Transcriptomic analysis of Medicago truncatula calli with MtWOX9-1 overexpression
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ABSTRACT:
ORGANISM(S): Medicago truncatula
PROVIDER: GSE201314 | GEO | 2022/04/25
REPOSITORIES: GEO
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Project description:Somatic embryogenesis (SE), a morphogenic process that takes advantage of the regenerative potential of plants to replicate whole plants starting from somatic explants, can be a source of variation with potential applications in plant breeding. It is one of the most suitable tools to apply functional genomics studies and genetic improvement in plants. However, beyond a few pioneering works mainly focused on model plants, the molecular characterization of SE mechanisms is still elusive, especially for woody species. In grapevine, this process is affected by many factors such as explant type, culture conditions and, most importantly, genotype. Many cultivars, in fact, have shown recalcitrance to tissue culture and transformation, and very low SE competence. Thus, the understanding of SE competence behind the regenerative aptitude is fundamental to the widespread application of the so-called “next-generation breeding techniques”, such as cisgenesis and genome editing, in grapevine. Here, we explored genetic and epigenetic features of the SE process in grapevine by investigating the behavior of two genotypes showing opposite SE competence. Embryogenic tissues were induced from immature stamens excised from field-collected flower clusters of Sangiovese (highly competent for embryogenesis) and Cabernet Sauvignon (poorly competent for embryogenesis). A multilayered approach was used to profile mRNA, smallRNAs and methylated DNA with high-throughput sequencing technologies in the initial explants, on undifferentiated calli induced after 40 days of culture, and in embryogenic and non-embryogenic calli after 3 months of culture. A comprehensive comparison of transcriptomes of the different types of calli with the grapevine gene expression atlas revealed that, in grapevine, the dedifferentiation to the callus formation during the embryogenesis process occurs via a ‘berry’ developmental pathway. This is dissimilar from that shown in Arabidopsis, in which dedifferentiated calli are more similar to the tip of a root meristem. Interestingly, a Gene Ontology (GO) analysis revealed that secondary metabolism and gene expression regulation/epigenetics are the enriched functional categories of genes differentially down- and up-regulated in embryogenic vs non-embryogenic calli, respectively. These results prompted us to define the epigenetic landscape dynamics during SE in grapevine, revealing a significant increase in DNA methylation, especially in intergenic regions, in the embryogenic calli tissues. Finally, we proposed potential key regulators of SE in different genotypes that could represent putative targets of next-generation breeding techniques in grapevine.
2021-12-28 | GSE152520 | GEO
Project description:Somatic embryogenesis (SE), a morphogenic process that takes advantage of the regenerative potential of plants to replicate whole plants starting from somatic explants, can be a source of variation with potential applications in plant breeding. It is one of the most suitable tools to apply functional genomics studies and genetic improvement in plants. However, beyond a few pioneering works mainly focused on model plants, the molecular characterization of SE mechanisms is still elusive, especially for woody species. In grapevine, this process is affected by many factors such as explant type, culture conditions and, most importantly, genotype. Many cultivars, in fact, have shown recalcitrance to tissue culture and transformation, and very low SE competence. Thus, the understanding of SE competence behind the regenerative aptitude is fundamental to the widespread application of the so-called “next-generation breeding techniques”, such as cisgenesis and genome editing, in grapevine. Here, we explored genetic and epigenetic features of the SE process in grapevine by investigating the behavior of two genotypes showing opposite SE competence. Embryogenic tissues were induced from immature stamens excised from field-collected flower clusters of Sangiovese (highly competent for embryogenesis) and Cabernet Sauvignon (poorly competent for embryogenesis). A multilayered approach was used to profile mRNA, smallRNAs and methylated DNA with high-throughput sequencing technologies in the initial explants, on undifferentiated calli induced after 40 days of culture, and in embryogenic and non-embryogenic calli after 3 months of culture. A comprehensive comparison of transcriptomes of the different types of calli with the grapevine gene expression atlas revealed that, in grapevine, the dedifferentiation to the callus formation during the embryogenesis process occurs via a ‘berry’ developmental pathway. This is dissimilar from that shown in Arabidopsis, in which dedifferentiated calli are more similar to the tip of a root meristem. Interestingly, a Gene Ontology (GO) analysis revealed that secondary metabolism and gene expression regulation/epigenetics are the enriched functional categories of genes differentially down- and up-regulated in embryogenic vs non-embryogenic calli, respectively. These results prompted us to define the epigenetic landscape dynamics during SE in grapevine, revealing a significant increase in DNA methylation, especially in intergenic regions, in the embryogenic calli tissues. Finally, we proposed potential key regulators of SE in different genotypes that could represent putative targets of next-generation breeding techniques in grapevine.
2021-12-28 | GSE152337 | GEO
Project description:Somatic embryogenesis (SE), a morphogenic process that takes advantage of the regenerative potential of plants to replicate whole plants starting from somatic explants, can be a source of variation with potential applications in plant breeding. It is one of the most suitable tools to apply functional genomics studies and genetic improvement in plants. However, beyond a few pioneering works mainly focused on model plants, the molecular characterization of SE mechanisms is still elusive, especially for woody species. In grapevine, this process is affected by many factors such as explant type, culture conditions and, most importantly, genotype. Many cultivars, in fact, have shown recalcitrance to tissue culture and transformation, and very low SE competence. Thus, the understanding of SE competence behind the regenerative aptitude is fundamental to the widespread application of the so-called “next-generation breeding techniques”, such as cisgenesis and genome editing, in grapevine. Here, we explored genetic and epigenetic features of the SE process in grapevine by investigating the behavior of two genotypes showing opposite SE competence. Embryogenic tissues were induced from immature stamens excised from field-collected flower clusters of Sangiovese (highly competent for embryogenesis) and Cabernet Sauvignon (poorly competent for embryogenesis). A multilayered approach was used to profile mRNA, smallRNAs and methylated DNA with high-throughput sequencing technologies in the initial explants, on undifferentiated calli induced after 40 days of culture, and in embryogenic and non-embryogenic calli after 3 months of culture. A comprehensive comparison of transcriptomes of the different types of calli with the grapevine gene expression atlas revealed that, in grapevine, the dedifferentiation to the callus formation during the embryogenesis process occurs via a ‘berry’ developmental pathway. This is dissimilar from that shown in Arabidopsis, in which dedifferentiated calli are more similar to the tip of a root meristem. Interestingly, a Gene Ontology (GO) analysis revealed that secondary metabolism and gene expression regulation/epigenetics are the enriched functional categories of genes differentially down- and up-regulated in embryogenic vs non-embryogenic calli, respectively. These results prompted us to define the epigenetic landscape dynamics during SE in grapevine, revealing a significant increase in DNA methylation, especially in intergenic regions, in the embryogenic calli tissues. Finally, we proposed potential key regulators of SE in different genotypes that could represent putative targets of next-generation breeding techniques in grapevine.
2021-07-07 | GSE133761 | GEO
Project description:Carotenoids have been demonstrated to be indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we utilized genomic expression investigation to gain a deep insight into the carotenoid-related biological processes in the citrus calli overexpressing CrtB. Abortive ovule embryogenic calli from four citrus genotypes were used in this study. They were derived from Star Ruby grapefruit (C. paradise Macf.), Marsh grapefruit (C. paradise Macf.), and Sunburst mandarin [Citrus reticulata Blanco M-CM-^W (C. paradisi Macf. M-CM-^W C. reticulata)], designated as RB, M, and SBT, respectively. Engineered cell models (ECMs) were established by over-expressing 35S::CrtB (tpM-bM-^@M-^SrbcSM-bM-^@M-^SCrtB) [CrtB protein, phytoene synthase from Erwinia herbicola (now known as Pantoea agglomerans), containing a Pea rbcS transit peptide] in citrus embryogenic calli. Twenty-day-old calli were harvested and used for RNA extraction and hybridization on Affymetrix microarrays.
2014-09-23 | E-GEOD-61633 | biostudies-arrayexpress
Project description:Phosphorylation is one of the most common post-translational modifications and is central to many cellular signaling events; however, little is currently known about the phosphorylation landscape during somatic embryogenesis (SE) for plant regeneration. Here, we systematically analyzed the phosphoproteomic profile of three typical developmentally staged cultures of SE, non-embryogenic calli (NEC), primary embryogenic calli (PEC), and globular embryos (GE), in cotton (Gossypium hirsutum L.), the pioneer crop for genetic biotechnology applications. Our data revealed a total of 6301 quantifiable phosphorylation sites in 2627 quantifiable phosphoproteins from 5548 modified peptides, of which 1105 phosphoproteins (2147 sites) were differentially phosphorylated. Functional enrichment analyses revealed that differentially regulated phosphoproteins (DRPPs) were significantly enriched in DNA mismatch repair and peroxisome during callus embryogenic differentiation (PEC vs. NEC) and somatic embryo initiation (GE vs. PEC), respectively. Notably, six dynamic trajectory patterns of DRPP enrichment were observed. In addition, preferentially activated DRPPs with specific phosphorylation patterns were identified at different developmental stages. These DRPPs were mainly involved in hormone-responsive and photosystem events during initiation of plant regeneration. Overall, this study identified a series of potential phosphoproteins responsible for SE trans-differentiation and plant regeneration, providing a valuable resource and molecular basis for understanding the regulatory pathways underlying cell totipotency at the post-translational modification level.
2025-03-18 | PXD061812 | Pride
Project description:Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (ECs) and nonembryogenic calli (NECs) of C. papaya using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. ECs and NECs presented remarkable differences in anatomical and histochemical characteristics. ECs showed greater reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate the role of mitochondrial metabolism in the embryogenic competence of C. papaya calli. Greater participation of alternative oxidase (AOX) enzymes in respiration, as well as stronger accumulation of mitochondrial stress response proteins, was observed in ECs. In addition, ECs showed a greater abundance of proteins related to oxidative phosphorylation and higher total respiration (TR). Auxin-responsive Gretchen Hagen 3 (GH3) family proteins may play an important role in decreasing the contents of free 2,4-dichlorophenoxyacetic acid (2,4-D) in ECs. The accumulation of stress response proteins among total proteins was observed in ECs. ECs also showed higher endogenous hydrogen peroxide (H2O2) contents. H2O2 is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos.
2022-06-28 | PXD026311 | Pride
Project description:Several factors influence the culture conditions and somatic embryogenesis responses, such as the role of plant growth regulators (PGRs) during the establishment of in vitro cultures. For instance, auxin is required for callus induction and the acquisition of embryogenic capacity in different species, but the removal of auxin is needed for the further differentiation of somatic embryos. Thus, we aimed to evaluate the effects of residual 2,4-dichlorophenoxyacetic acid (2,4-D) on the maturation of sugarcane somatic embryos. First, embryogenic calli were separated into two groups: the PGR-free group was cultured without 2,4-D and b) the control group was maintained on proliferation culture medium, which contains 2,4-D. After 21 days of culture in the dark, both groups were transferred to maturation culture medium under light. Our results showed that calli grown in PGR-free culture medium yielded more somatic embryos and exhibited a higher accumulation of protein and starch reserves. A proteomic analysis showed a decrease in the abundance of abscisic acid (ABA)-induced proteins in the control group. The levels of residual 2,4-D and endogenous 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, were higher in the control group than in the PGR-free group. Conversely, the level of ABA was higher in the PGR-free group than in the control group. A disruption in the ABA and ethylene levels might be responsible for the observed delay in the accumulation of storage reserves in the embryogenic calli of the control group, which might have affected the development of somatic embryos. Our results also showed that the efficient development of sugarcane somatic embryos appears to be preceded by an efficient accumulation of storage reserves in calli, which is related to hormone homeostasis.
2021-06-01 | PXD021987 | Pride
Project description:Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency.
2025-03-21 | PXD062095 | Pride
Project description:Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In order to investigate the related genes affecting the transformation efficiency of embryogenic calli of different rice cultivars, we used Affymetrix GeneChipM-BM-. Rice Genome Array to measure the global gene expression profiling just before transformation and at four different time points after transformation (1 h, 6 h, 12 h, 24 h) in both japonica rice cultivar Nipponbare and indica rice cultivar Zhenshan 97. The mature embryo-derived embryogenic calli of Nipponbare (Nip) and Zhenshan 97 (ZS) were infected by Agrobacterium. Calli of Nip and ZS were sampled just before infection (0 h) and 1h, 6h, 12 h and 24h after infection, respectively. Three independent biological replications for each time point of the two varieties were used. To avoid the influence of polymorphisms between the probe sequence on the array and the genomes of the varieties used, we used a genomic DNA (gDNA)-based probe-selection strategy based on the hybridization efficiency of gDNA from Nip and ZS with the PM oligonucleotide probes on the rice array. The genomic DNA of Nip and ZS were extracted and hybridized to the Affymetrix Rice Genome Arrays. Three biological replications per cultivar were performed.
2012-01-01 | E-GEOD-32426 | biostudies-arrayexpress
Project description:Somatic embryogenesis (SE) is a complex stress related process regulated by numerous biological factors. SE is mainly applicable to mass propagation and genetic improvement of plants through gene transfer technology and mutation breeding. In banana, SE is highly genome dependent as the efficiency varies with cultivars. To understand molecular mechanism of SE, proteomics approach was carried out to identify genes responsible for embryogenic calli (EC) induction, regeneration and germination of somatic embryos (se) in cv. Rasthali (AAB). In total, 70 spots were differentially expressed in various developmental stages of SE. Of which, 16 were uniquely expressed and 17 were highly abundant in EC than nonembryogenic calli and explant and four spots were also uniquely expressed in germinating se. Functional annotation of identified proteins revealed that calcium signaling along with stress and endogenous hormones related proteins played a vital role in EC induction and germination of se. Thus based on the outcome, callus induction media was modified and tested in five cultivars. In cv. Grand Naine (AAA), increased concentration of 3- IAA and tryptophan recorded highest EC induction of 24.28% while Red Banana with similar genome showed 18.96% in kinetin supplemented media. Similarly, in cultivars Monthan and Karpuravalli with ABB genome showed maximum EC induction in tryptophan supplemented media (8.54%) and CaCl2 enriched media (17.34%) respectively. In cv. Neypoovan (AB), higher concentration of tryptophan induced more EC. These results illustrated that EC formation is genome as well as cultivar dependent. Simultaneously, germination media was modified to induce proteins responsible for germination. In cv. Rasthali, media supplemented with 10 mM CaCl2 showed maximum increase in germination (51.79%) over control. Thus present study revealed that media modification based on proteomic studies can induce SE in recalcitrant cultivars and also enhance germination in cultivars amenable for SE.
2019-03-19 | PXD011392 | Pride