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Evaluation of the embryogenic response through 6-benzyladenine in Coffea canephora in primed explants with auxin (1-naphthaleneacetic acid) plus cytokinin (kinetin) and protein profile.

ABSTRACT: Plant somatic embryogenesis encompasses somatic cells switch into embryogenic cells that can later produce somatic embryos with the ability to produce plantlets. Using defined in vitro culture settings for the somatic embryogenesis process of Coffea canephora that consist of an adequation of plantlets with auxin plus cytokinin followed by cut-leaf explant cultivation with cytokinin, producing embryos with the ability to regenerate plantlets, the protein profile was explored in primed and control explants. We confirmed that cultivating cut-leaf explants with cytokinin is sufficient to promote the proliferation of somatic embryos and the high yield of somatic embryos in the protocol requires the adequation of plantlets with auxin plus cytokinin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels reveal auxin- plus cytokinin-dependent regulated proteins in plantlets with up and down abundance. Chitinase A class III, proteins involved in the metabolism and folding of proteins, photosynthesis, antioxidant activity, and chromatin organization were identified. The RPN12 protein, which is a subunit of the proteasome 26S, has an abundance that is not associated with transcript changes, suggesting post-translational regulation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Coffea Canephora

TISSUE(S): Leaf

SUBMITTER: Victor Aguilar-Hernandez

LAB HEAD: Victor Aguilar-Hernández

PROVIDER: PXD055039 | Pride | 2024-10-02

REPOSITORIES: Pride

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Project description:In Cichorium intybus, macroscopic and histological modifications were identified during in vitro cell reactivation and morphogenesis events. The relationships between these modifications and transcript profiles of genes were investigated. Two chicory genotypes derived from the Hungarian landrace Koospol, were tested: K59 is a responsive genotype (embryogenic), as it undergoes cell de- and re-differentiation that leads to somatic embryogenesis (SE). C15 is an unresponsive genotype (nonembryogenic), unable to express this morphogenetic pattern. Previously studies showed that addition of beta-D-glucosyl Yariv reagent (BGlcY), a synthetic phenyl-glycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in chicory root explants in a concentration-dependent manner with complete inhibition of induction occurring at 250 ÂµM of BGlcY (Chapmann et al. 2000a). A putative AGP (DT212818) encoding gene was previously found significantly over-expressed in the K59 as compared to the C15 and was suggested to be involved in chicory re-differentiation. To better understand the cell reactivation events, a treatment with BGlcY was applied in order to precipitate AGPs in muro and to block the in vitro cell re-differentiation processes. The two different genotypes together with BGlcY represented an original tool to discriminate cell reactivation events from cell fate determination. Microscopy, biochemical and transcriptome analyses showed that in vitro SE in chicory was blocked during BGlcY-treatment but cell reactivation still occurred. Consequently, AGP (DT212818) seems to be only important in cell fate determination leading to SE commitment. Gene expression profiles showed in addition to the AGP, other proteins could play a role in SE determination: 26S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein 1 (MT1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Cell wall events during BGlcY-treatment were observed and discussed. Gene expression of K59 explants induced (d4) was compared with gene expression of K59 explants without induction (d0). Gene expression of K59 explants induced in the presence of BGlcY (Y+; d4) was next compared with gene expression of K59 explants induced in the absence of BGlcY (Y-; d4). Similar comparisons were made for C15 leaf explants. To link up both genotypes, direct comparison was done between gene expression of K59 explants without induction (d0) and C15 explants without induction (d0). Three biological replicates were used for each analysed condition. Dye labelling for each paired sample was reversed in two subsequent individual hybridizations giving rise to a total of six hybridizations per condition except for the K59_D0_vs_C15_D0 condition.

Project description:This model is from the article: The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development Muraro D, Byrne H, King J, Voss U, Kieber J, Bennett M. J Theor Biol.2011 Aug 21;283(1):152-67. PMID: 21640126, Abstract: Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.

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	S17_S890.msf	Msf
	S17_S890.raw	Raw
	S17_S9.msf	Msf
	S17_S9.raw	Raw
	S1_S69.msf	Msf

Dataset Information

Evaluation of the embryogenic response through 6-benzyladenine in Coffea canephora in primed explants with auxin (1-naphthaleneacetic acid) plus cytokinin (kinetin) and protein profile.

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