Project description:Wounding is a primary trigger of organ regeneration but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study we combined the transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis thaliana. Our time-course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes that can be categorized into five clusters with distinct temporal patterns. Gene ontology analyses uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signalling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin has major contribution in wound-induced callus formation. We further demonstrate that type-A ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signalling regulates the expression of CYCLIN D3;1 (CYCD3;1) and mutations in CYCD3;1 and its homologs CYCD3;2-3 cause defects in callus formation. Our transcriptome data, in addition, showed that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 (ERF115) and PLETHORA3 (PLT3), PLT5, PLT7 in wound-induced callus formation. Together, this study provides novel mechanistic insights into how wounding reactivates cell proliferation during callus formation.
Project description:Unlike most animal cells, plant cells can easily regenerate new tissues from a wide variety of organs when properly cultured. The common elements that provide varied plant cells with their remarkable regeneration ability are still largely unknown. Here we describe the initial process of Arabidopsis in vitro regeneration, where a pluripotent cell mass termed callus is induced. We demonstrate that callus resembles the tip of a root meristem, even if it is derived from aerial organs such as petals, which clearly shows that callus formation is not a simple reprogramming process backwards to an undifferentiated state as widely believed. Furthermore, callus formation in roots, cotyledons and petals is blocked in mutant plants incapable of lateral root initiation. It thus appears that the ectopic activation of a lateral root development program is a common mechanism in callus formation from multiple organs.
Project description:To identify key miRNAs involved in root meristem formation in M. truncatula, deep sequencing was used to compare the miRNA populations dreived from four tissues. These were; root tip tissue, containing the root apical meristem, elongation zone tissue, root forming callus tissue and non-root forming callus tissue. We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families mir165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC.
Project description:Unlike most animal cells, plant cells can easily regenerate new tissues from a wide variety of organs when properly cultured. The common elements that provide varied plant cells with their remarkable regeneration ability are still largely unknown. Here we describe the initial process of Arabidopsis in vitro regeneration, where a pluripotent cell mass termed callus is induced. We demonstrate that callus resembles the tip of a root meristem, even if it is derived from aerial organs such as petals, which clearly shows that callus formation is not a simple reprogramming process backwards to an undifferentiated state as widely believed. Furthermore, callus formation in roots, cotyledons and petals is blocked in mutant plants incapable of lateral root initiation. It thus appears that the ectopic activation of a lateral root development program is a common mechanism in callus formation from multiple organs. Four sets of biologically independent tissue samples were collect for root, cotyledon and petal explants just after being excised from plants (d0) or after ten days on callus-inducing medium (CIM, d10). Samples derived from the same organ were co-hybridized in the array experiments. Dyes used for labeling the RNA populations derived from the individual samples were switched in the replicate experiments to reduce dye-related artefacts.
Project description:Proper functioning of the nuclear auxin pathway is essential for regulating plant growth and development by maintaining auxin homeostasis. To understand better physiological mechanisms involved in auxin signaling pathways we investigated the localization and effect of accumulation of auxin coreceptor IAA17/AXR3 in root. We demonstrate that the accumulation of stable nuclear AXR3-1 protein interferes with auxin homeostasis, causing auxin insensitivity and increased rapid root cell elongation followed by detained growth. This growth pattern is associated with changes in phytohormone gene expression. Data from transcriptomic screen combined with reporter lines and mutant studies declare essential role of auxin homeostasis in maintaining optimal root growth rate and development. We proposed a model in which rapid cell elongation is caused by combination of AXR3-1-dependent auxin insensitivity associated with unblocked gibberellin effect on root. This study demonstrate that plants coordinate gibberellin homeostasis by the auxin signaling pathway, contributing to avoid excessive root elongation.
Project description:To identify key miRNAs involved in root meristem formation in M. truncatula, deep sequencing was used to compare the miRNA populations dreived from four tissues. These were; root tip tissue, containing the root apical meristem, elongation zone tissue, root forming callus tissue and non-root forming callus tissue. We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families mir165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Examination and comparison of the microRNA population of four Medicago truncatula tissue types
Project description:Callus formation is usually a necessary step in regenerating a new plant from detached plant tissues, and the nature of the callus is similar to that of the root meristem. In this study, we intended to address the molecular basis that directs different plant tissues to form the root-meristem-like callus. We found that leaves, but not roots, of the Polycomb group (PcG) double mutant curly leaf-50 swinger-1 lost the ability to form a callus. Using ChIP-chip analysis, we identified genes that are changed markedly in the histone H3 lysine 27 trimethylation (H3K27me3) levels during callus formation from leaf explants. Among these genes, a number of leaf-regulatory genes were repressed through PcG-mediated H3K27me3. Conversely, certain auxin pathway genes and many root-regulatory genes were derepressed through H3K27 demethylation. Our data indicate that genome-wide H3K27me3 reprogramming, through the PcG-mediated H3K27me3 and the H3K27 demethylation pathways, is critical in directing cell fate transition. This submission represents the gene expression component of the study
Project description:In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.
Project description:Callus formation is usually a necessary step in regenerating a new plant from detached plant tissues, and the nature of the callus is similar to that of the root meristem. In this study, we intended to address the molecular basis that directs different plant tissues to form the root-meristem-like callus. We found that leaves, but not roots, of the Polycomb group (PcG) double mutant curly leaf-50 swinger-1 lost the ability to form a callus. Using ChIP-chip analysis, we identified genes that are changed markedly in the histone H3 lysine 27 trimethylation (H3K27me3) levels during callus formation from leaf explants. Among these genes, a number of leaf-regulatory genes were repressed through PcG-mediated H3K27me3. Conversely, certain auxin pathway genes and many root-regulatory genes were derepressed through H3K27 demethylation. Our data indicate that genome-wide H3K27me3 reprogramming, through the PcG-mediated H3K27me3 and the H3K27 demethylation pathways, is critical in directing cell fate transition. This submission represents the gene expression component of the study Leaves from 20-day-old seedlings of wild-type Col-0 and 20-DAC calli were used for RNA preparation.
Project description:In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.