany and France (and Australia) are beating them in that game.
Oh, "the irony of the rant".


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Lizard Lake fire update-
The Lizard Lake fire is starting to quiet down. A combination of water dropping and high humidity is helping.
Early fall means the loons are gathering-
Chel Anderson is a botanist and plant ecologist for the Minnesota Department of Natural Resources.
Plein Air painting competition under way in Grand Marais-
Artists Neil Sherman and Brian Stewart visited The Roadhouse Aug.
EPA seeks comments on Great Lakes restoration-
The U.S. Environmental Protection Agency (EPA) is looking for input from the public on cleaning up and protecting the Great Lakes.
Water Dropping Aircraft To Be Used Today On Lizard Lake Fire-
The following information has been received from the US
Sunday Morning Update on Lizard Lake Fire Suppression-
The US Forest Service has been providing updates on the Lizard Lake fire in the Superior National Forest during the weekend. This latest report came at 10:40 AM, Sunday, August 29.
Local Fishing Report for Aug. 28-
Weekend News Wrap for Aug. 28-
Each weekend the WTIP News Department assembles the week's important news stories to play here on North Shore Weekend.
Fire smolders near Lizard Lake-
Smoke was spotted on Aug. 26 by United States Forest Service (USFS) fire personnel traveling on the Gunflint Trail.
Wildersmith August 27-
The upper Gunflint finally received some rain that is worthy of mention.
Dog attack reported at PincushionJay Andersen-
On Wednesday, the Cook County Sheriff's Office investigated the report of a 19 year old Grand Marais woman who was attacked by a stray dog near the Pincushion Trail system.
ISD166 prepares for levy referendumJay Andersen-
In the past few years, the way public schools are funded has led to some far reaching changes at Independent School District 166.


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Resource website for students of Abdulahad Ikram. Students can find lectures, handouts, and other useful resources for their courses here.
Beautiful Scene
April 2005: One of the most beautiful evenings, sitting at the banks of river Swat, in Mingora, doing nothing, just admiring the picturesque beauty of this peaceful valley. At the elevation of 990 meters (3,250 feet) above sea level, Mingora and Saidu Sharif are twin-towns merged into each other. Mingora is the biggest market town in Swat with all the main markets, buses and coaches stands, hotels, restaurants and shopping places; while Saidu Sharif, to the South is the administrative capital of Swat. #Mingora #Swat #KP #Pakistan #nature #river #mountains #valley #clouds #evening #sunset
A photo posted by Abdulahad Ikram (@aliphbaypay) on


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Ishrat case: CBI sticks to guns on role of ex-IB manNew Delhi, Feb 19 (PTI) Seeking sanction to prosecute the then Joint Director of Intelligence Bureau, Rajinder Kumar, in the Ishrat Jahan encounter case, CBI has sent a clarification to the Home Ministry reiterating its position that there was ample ... Press Trust of India, 1 month ago
Ishrat Jahan case: CBI sticks to guns on role of ex-IB man - Economic Times, 1 month ago
Ishrat Jahan case CBI sticks to guns on IB mans role - Calcutta News.Net, 1 month ago
4 images for rajinder kumar
Kanungo-Patwar Union strike enters 8th day
Attack on Patwari Ashok Kumar and Kanungo Rajinder Singh on December 30 along with other officers in Mand Chaunta village, in Koomkalan sub-division of Ludhiana has resulted in a strike of revenue department since December 31. The strike entered ...Indian Express, 2 months ago
National mark by Rajinder in javelin; Gold for Renjith
Thiruvananthapuram: Rajinder Singh smashed the national record in men's javelin throw, while former Asian champion Renjith Maheshwary won his maiden National Games gold with a new meet record in triple jump, as the athletes arena continued to ...Calcutta Telegraph, 1 month ago Track set ablaze in Kerala New Delhi Pioneer, 1 month ago National Games: Renjith Maheshwary Wins Maiden Gold, Preeja Sreedharan Signs Off with Silver NDTV, 1 month ago 35th National Games: Renjith wins maiden gold, Preeja signs off with silver ZeeNews.com, 1 month ago
Social status
| Shrabasti Mallik Veteran director Mala Kumar's latest presentation was an adaptation of Vijay Tendulkar's classic play Jat Hi Poochho Sadhu Ki . She said that casteism is as relevant today in villages as it was decades ago. By Shrabasti ...New Delhi Pioneer, 3 weeks ago
Faridkot's former MC vice-president arrested
Faridkot police arrested former municipal council vice-president and ex-Akali Dal leader Devinder Kumar alais Tintu for allegedly conspiring to kill truck union leader Rajinder Kumar alias Simpi.Times of India, 3 weeks ago Former Akali leader booked for truck union leader's murder Times of India, 3 weeks ago
Outside In | Whose land is it anyway
On the day India's Parliament began its budget session this week, Rajinder Oraon, Ram Prasad Oraon and Sarju Ram Oraon—men who looked to be in their forties—sat down on a kerb close by the stage at New Delhi's protest square, located just over a ...Livemint.com, 1 month ago
48 hrs to go, the battlelines harden — between Aam Garib Aadmi and rest
AAP supporters listen to party convener Arvind Kejriwal at an election campaign meeting in old Rajinder Nagar, in New Delhi on Monday. (PTI Photo) In this city of many cities, this Assembly election may be drawing out a blunt line. Between poor and ...Indian Express, 1 month ago Probe us and the BJP, Cong: AAP to Supreme Court on funding Rediff.com, 1 month ago AAP MLAs ELECT KEJRIWAL THEIR LEADER India Today, 1 month ago
Delhi Assembly Elections 2015 Cons..
ConstituencyNo. Constituency Name Winning Party Winning Candidate 1 Narela AAP Sharad Kumar 2 Burari AAP Sanjeev Jha 3 Timarpur AAP Pankaj Pushkar 4 Adarsh Nagar AAP Pawan aumar Sharma 5 Badli ...Samaylive, 1 month ago Complete Results: Delhi State Elections 2015 Sify, 1 month ago Junior engineer sacked for Delhi lawyer's death Times of India, 2 months ago Delhi Election Results 2015 Live Updates: Winners list Tehelka, 1 month ago
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Rajinder Puri, ace cartoonist and fierce critic of Emergency, passes away at 80
Veteran cartoonist, columnist and political activist Rajinder Puri died on Sunday night after a brief illness. His friends and family said Puri, who was the founding General Secretary of the Janata Party in 1977, had died peacefully in his sleep. ...Indian Express, 1 month ago
National Games: Rajinder nails javelin record
Haryana's Rajinder Singh shows off the reason for his excellent throw which landed him the javelin gold. Photo: S. Mahinsha The Hindu Renjith Maheswary triple jumps his way to a new record at the 35th National Games. Photo: S Mahinsha TOPICS ...The Hindu, 1 month ago
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Countertop Screwup + soapstone question
So I have a large kitchen remodel and I was at the stage of getting the counters in. Soapstone on the perimeter with a large quartzite island top. I received a message today that they made and error while cutting the soapstone and half my slab is now unusable. They do not have any more from my lot (I have had my slab reserved for 1 1/2 years). They have one that is close to matching and when oiled I shouldn't be able to tell. However I am not planning to oil it all the time - see picture. The other option is to select from a different lot, but they only have small slabs (so more seams) and the color is lighter with beige veins (not what I want). I have come to the conclusion they will have to install my island and the perimeter will be plywood until they get in some soapstone I like (I may have plywood for quite some time). I guess I should get my money back for the perimeter counter (I've already paid in full).
For the soapstone people out there - I have a 2" bumpout at my cooktop and they said the smallest corner radius possible is 1.8". I want something more square. They said they could do that, but it would have to be cut by hand and it would not look perfectly straight and the stone would feel wavy. Anyone have experience with this?
Sorry to hear your situation. I've been reading GW kitchens just over a year now, and I am amazed by the number of fabrication and install issues people report. How do these fabricators stay in business? It seems there are many "mistakes" made either templating, (or lack of), fabricating, and or installing... Anyway, hold out for the stone you want, although paying in full up front does not give you much leverage :( hope you get it resolved soon!
As to your question on the inside corner radius, here's one on my counter that is almost square, maybe a 1/8" radius at most, so it can be done... Soapstone is like wood, if the corner is not smooth, it can be sanded until it is
This post was edited by ctycdm on Mon, Jan 13, 14 at 20:07
I'm confused. If what your pic is is what they are proposing, then what don't you like about it? It seems perfectly lovely. As far as the radius goes, you really don't want under 3/4" as that makes a stressed point that is more prone to cracking.
I think you're over-worrying; the picture looks perfectly fine to me.
These guys are profiling this stone with a CNC machine and their tooling limits the size of the radius they can cut. A competent fabricator should be able to make a 3/4" radius (I'd go no smaller) that is indistinguishable from the CNC profile, especially in soapstone.
I would be happy with a 3/4" or even 1" radius at the corners. But they say they can only do an almost 2" radius and on the 2" bumpout that bugs me - it looks "swoopy" and undefined if that makes sense.
Also the reason I don't like the new replacement piece is because it has a touch of green in it with more beige in the veins. If I can see that in the small picture then I'll really notice it with the full size counters.
I understand your disapointment in the soapstone mishap especially since you have had the slab on hold for so long. It took me a year and a half to find the counters I wanted and I lived with plywood countertops for a few months waiting for the install. It wasn't a hardship. My husband bought laminated plywood that had black laminate on one side. It wasn't much more than regular plywood and it gave a more finished look to the counter and was easy to wipe clean.
I looked at several different soapstones until I found the one I wanted and I was prepared to wait until the right one came along.
Don't settle just to get the job finished.


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Click here to print the obituary below
Patricia R. Raczkowski
RACZKOWSKI, Patricia R., 90 years old. Born July 22, 1922 to Emma S. and Jesse J. Porter in Marianna, Florida. Graduated Crystal River High School in 1941. Worked at Wainwright Shipyard in Panama City, Florida during
World War II
. Later served in the United States Air Force, Pope Field, North Carolina, 1945 moved to Avon Park, Florida, 1949 graduated from Orange County Beauty College, Orlando, Florida. 1978 graduated from South Florida Junior College, Practical Nursing, Avon Park, Florida. Retired from nursing at Royal Care Nursing Home 1991. She taught Sunday School Senior Ladies since the early 50's and Charter Member of the Fellowship Baptist Church. Family members: two sons-Don "Rod" (Barbara) and Dennis "Ski" (Kimmi) Raczkowski, Avon Park, Florida; four grandchildren: Trisha and husband Gabe Crespo of Naples, Jennifer Robinson of Sebring, Josh and wife Becky Raczkowski of Orlando, Jessica Huerta-Garcia of Avon Park. 12 great-grandchildren. Two sisters-Stella McGinniss of Sarasota, Joy Hunter of Walhulla, South Carolina and three nieces, four great nieces and three great nephews. Memorial services Wednesday, May 8, 2013 at 3:00 p.m. at the Fellowship Baptist Church with Rev. R.L. Oquinn officiating. Memorial contributions may be made to the Children's Home in Lakeland through the Fellowship Baptist Church. Stephenson-Nelson Funeral Home - Avon Park www.stephensonnelsonfh. com
Funeral Home
Stephenson-Nelson Funeral Home of Avon Park
111 East Circle Street
Avon Park, FL 33825
(863) 453-3101
Published in TBO.com on May 7, 2013


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March 17, 2014
To respond to the increasing number of women choosing careers as professional truck drivers and to inspire more women to consider the transportation industry as a career, Ryder System, announced a partnership with Women In Trucking, a non-profit organization established to encourage the employment of women in the trucking industry.
December 27, 2013
The Women in Trucking Association and Navistar are seeking candidates for the 2014 Influential Woman In Trucking award.
July 25, 2013
The Cleveland Indians are partnering with the Women In Trucking Association to honor all women in transportation careers. The event will take place at Progressive Stadium during the Cleveland Indians versus Texas Rangers game on July 28.
February 27, 2013
Women in the trucking industry are becoming more visible and more welcome as leaders. For the third consecutive year, the Women In Trucking association, with the support of Navistar Inc., will highlight the achievements of female role models and pioneers in the trucking industry with the Influential Woman In Trucking award.
January 10, 2013
The Women in Trucking Association is now seeking candidates and accepting nominations for the 2013 Influential Woman in Trucking award
February 1, 2007
This group works to generate interest in trucking careers and improve industry relations.
We offer e-newsletters that deliver targeted news and information for the entire fleet industry.
The expert, Mark Betner from CITGO, will answer your questions
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Drones And the Future of Package Delivery
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Ultimate Test Drive Video: Brain and Brawn
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Crowdfund A Numenera Short Film, Microscopic Fantasy, And More!
Fantasy of all kinds rules the roost in this weeks look at all things crowdfunding - with everything from short films to books to comics giving us a dose of the fantastical. Let's dig in and have a look, shall we? »


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4 members Latest Activity: Jun 6, 2012 A group for those who enjoy the Texas Gulf Coast. All are welcome. Great place to link up with kayaking/outdoor activity buddies!
78 members Latest Activity: Mar 21, 2014 Into running whitewater and exploring rivers? Join us!
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The Great Railroad Revolution
The History of Trains in America
From the critics
Community Activity
CommentAdd a Comment
Read it for a class. A long book for a few interesting and insightful ideas.
The origins, financial machinations, political oversight and shear volume of assets and freight involved in the development of the American railroads up to 2012. A better book than I expected.
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Quiz: Remember the definition of mal de mer?
medical Latin, from Latin urtica "nettle," from urere "to burn." The native name is nettle-rash.
urticarial ur·ti·car·i·al (ûr'tĭ-kâr'ē-əl)
Relating to or marked by urticaria.
urticaria ur·ti·car·i·a (ûr'tĭ-kâr'ē-ə)
A skin condition characterized by welts that itch intensely, caused by an allergic reaction, an infection, or a nervous condition. Also called hives, nettle rash.
See hives.


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MOVING COMFORT Women's Flex Tank, Pixie Heather
Item: 27264876 Product Description >
Regular Price: $42.00 SALE $29.98
Buy one outlet clothing or footwear item, get a second for 50% OFF! This item is Eligible - Get Details
MOVING COMFORT Women's Flex Tank, Pixie HeatherIn a sea of racerbacks, Moving Comfort's seamless Flex Tank stands out with a great cross-back design and DriLayer fabric.
- DriLayer Seamless fabric: jacquard mesh knit of nylon, polyester, and spandex for breathability
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- Wicks moisture to keep you comfortable
- Engineered knit with jacquard design
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Country of Origin: Imported
MOVING COMFORT Women's Flex Tank, Pixie Heather
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Reviewed by 1 customer
- Nice Fabric Looked Good
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Best Uses
Comments about MOVING COMFORT Women's Flex Tank, Pixie Heather:
I liked the look and fit of this, but not enough to justify the price -- I returned it
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MMA: Superstar trio Jones, Silva and St-Pierre unlikely to come to Singapore
Published on Apr 28, 2014 11:01 PM
MIXED martial arts (MMA) superstars Jon Jones, Anderson Silva and Georges St-Pierre are unlikely to be coming to Singapore after all, after organisers LAMC Productions and Ch'i Life Studio Asia announced that they are distancing themselves from the event.
"Due to circumstances beyond our control, we will not be promoting this event," they said in a joint statement to The Straits Times. "Should there be an event taking place, we will no longer be involved."
The statement is the latest in a series of twists concerning the Fighters World Tour, an 11-city global tour of which the Singapore event was to be a part. The three MMA superstars were scheduled to be in town on May 31 for a two-hour, hands-on training clinic, as well as a meet-and-greet fan session at the Marina Bay Sands' ArtScience Museum.
But it seems their latest promotion was no-go from the start, with the Ultimate Fighting Championship (UFC), the sport's biggest promoter, referring to the event as "false advertising". Nike also responded on Sunday to a Straits Times query, saying that it had neither "been approached nor is even aware of a global, multi-city tour" involving the MMA trio.
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Miele PW6065W Washing Machine - rating?
I'm needing a new washer and dryer (actually, a whole new house, as ours burned down). I am not big on front loaders, but I will purchase whichever washer and dryer get our clothes the cleanest without shredding them, as the whirlpool in our current rental home does (shreds, not cleans). I do not care a bit about water efficiency or energy efficiency. That is all negated when I have to wash the clothes 4 times to get them close to clean, or when I have to replace the clothes because they are ruined. I am looking at the PW6065W Miele Washing Machine only because it boasts more power and higher heat (theoretically). Anybody used this machine? It's so pricey that I want to be sure the money is buying me more cleaning power. I have a large family, so would like large capacity. I do not care one bit about how long the cycle takes - if it gets the clothes cleaner, it could take hours for all I care... it's not like I have to sit there and watch it! I would like the 'steam' feature due to allergies, but if the clothes aren't clean, features don't do me any good. Ideas?
First, I am so so sorry to hear about your house. I cannot imagine losing one to fire. Hope your rebuilding or new home purchase will go forward quickly and without incident.
I have a year-old Miele 3035, which is the 24" (standard size) model. It is a fabulous machine and does an excellent job. I'm thrilled with it and feel it was well worth the money.
It's the little sister of the one you are considering.
Used properly, it takes out stains with no pretreating and cleans clothes beautifully -- including wool sweaters, fleece jackets, quilts etc. Some of DHs shirts come out and can be hung to dry without ironing. Anything that's stretched out returns to its original shape.
Yes, some of the cycles are long but I've never rewashed and there are so many options. One thing I love is the delay start which is so efficient -- I can put in a load and set it to be finished when I wake up.
My machine goes to 165F, which is as hot as I need on a regular basis for any laundry. My previous front loader went to 205F (almost boiling water) but I rarely used that setting. It was hotter than needed. My machine has a "Sanitize" cycle which does a great job because there's a cold prewash, I can set it for a longer main wash if I like and I can get as many as 3 rinses on that cycle. I don't need to use very much soap either.
This machine will do what you need but the trade off is the learning curve it takes to use it. Two of us got together and did a cheat sheet which includes all the options and some user intelligence. You might want to take a look as it will give you a window on how Miele washers work in general.
Here is a link that might be useful: Miele washer cheat sheet


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List Price: US$1.30
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Pros: Useful stuff, you dont need to messy your mobile's screen or your tablet's screen with your fingers. Cons: Very light build. A bit short I think.
Ce petit stylet est vraiment parfait, il ne raye pas l'écran, ne le graisse pas contrairement au doigt, et il fonctionne à merveille. Que demander de plus !
Pros: Facilita muito no uso do Smartphone e Iphone. Não prejudica a tela fazendo arranhão algo do gênero. Cons: Nada mesmo. Recomendo à todos a comprarem não vão se arrepender.
Works on any touchscreen capacitive and resistive both, is of great quality and does not scratch the screen.
I use it for my tablet and work well. It is small but not too smal and is very usefull for the portable tablets regards
This pen is very cheap. It has a nice design and looks good. It's nice to hold. It has a practical clip. Functionality is zero. Doesn't I... Read more
Pros: Useful stuff, you dont need to messy your mobile's screen or your tablet's screen with your fingers. Cons: Very light build. A bit... Read more
Ce petit stylet est vraiment parfait, il ne raye pas l'écran, ne le graisse pas contrairement au doigt, et il fonctionne à merveille. Que... Read more
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how to remove glued on fabric...
hello,this is my first posting here,hope it is the right place.i inherited an old desk with drawers.the drawers have glued on fabric in them.the drawers smell real bad and i don't like the red color.does anyone know how to remove this fabric from the drawers,i forgot to say that the fabric is felt.thank you for any ideas.
I hope someone knows the answer to this. I need to remove 1970's flocked wallpaper from cabinet wood door panels and this stuff is on there like cement! I don't think wallpaper remover is going to work.
This site is for woodworking, but glue is glue, right? It suggests heat and/or steam, so my suggestion is to try using your iron on the felt, using the hottest steam setting. Good luck!
Here is a link that might be useful: Removing old glue


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Mitt Romney Solidifies Link With Newly Voted GOP Leaders Ex- Republican presidential rival Mitt Romney solidified his link with the newly-voted GOP leaders just when he made phone calls to congratulate them on their victories. The Washington Post reported that Romney called more than 80 … Read more on Gatekeeper Liberty Voice
Why Is The Weather Channel Mocking Mitt Romney?
Why Is The Ride out Supervise over Contemptuous Mitt Romney? Appearing on the daylight AMHQ w/ Sam Champion curriculum, self-described "tropical storm/storm specialist" Carl Parker talked about his Halloween clothes with host Sam Champion and joked "I reckon I'm going to be Huge Bird. Try to irritate Mitt Romney." … Read more on NewsBusters (blog)
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Pols & Politics: Vintage '68 battle poster find delights Mitt Romney The prove of Romney's presidential run reappeared last week in city focal point Boston. At a fundraiser headlined by Mitt Romney, House Underground Chief Brad Jones unveiled deMacedo's poster — a clean touch to Mitt's advent and, for Jones, a small toll … Read more on […]
Jason Chaffetz: Mitt Romney is leaving door open to another run
Jason Chaffetz: Mitt Romney is leave-compelling door open to a bonus run SALT LAKE CITY — Mitt Romney is leave-compelling the door open to a third run for head if no apt Republican enters the race, according to one of his key supporters, Rep. Jason Chaffetz, R-Utah. "I reckon that's right. He to be sure hasn't place […]
Mitt Romney News
Does America like its candidates poor? As an adviser to the super PAC that hammered Mitt Romney's topic confirmation, let me give reasons for why Clinton's case is uncommon. The issue was never how much money Romney had; it was how he made that money. When you amass a megafortune in part by … Read more […]
Will you remember the name " Mitt Romney" by this time tommorrow?
Investigation by ROC-D: Will you scour up the name " Mitt Romney" by this time tommorrow? Best swear an oath(s): Swear an oath by Pancho VillaRomney who? Swear an oath by Moki Hunter'Way. He was our only hope. Swear an oath by chloeWho? Swear an oath by Michael B – Void Prop. 8!Romnesia will be gone and we won't have to take note to his […]
Mitt Romney News
Head owes Mitt Romney an description Given the actions of the last 20 months since their presidential line of reasoning, I reckon Head Obama owes Mitt Romney an description. Barack Obama, like George W. Bush before to to him, has been so reckless to champion a strategic reset with Russia that he is the … Read more on […]
Mitt Romney 2012 Bumper Sticker Decal Reviews
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Mitt Romney Donates $10000 To Bob McDonnell's Legal Defense Fund
Mitt Romney Donates 000 To Bob McDonnell's Legal Safeguard Fund Kaufman noted that McDonnell did not beg the donation, even if McDonnell and Romney do speak irregularly. "Announce near [McDonnell] brought it to Mitt's concentration," Kaufman told the Post, adage Romney "did it on his own choice." McDonnell … Read more on Huffington Post
Posted in Politics Tagged 10000, Defense, donates, Fund, legal, McDonnell's, Mitt, Romney Leave a comment
Is the Mitt Romney "A Celebrity on Yahoo! Answers" really him?
Investigation by heThatDoesNotWantToBeNamed: Is the Mitt Romney "A Star on Yahoo! Answers" really him? He posted one investigation 4 weeks ago, did not pick a best swear an oath, and has not answered any questions. I want to get more supervise over infromation from him, and not the spin doctors in succession his campagin. Mitt, do not […]
LDS community: will "Brother Romney" commercials humanize him?
Investigation by Grayson B.: LDS union: will "Brother Romney" commercials make more humane him? As chief of the Boston / Massachusetts Place of worship of Jesus Christ of End-Day Saints, will this help America to learn more about his belief in the power of prayer ? ? ? tales references Romney's deep conviction in a […]
Posted in Politics Tagged Brother, commercials, Community, humanize, Romney Leave a comment
Harmony Ball Pot Belly Mitt Romney
Harmony Ball Pot Belly Mitt Romney Harmony Ball collectible appx. 2.5″ tall Mitt Romney Outline Breviate Mitt Romney was born on March 12, 1947 in Detroit and raised in Bloomfield Hills, Michigan. The son of George Romney (Michigan authoritative and Republican presidential contender who was defeated by Richard Nixon in 1968), Mitt attended the lofty […]
Chris Christie Is No Mitt Romney. Unfortunately.
Chris Christie Is No Mitt Romney. Sorry to say. Many pundits were dainty that Mitt Romney would never be nominated since his shape-care confirmation in Massachusetts would make him radioactive to Obamacare-hating Tea Party voters in Republican primaries, or since of his flip-flops on mutual issues, … Read more on Bloomberg View
Posted in Politics Tagged Chris, Christie, Mitt, Romney, Unfortunately. Leave a comment
Is it fair to say that Mitt Romney "unearns $57K a day"?
Investigation by GOP-shaped Regime: Is it reliable to say that Mitt Romney "unearns K a day"? That's technically assess, but it still sounds inaccurate to my ear. What am I gone? Best swear an oath(s): Swear an oath by Righter of wrongsI don't know if ample announce say it ample time it will start to sound right. . Swear an oath […]
Romney says hes not running for president in 2016
Romney says hes not in succession for head in 2016 Mitt Romney once again said on Sunday he's not in succession for head in 2016, puffiness that he's excited about the Republican field of the makings nominees. "I'm plotting about the announce who I want to see in succession for head," he said on CBS's […]
I'll Take the Mormon (mitt romney) over the Moron Bumper (Obama) Sticker Reviews
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Why are the British people calling Romney "Mitt the Twitt"?
Investigation by : Why are the British announce mission Romney "Mitt the Twitt"? Best swear an oath(s): Swear an oath by magickSince they don't want to circulate a$ $ hole . Swear an oath by doctdonWho really cares what they are adage? Best than being plotting of as a weak fool like Obama. Swear an oath by LibertarianRepublicanWhy are the American announce mission […]
I have a great nickname for Romney, "Mitt Wrongney", do you like it?
Investigation by Composer: I have a fantastic nickname for Romney, "Mitt Wrongney", do you like it? If you do, delight use it as much as attainable and make it go viral. EDIT: "Obamanure" are you kidding? He by now has one built in, just say "Obama" and "State" commonly really quick. See I can make […]
Mitt Romney backs Joni Ernst in Iowa race
Mitt Romney backs Joni Ernst in Iowa race Mitt Romney refined state Sen. Joni Ernst Wednesday in a crowded Republican essential for Iowa's open Assembly seat, mission her a "proven conservative" with broad fascinate to win a all-gathering appointment. It's the newest wits of Romney's biased … Read more on Politico
Mitt Romney News
Op-ed Mitt Romney on the inaccurate side of the group campaigns I am very disappointed but not bowled over by Mitt Romney's contemporary declaration, the Count My Vote (aka Buy My Vote) beg, which, if affluent, would mean the end of the most accessible biased appointment systems in America get help to bring. It is […]


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what kind of fruit tree do i have growing in my yard?
i have this (fruit?) tree that grows in my backyard and am a loss to tell what it is. at first i thought it was from the citris family but now upon closer inspection...it looks more like a tomato. i know this can not be so because it grows on a tree about 10 feet tall. they are green at first and then turn bright orange. they also have a flower type petal at the top of the ? someone please help me identify my plant! i have a picture but am not sure how to get it out!
thank you
The fruit you describe could be a Persimmon but their flowers are almost unnoticeable. What color is the flower? If your picture is digital (jpeg) you could open a free account on Photobucket and direct link it here. After you open the account just copy the direct link and paste it into your post.
Here is the link to Photobucket http://photobucket.com/