Project description:Background: Maize (Zea Mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos. Results: We observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed. Conclusions: These data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.
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:Transcriptional profiling of age-related change of callus formation capability in Arabidopsis hypocotyls Organogenesis in vitro consists of many aspects such as phytohormone perception, dedifferentiation of differentiated cell to acquire organogenic competence, and re-entry of quiescent cells into cell cycle. In this study, we established an in vitro experimental system to study the age-dependent callus formation capacity in Arabidopsis. Interestingly, mature (35- to 38-day-old) hypocotyl explants exhibited better callus-forming potential than that of juvenile (7- to 10-day-old), determined by callus growth rates. To explore genome-wide expression changes underlying the phenomenon of age-dependent callus formation, a transcriptome-based analysis was performed. Gene expression profiling indicated that age-dependent callus formation capacity was associated with changes in phytohormone (auxins, cytokinins, abscisic acid, brassinosteroids and gibberellins) homeostasis, epigenetic mechanism and the cell cycle regulation. Besides, we identified two groups of genes involved in age-dependent callus formation capacity: (1) positive regulatory and (2) negative regulatory categories, i.e. genes that were significantly up- or down-regulated during callus formation derived from mature explants, respectively. One gene encoding DNA-binding protein (VARIANT IN METHYLATION 1, VIM1) belonging to the positive regulatory category was selected for functional analysis and assessment of age-dependent callus formation capacity. Indeed, vim1 reduced the efficiency of callus formation in mature explants, but not in juvenile. The result suggests that VIM1 plays an important role in regulating age-dependent callus formation capacity. Taken together, the investigation will help to better understand the molecular regulatory mechanism of age-dependent callus formation. Comparison of young and mature Arabidopsis hypocotyls either with or without auxin treatment for 1 day
Project description:Transcriptional profiling of age-related change of callus formation capability in Arabidopsis hypocotyls Organogenesis in vitro consists of many aspects such as phytohormone perception, dedifferentiation of differentiated cell to acquire organogenic competence, and re-entry of quiescent cells into cell cycle. In this study, we established an in vitro experimental system to study the age-dependent callus formation capacity in Arabidopsis. Interestingly, mature (35- to 38-day-old) hypocotyl explants exhibited better callus-forming potential than that of juvenile (7- to 10-day-old), determined by callus growth rates. To explore genome-wide expression changes underlying the phenomenon of age-dependent callus formation, a transcriptome-based analysis was performed. Gene expression profiling indicated that age-dependent callus formation capacity was associated with changes in phytohormone (auxins, cytokinins, abscisic acid, brassinosteroids and gibberellins) homeostasis, epigenetic mechanism and the cell cycle regulation. Besides, we identified two groups of genes involved in age-dependent callus formation capacity: (1) positive regulatory and (2) negative regulatory categories, i.e. genes that were significantly up- or down-regulated during callus formation derived from mature explants, respectively. One gene encoding DNA-binding protein (VARIANT IN METHYLATION 1, VIM1) belonging to the positive regulatory category was selected for functional analysis and assessment of age-dependent callus formation capacity. Indeed, vim1 reduced the efficiency of callus formation in mature explants, but not in juvenile. The result suggests that VIM1 plays an important role in regulating age-dependent callus formation capacity. Taken together, the investigation will help to better understand the molecular regulatory mechanism of age-dependent callus formation.
Project description:Purpose: Maize somatic embryogenesis is usually required to achieve genetic transformation and represents an important alternative in plant development. Although many embryogenesis-related genes have been studied in this model, the molecular mechanisms underlying cell dedifferentiation and further plant regeneration are not completely understood. Methods: Immature embryos smRNA profiles of 15-day-after-pollination (IE) and Embryogenic Callus from one (C1), four (C4), and ten months (C10) were generated by deep sequencing, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed with two methods: Bowtie 1.1.2 and ShortStack 3.4. qRT–PCR validation for selected miRNAs was performed using SYBR Green assays. Results: We used high throughput sequencing to explore the sRNA populations during maize embryogenic callus induction and established subcultures from the Mexican cultivar VS-535, Tuxpeño landrace. We detected readjustments in 24 nt and 21-22 nt sRNA populations during the embryogenic callus establishment and maintenance. miRNAs related to stress response substantially increased upon callus proliferation establishment, correlating with a reduction in some of their target levels. On the other hand, while 24 nt-long hc-siRNAs derived from transposable retroelements transiently decreased in abundance during the embryogenic callus establishment, a population of 22 nt- hc-siRNAs increased. This was accompanied by reduction in transposon expression in the established callus subcultures. Conclusions: Stress- and development-related miRNAs are highly expressed upon maize EC callus induction and during maintenance subcultures, while miRNAs involved in hormone response only transiently increase during induction. The establishment of proliferative maize embryogenic callus is accompanied by important readjustments in the length of hc-siRNAs mapping to LTR retrotransposons, and their expression regulation.
Project description:Transcriptome analysis of small RNA was performed using pollen and embryonic callus, and vegetative tissues, needles of Picea abies to address differences in small RNA profiles between reproductive tissues and vegetative tissues in gymnosperm.
Project description:The applications of plant callus regeneration has been widely spreaded in agricultural improvement. By using immature sorghum embryos as explants, progress in successful genetic transformation has been made in sorghum. However, the underlying mechanism of callus differentiation is still largely unknown in sorghum. Here, we described three types of callus with different abilities of redifferentiation (Callus I-III), undergoing distinct induction from immature embryo in the variety of Hiro-1. In comparison to the non-embryonic Callus III who lost the ability of regeneration, the Callus I produced only some characterized adventitious roots and the embryonic Callus II is sufficient to regenerate whole plants. Genome-wide transcriptome profiles were performed to reveal the underlying mechenisms. The numbers of differentially expressed genes for the three types of callus vary from 5906 to 8029. Principal component analysis analysis demonstrated that gene expression patterns of Callus I and II were totally different from that of Callus III and differential leaves from Callus II, indicating that the compassions of Callus I and II provide clues for revealing regulations of regeneration in sorghum callus. Notably, KEGG and GO analysis showed that plant ribosome, lignin metabolic process, and metabolism of starch and sucrose are main processes that are associated with callus differentiation. Taken together, the results contributed the elucidation of molecular regulation in three types of callus with several regeneration abilities in sorghum.
Project description:We report the role of sRNAs populations during the induction of callus tissues from VS-535 maize embryos displaying contrasting in vitro embryogenic potential; characterized through Next-generation sequencing (NGS). We conclude that the Embryogenic Response during Maize Somatic Embryogenesis induction is closely related to sRNAs regulation and depends on the developmental stage of the explant.
Project description:Plants can regenerate from a variety of tissues on culturing in appropriate media. However, the metabolic shifts involved in callus formation and shoot regeneration are largely unknown. The metabolic profiles of callus generated from tomato (Solanum lycopersicum) cotyledons and that of shoot regenerated from callus were compared with the pct1-2 mutant that exhibits enhanced polar auxin transport and the shr mutant that exhibits elevated nitric oxide levels. The transformation from cotyledon to callus involved a major shift in metabolite profiles with denser metabolic networks in the callus. In contrast, the transformation from callus to shoot involved minor changes in the networks. The metabolic networks in pct1-2 and shr mutants were distinct from wild type and were rewired with shifts in endogenous hormones and metabolite interactions. The callus formation was accompanied by a reduction in the levels of metabolites involved in cell wall lignification and cellular immunity. On the contrary, the levels of monoamines were upregulated in the callus and regenerated shoot. The callus formation and shoot regeneration were accompanied by an increase in salicylic acid in wild type and mutants. The transformation to the callus and also to the shoot downregulated LST8 and upregulated TOR transcript levels indicating a putative linkage between metabolic shift and TOR signalling pathway. The network analysis indicates that shift in metabolite profiles during callus formation and shoot regeneration is governed by a complex interaction between metabolites and endogenous hormones.
Project description:Histone modification H3K27me3 profilings by the CUT&RUN method (Skene et al., 2017) were performed using embryonic callus and non-embryonic callus of Picea abies to identify genes related to somatic embryogenesis capacity.