Project description:To identify genes involved in the early phases of lateral root initiation, we profiled the transcriptomes of plants synchronously induced for lateral root initiation after 0, 1, 2, 4 and 6h of auxin treatment in conditions where IAA14 or IAA3-dependent auxin signaling is blocked. For this we used seedlings expressing non-degradable versions of the AUX/IAAs IAA14 (slr-1) or IAA3 (shy2-2) fused to the glucocorticoid receptor domain (slr-1:GR or shy2-2:GR) under the control of the pericycle and founder cell specific GATA23 promoter. Treatment with dexamethasone induces, specifically in pericycle cells, the nuclear translocation of the non-degradable AUX/IAA that acts as a dominant repressor of auxin signaling resulting in a complete block of lateral root formation
Project description:Lateral root initiation was used as a model system to study the mechanisms behind auxin-induced cell division. Genome-wide transcriptional changes were monitored during the early steps of lateral root initiation. Inclusion of the dominant auxin signaling mutant solitary root1 (slr1) identified genes involved in lateral root initiation that act downstream of the AUX/IAA signaling pathway. Interestingly, key components of the cell cycle machinery were strongly defective in slr1, suggesting a direct link between AUX/IAA signaling and core cell cycle regulation. However, induction of the cell cycle in the mutant background by overexpression of the D-type cyclin (CYCD3;1) was able to trigger complete rounds of cell division in the pericycle that did not result in lateral root formation. Therefore, lateral root initiation can only take place when cell cycle activation is accompanied by cell fate respecification of pericycle cells. The microarray data also yielded evidence for the existence of both negative and positive feedback mechanisms that regulate auxin homeostasis and signal transduction in the pericycle, thereby fine-tuning the process of lateral root initiation. Experiment Overall Design: Seedlings of both wild type (Col-0) and the lateral root defective mutant (slr-1) were germinated on MS medium supplemented with 10μM NPA (=auxin transport inhibitor). Three days after germination, such seedlings were transferred to MS supplemented with 10μM NAA for 0h, 2h and 6h respectively. The segment between root meristem and root-hypocotyl junction was harvested from about 1500 seedling per time point. All treatments were repeated biologically. 5.8 μg total RNA was used for the preparation of biotinylated cRNA. Labeled RNA was hybridised to ATH1 Affymetrix chips. The resulting data was MAS5.0 normalised.
Project description:The goal of this experiment was to identify the downstream targets of the GOLVEN6 peptide signaling pathway in Arabidopsis thaliana, specifically during lateral root initiation. Using an estradiol inducible GLV6 overexpression construct in wildtype and rgi1rgi5 double mutant (mutant in receptors for the GLV6 peptide) backgrounds, in combination with gravistimulation induced lateral root formation, the RGI receptor dependent transcriptional effects of GLV6 overexpression were characterized. An estradiol inducible GLV6 overexpression line in a wildtype (iGLV6) and in an rgi1rgi5 double receptor mutant background (rgi1rgi5/iGLV6) were used. 4-day old seedlings of both lines were gravistimulated (vertically grown seedlings were turned counterclockwise by 90°) to induce lateral root initiation in the resulting root bends. 8h after gravistimulation, seedlings of both lines were treated with 2µM of estradiol to induce GLV6 overexpression, or DMSO as a mock treatment. 3h and 6h after treatment, root bends were dissected and collected for RNA-sequencing. This yielded a total of 8 samples per replicate; 3h mock treated iGLV6 (IM3), 3h estradiol treated iGLV6 (IE3), 3h mock treated rgi1rgi5/iGLV6 (RM3), 3h estradiol treated rgi1rgi5/iGLV6 (RE3), 6h mock treated iGLV6 (IM6), 6h estradiol treated iGLV6 (IE6), 6h mock treated rgi1rgi5/iGLV6 (RM6), 6h estradiol treated rgi1rgi5/iGLV6 (RE6). For each sample, 4 replicates were obtained. This setup enabled the comparison of the GLV6 induced transcriptional effects between wildtype and rgi1rgi5 mutants at 2 time points after treatment, in samples that are strongly enriched for lateral root initiation events.
Project description:De novo shoot organogenesis (DNSO) is a commonly used pathway for plant biotechnology, and is a hormonally regulated process, where auxin and cytokinin coordinates suites of genes encoding transcription factors, general transcription factors, and RNA metabolism machinery genes. Here we report that silencing Arabidopsis thaliana CTD phosphatase-like 4 (CPL4RNAi), which increases phosphorylation level of RNA polymerase II (pol II) CTD, altered lateral root development and DNSO efficiency of the host plants, suggesting an importance of precise control of pol II activities during DNSO. Under standard condition, roots of CPL4RNAi lines produced no or few lateral roots. When induced by high concentration of auxin, CPL4RNAi lines failed to produce focused auxin maxima at the meristem of lateral root primordia, and produced fasciated lateral roots. By contrast, root explants of CPL4RNAi lines were highly competent for DNSO. Efficient DNSO of CPL4RNAi lines were observed even under 10 times less cytokinin required for wild type explants. Transcriptome analysis showed CPL4RNAi but not wild type explants expressed high levels of shoot meristem related genes during priming by high auxin/cytokinin ratio, and subsequent shoot induction with cytokinin. These results indicate that CPL4 functions as a repressor of the early stage of DNSO, during acquisition of competency by high auxin/cytokinin ratio, perhaps via regulation of pol II activities.
Project description:The plant hormone auxin represents an important regulator of growth and development. Significant insight into the mechanisms of auxin action have been obtained from studies of auxin resistant mutants such as aux1 and axr3. The Arabidopsis axr4 mutant was identified in a screen for auxin resistant root growth. In addition to the root growth of axr4 being resistant to exogenous auxin, there is also a 50% reduction in the number of lateral roots that form. The double axr4/aux1 mutant shows an additive effect in reducing lateral root numbers to 10% of wild-type. Gaining further information about the potential interaction between AUX1 and AXR4 may provide important insight into auxin regulated plant growth. Mapping experiments have placed the AXR4 gene on the lower arm of chromosome 1 between the ch1 and le markers (Hobbie and Estelle 1995). However, the AXR4 gene remains to be cloned. Identifying the AXR4 gene will help in elucidating the function of the protein. A transcript analysis of axr4 mutant seedlings will be used in 2 ways. Firstly, the transcription level of genes in the locality of the axr4 map position will be examined to identify those which are absent or significantly reduced in axr4 compared to the Col0 control. If the lesion causing the axr4 mutation results in a highly unstable mRNA or abolishes transcription then the signal will be dramatically reduced. Potential candidate genes identified in this way will be further analysed using a combination of RT-PCR and sequencing to identify the AXR4 gene. Secondly, the transcriptomics data obtained from axr4 and Col0 will be compared to identify genes which show significant transcript level differences and therefore represent targets for either direct or indirect regulation by AXR4. Hobbie, L. and Estelle, M. (1995) The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation. Plant J. 7 211-220
Project description:Lateral root initiation was used as a model system to study the mechanisms behind auxin-induced cell division. Genome-wide transcriptional changes were monitored during the early steps of lateral root initiation. Inclusion of the dominant auxin signaling mutant solitary root1 (slr1) identified genes involved in lateral root initiation that act downstream of the AUX/IAA signaling pathway. Interestingly, key components of the cell cycle machinery were strongly defective in slr1, suggesting a direct link between AUX/IAA signaling and core cell cycle regulation. However, induction of the cell cycle in the mutant background by overexpression of the D-type cyclin (CYCD3;1) was able to trigger complete rounds of cell division in the pericycle that did not result in lateral root formation. Therefore, lateral root initiation can only take place when cell cycle activation is accompanied by cell fate respecification of pericycle cells. The microarray data also yielded evidence for the existence of both negative and positive feedback mechanisms that regulate auxin homeostasis and signal transduction in the pericycle, thereby fine-tuning the process of lateral root initiation. Keywords: time-course wild type vs mutant comparison
Project description:Root architecture is vital for plant growth and largely depends on primary root growth and lateral root development. Several plant hormones have been shown to affect root architecture among which auxin has been granted a central role. Lately, small signalling peptides also emerged as potential molecular components regulating root growth and development. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, phloem poleexpressed paracrine signal for lateral root initiation. Our genetic, biochemical and pharmacological results show that CEP5 counteracts auxin signalling by stabilizing AUXIN/INDOLE ACETIC ACID (AUX/IAA) transcriptional repressors, suggesting the existence of an additional control mechanism through which plants can attenuate auxin signalling in a developmental context. Reducing CEP5 expression levels resulted in an increased auxin response and subsequently interfered with the normal progression through lateral root developmental stages.
Project description:Auxin is a key phytohormone regulating central processes in plants that include embryo development, lateral root growth and flower maturation among others. Auxin is sensed by a set of F-Box proteins of the TIR1/AFB3 family triggering auxin dependent responses by a pathway that involves an interplay between the Aux/IAA transcription repressors and the ARF transcription factors. We have previously shown that the AFB3 auxin receptor has a specific role in coordinating primary and lateral root growth to external and internal nitrate availability (Vidal et al., 2010). In this work, we used an integrated genomics, bioinformatics and molecular genetics approach to dissect regulatory networks acting downstream AFB3 that are activated by a transient nitrate treatment in Arabidopsis roots. Our systems approach unraveled key components of the AFB3 regulatory network leading to changes in lateral root growth in response to nitrate. Arabidopsis seedlings of the Ws and afb3-1 genotypes were grown on hydroponic medium containing 1X MS salts without Nitrogen, supplemented with 0.5 mM ammonium succinate as Nitrogen source and 3 mM sucrose on a Percival chamber under a photoperiod of 16 hours of light (100 μE/m2/sec) and 8 hours of dark at 22°C for 14 days. The plants were treated at the onset of the light cycle with 5 mM KNO3 or 5 mM KCl as control for 2 hours. Whole roots were cut from seedlings and frozen on liquid Nitrogen. Total RNA was extracted using the TriZol reagent. 3 independent biological replicates were performed.
Project description:The main purpose of this work is to clarify the effect of bacterial PLR on lateral root development in Arabidopsis, especially focusing on the fluctuation of auxin signaling in plants, so as to explain that PLR promotes lateral root development by promoting auxin signaling in plants.