Enhancement of cell wall protein SRPP expression during emergent root hair development in Arabidopsis.
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ABSTRACT:
SUBMITTER: Uno H
PROVIDER: S-EPMC5647948 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation.
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Project description:In plants, the cell wall is a major determinant of cell morphogenesis. Cell enlargement depends on the tightly regulated expansion of the wall, which surrounds each cell. However, the qualitative and quantitative mechanisms controlling cell wall enlargement are still poorly understood. Here, we report the molecular and functional characterization of LRX1, a new Arabidopsis gene that encodes a chimeric leucine-rich repeat/extensin protein. LRX1 is expressed in root hair cells and the protein is specifically localized in the wall of the hair proper, where it becomes insolubilized during development. lrx1-null mutants, isolated by a reverse-genetic approach, develop root hairs that frequently abort, swell, or branch. Complementation and overexpression experiments using modified LRX1 proteins indicate that the interaction with the cell wall is important for LRX1 function. These results suggest that LRX1 is an extracellular component of a mechanism regulating root hair morphogenesis and elongation by controlling either polarized growth or cell wall formation and assembly.
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Project description:Karrikins are smoke-derived compounds presumed to mimic endogenous signalling molecules (KAI2-ligand, KL), whose signalling pathway is closely related to that of strigolactones (SLs), important regulators of plant development. Both karrikins/KLs and SLs are perceived by closely related α/β hydrolase receptors (KAI2 and D14 respectively), and signalling through both receptors requires the F-box protein MAX2. Furthermore, both pathways trigger proteasome-mediated degradation of related SMAX1-LIKE (SMXL) proteins, to influence development. It has previously been suggested in multiple studies that SLs are important regulators of root and root hair development in Arabidopsis, but these conclusions are based on phenotypes observed in the non-specific max2 mutants and by use of racemic-GR24, a mixture of stereoisomers that activates both D14 and KAI2 signalling pathways. Here, we demonstrate that the majority of the effects on Arabidopsis root development previously attributed to SL signalling are actually mediated by the KAI2 signalling pathway. Using mutants defective in SL or KL synthesis and/or perception, we show that KAI2-mediated signalling alone regulates root hair density and root hair length as well as root skewing, straightness and diameter, while both KAI2 and D14 pathways regulate lateral root density and epidermal cell length. We test the key hypothesis that KAI2 signals by a non-canonical receptor-target mechanism in the context of root development. Our results provide no evidence for this, and we instead show that all effects of KAI2 in the root can be explained by canonical SMAX1/SMXL2 activity. However, we do find evidence for non-canonical GR24 ligand-receptor interactions in D14/KAI2-mediated root hair development. Overall, our results demonstrate that the KAI2 signalling pathway is an important new regulator of root hair and root development in Arabidopsis and lay an important basis for research into a molecular understanding of how very similar and partially overlapping hormone signalling pathways regulate different phenotypic outputs.
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Project description:The epidermis of Arabidopsis wild-type primary roots, in which some cells grow hairs and others remain hairless in a position-dependent manner, has become an established model system to study cell differentiation. Here we present a molecular analysis of the RHL1 (ROOT HAIRLESS 1) gene that, if mutated, prevents the formation of hairs on primary roots and causes a seedling lethal phenotype. We have cloned the RHL1 gene by use of a T-DNA-tagged mutant and found that it encodes a protein that appears to be plant specific. The predicted RHL1 gene product is a small hydrophilic protein (38.9 kD) containing putative nuclear localization signals and shows no significant homology to any known amino acid sequence. We demonstrate that a 78-amino-acid sequence at its amino terminus is capable of directing an RHL1-GFP fusion protein to the nucleus. The RHL1 transcript is present throughout the wild-type plant and in suspension culture cells, but in very low amounts, suggesting a regulatory function for the RHL1 protein. Structural evidence suggests a role for the RHL1 gene product in the nucleolus. We have examined the genetic relationship between RHL1 and GL2, an inhibitor of root hair initiation in non-hair cells. Our molecular and genetic data with double mutants, together with the expression analysis of a GL2 promoter-GUS reporter gene construct, indicate that the RHL1 gene acts independently of GL2.
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Project description:Two class I family teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins from allotetraploid cotton are involved in cotton fiber cell differentiation and elongation and root hair development. However, the biological function of most class II TCP proteins is unclear. This study sought to reveal the characteristics and functions of the sea-island cotton class II TCP gene GbTCP4 by biochemical, genetic, and molecular biology methods. GbTCP4 protein localizes to nuclei, binding two types of TCP-binding cis-acting elements, including the one in its promoter. Expression pattern analysis revealed that GbTCP4 is widely expressed in tissues, with the highest level in flowers. GbTCP4 is expressed at different fiber development stages and has high transcription in fibers beginning at 5 days post anthesis (DPA). GbTCP4 overexpression increases primary root hair length and density and leaf and stem trichomes in transgenic Arabidopsis relative to wild-type plants (WT). GbTCP4 binds directly to the CAPRICE (CPC) promoter, increasing CPC transcript levels in roots and reducing them in leaves. Compared with WT plants, lignin content in the stems of transgenic Arabidopsis overexpressing GbTCP4 increased, and AtCAD5 gene transcript levels increased. These results suggest that GbTCP4 regulates trichome formation and root hair development in Arabidopsis and may be a candidate gene for regulating cotton fiber elongation.
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Project description:Using cell sorting technique, we collected Arabidopsis root hair protoplasts (transgenic line harboring root hair specific gene promoter ::GFP, here named GFP) and nonGFP protoplasts( whole root except root hair). Then, we extracted total proteins and RNA for proteome and RNASeq. RNAseq data have already been deposited into NCBI (accession numbers SRR364677 and SRR364678). Total proteins were separated by 1D SDS-PAGE, followed by gel slicing, in gel digested and run ESI-LC-MS/MS on LTQ Orbitrap Velos. We set two biological repeats and each sample was scanned two times (two technique repeats). We then combined all ms/ms raw data from all fractions and technique repeats from one biological sample into one file to indentify proteins by searching Arabidopsis protein database with software Proteome Discoverer (with two searching engines mascot and sequester). Protocol: Protoplasts from EXP7 and non-GFP protoplastswere stored at -80°C, thawed on ice for 5 min, vortexed several times and suspended in 5 x volume of pre-cooled acetone (-20°C) containing 10% (v/v) trichloroacetic acid (TCA) and 0.07% (v/v) 2-mercaptoethanol. Proteins were then precipitated for 2 h at −20°C after thorough mixing. Proteins were collected by centrifuging at 35,000 g (JA-20 108 rotor, Beckman J2-HS) at 4°C for 30 min. The supernatant was removed and the protein pellets were washed three times with cold acetone containing 0.07% (v/v) 2-mercaptoethanol and 1 mMphenylmethanesulfonylfluoride. Protein pellets were dried by lyophilization and stored at -80°C, or immediately extracted using Laemmli buffer (63 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue). Protein concentration was determined using Pierce protein assay kit (Pierce, Rockford). Proteins from each sample were separated using a Bis-Tris precast gradient gel (4−16%, Bio-Rad) according to the manufacturer’s instructions. After electrophoresis, the gel was Coomassie-stained using the Colloidal Blue Staining Kit (Invitrogen). The protocol used for in-gel trypsin digestion of proteins in gels was adapted from the method described in Shevchenko et al. (1996). Briefly, protein bands were manually excised and each band was cut into small pieces (~0.5 mm). The gel pieces were washed three times with a solution containing 50% methanol and 5% acetic acid for 2 h, twice with a solution containing 25 mM NH4HCO3 in 50% acetonitrile for 10 min each, and then the gel pieces were dried in a vacuum centrifuge. Reduction of proteins in gel pieces was performed with DTT and alkylation with iodoacetamide, and the gel pieces were washed and dried in a vacuum centrifuge. A trypsin solution in 25 mM NH4HCO3 containing 75 to 100 ng of sequencing grade modified trypsin (Promega) in 25-40 µl was added and incubated with gel pieces for 12-16 h at 37°C. To recover the tryptic peptides, a solution of 30 µl containing 5% formic acid and 50% acetonitril was added to the gel pieces, agitated in a vortex for 30-60 min and withdrawn into a new tube. The recovery was repeated once with 15 µl solution and the resulting two recovers were combined and dried in a vacuum centrifuge. The dried pellet was re-dissolved in 10-20 µl of 0.1% formic acid for LC-MS/MS analysis.
2013-05-28 | PXD000195 | Pride
Project description:The gain-of-function mutant edt1 shows significantly enhanced drought tolerance and a well-developed root system including deeper primary roots and more lateral roots. To explore the molecular mechanisms underlying the improved root system of edt1, we performed transcriptome comparison between the wild-type and edt1 roots. One of the interesting findings from the analysis was that several gene families of cell-wall-loosening proteins were upregulated in the mutant roots, including expansins, extensins, xyloglucan endotransglucosylase/hydrolases (XTHs), pectin-related enzymes, and cellulases. Most of these genes contain HD-binding cis-elements in their promoters predominantly with the TTTAATTT sequence, which can be bound by HDG11 in vitro and in vivo. The coordinated expression of these gene families overlaps fast root elongation. Furthermore, overexpression of AtEXPA5, which was dramatically upregulated in edt1, resulted in longer primary roots because cells were more extended longitudinally. When combined by crossing the AtEXPA5-overexpression lines with one pectin methylesterase inhibitor family protein (PMEI) gene (At5g62360)- or one cellulase (CEL) gene (At2g32990)-overexpression lines, the primary roots of the progeny even exceeded both parents in length. Our results demonstrate that HDG11 directly upregulates cell-wall-loosening protein genes, which is correlated with altered root system architecture, and confirm that cell-wall-loosening proteins play important roles in coordinating cell-wall extensibility with root development. The results of transgene experiments showed that expansin works together with PMEI and CEL to generate synergistic effects on primary root elongation, suggesting that different cell-wall-loosening protein families may function in combination to generate optimal effects on root extensibility.
| S-EPMC4112634 | biostudies-literature
Project description:Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein-protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development.
| S-EPMC3344980 | biostudies-literature
Project description:The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes-termed herein as dancing-endosomes-which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth.
| S-EPMC4731515 | biostudies-other
Project description:BACKGROUND: Phosphorus (P) is one of the essential but often limiting elements for plants. Based on transcriptional profiling we reported previously that more than 3,000 genes are differentially expressed between phosphate (Pi)-deficient and Pi-sufficient Arabidopsis roots (MCP 11(11):1156-1166, 2012). The current study extends these findings by focusing on the analysis of genes that encode protein kinases (PK) and phosphatases (PP) by mining PK and PP genes that were differentially expressed in response to Pi deficiency. RESULTS: Subsets of 1,118 and 205 annotated PK and PP genes were mined on the basis of the TAIR10 release of the Arabidopsis genome. Analysis of RNA-seq data showed that 92 PK and 19 PP genes were not detected in roots (zero reads in three biological repeats); 96 PK and 10 PP showed low abundance (? 10 reads). Gene ontology analysis revealed that the 188 PK genes with no or low expression level in Arabidopsis roots are mainly involved in pollen recognition, pollen tube growth or other processes not relevant for root hair formation. More than 50% of the cysteine-rich RLK (receptor-like protein kinase) subfamily genes belong to this group. Among the 29 PP genes with no or low expression level, purple acid phosphatases, haloacid dehalogenase-like hydrolases, and PP2C genes with functions in the dephosphorylation of RNA polymerase II C-terminal domain and mRNA capping were enriched. Subsets of 173 PK and 35 PP genes were differentially expressed under Pi-deficient conditions. Putative functional modules (clusters) of these PK and PP genes were constructed based on co-expression analysis using the MACCU toolbox. A co-expression network comprising 65 known or annotated PK and PP genes (60 PK and 5 PP genes, respectively) was subdivided into several highly co-expressed gene sub-clusters. The largest sub-cluster was composed of 22 genes, most of which have been assigned to the RLK superfamily and were associated with cell wall metabolism, pollen tube and/or root hair development and growth. CONCLUSIONS: We here provide comprehensive 'digital' transcriptional information on PK and PP genes in Arabidopsis roots. The co-expression network derived from our data mining approach sets the stage for follow-up experimentation that helps to complete our understanding of the post-translational regulation of Pi deficiency-induced changes in root hair morphogenesis.
| S-EPMC3636113 | biostudies-literature