Current insights into hormonal regulation of microspore embryogenesis.
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SUBMITTER: Zur I
PROVIDER: S-EPMC4462098 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Key messageEffective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11-39 nmol g(-1) DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121-424 pmol g(-1) DW) and zeatin ryboside (cZR, 146-432 pmol g(-1) DW). Surprisingly, a relatively high level (10-64 pmol g(-1) DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of 'responsive' DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with 'recalcitrant' genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential.
| S-EPMC4282712 | biostudies-literature
Project description:Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5-10 μM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).
| S-EPMC8593058 | biostudies-literature
Project description:The use of doubled haploid (DH) technology enables the development of new varieties of plants in less time than traditional breeding methods. In microspore embryogenesis (ME), stress treatment triggers microspores towards an embryogenic pathway, resulting in the production of DH plants. Epigenetic modifiers have been successfully used to increase ME efficiency in a number of crops. In wheat, only the histone deacetylase inhibitor trichostatin A (TSA) has been shown to be effective. In this study, inhibitors of epigenetic modifiers acting on histone methylation (chaetocin and CARM1 inhibitor) and histone phosphorylation (aurora kinase inhibitor II (AUKI-II) and hesperadin) were screened to determine their potential in ME induction in high- and mid-low-responding cultivars. The use of chaetocin and AUKI-II resulted in a higher percentage of embryogenic structures than controls in both cultivars, but only AUKI-II was superior to TSA. In order to evaluate the potential of AUKI-II in terms of increasing the number of green DH plants, short and long application strategies were tested during the mannitol stress treatment. The application of 0.8 µM AUKI-II during a long stress treatment resulted in a higher percentage of chromosome doubling compared to control DMSO in both cultivars. This concentration produced 33% more green DH plants than the control in the mid-low-responding cultivar, but did not affect the final ME efficiency in a high-responding cultivar. This study has identified new epigenetic modifiers whose use could be promising for increasing the efficiency of other systems that require cellular reprogramming.
| S-EPMC10975478 | biostudies-literature
Project description:Antibiotics are widely applied for plant cultivation in vitro to eliminate bacterial contamination. However, they can have both positive and negative effects on the cells of cultivated plants, and these effects largely depend on the type antibiotic used and its concentration. The objective of the present study was to estimate the effect of ?-lactam antibiotics ampicillin (Amp) and cefotaxime (Cef) on microspore embryogenesis induction in vitro in the Brassica species. The performed experiments confirmed cefotaxime inhibits microspores in B. napus and B. oleracea, even in concentrations as low as 50 mg/L. The highest embryo yield was obtained for B. napus in the NLN-13 medium with added ampicillin in concentrations of 50-100 mg/L as an antimicrobial agent. This embryo yield was significantly higher than that obtained in a medium without supplemented antibiotics and two times higher than in the medium with added cefotaxime. Analogous results were obtained for B. oleracea and B. rapa.
| S-EPMC7238966 | biostudies-literature
Project description:Reprogramming of microspores development towards embryogenesis mediated by stress treatment constitutes the basis of doubled haploid production. Recently, compounds that alter histone post-translational modifications (PTMs) have been reported to enhance microspore embryogenesis (ME), by altering histones acetylation or methylation. However, epigenetic mechanisms underlying ME induction efficiency are poorly understood. In this study, the epigenetic dynamics and the expression of genes associated with histone PTMs and ME induction were studied in two bread wheat cultivars with different ME response. Microspores isolated at 0, 3 and 5 days, treated with 0.7M mannitol (MAN) and 0.7M mannitol plus 0.4µM trichostatin A (TSA), which induced ME more efficiently, were analyzed. An additional control of gametophytic development was included. Microspores epigenetic state at the onset of ME induction was distinctive between cultivars by the ratio of H3 variants and their acetylated forms, the localization and percentage of labeled microspores with H3K9ac, H4K5ac, H4K16ac, H3K9me2 and H3K27me3, and the expression of genes related to pollen development. These results indicated that microspores of the high responding cultivar could be at a less advanced stage in pollen development. MAN and TSA resulted in a hyperacetylation of H3.2, with a greater effect of TSA. Histone PTMs were differentially affected by both treatments, with acetylation being most concerned. The effect of TSA was observed in the H4K5ac localization pattern at 3dT in the mid-low responding cultivar. Three gene networks linked to ME response were identified. TaHDT1, TaHAG2, TaYAO, TaNFD6-A, TabZIPF1 and TaAGO802-B, associated with pollen development, were down-regulated. TaHDA15, TaHAG3, TaHAM, TaYUC11D, Ta-2B-LBD16 TaMS1 and TaDRM3 constituted a network implicated in morphological changes by auxin signaling and cell wall modification up-regulated at 3dT. The last network included TaHDA18, TaHAC1, TaHAC4, TaABI5, TaATG18fD, TaSDG1a-7A and was related to ABA and ethylene hormone signaling pathways, DNA methylation and autophagy processes, reaching the highest expression at 5dT. The results indicated that TSA mainly modified the regulation of genes related to pollen and auxin signaling. This study represents a breakthrough in identifying the epigenetic dynamics and the molecular mechanisms governing ME induction efficiency, with relevance to recalcitrant wheat genotypes and other crops.
| S-EPMC9868772 | biostudies-literature
Project description:Among various methods stimulating biological progress, double haploid (DH) technology, which utilizes the process of microspore embryogenesis (ME), is potentially the most effective. However, the process depends on complex interactions between many genetic, physiological and environmental variables, and in many cases, e.g., winter wheat, does not operate with the efficiency required for commercial use. Stress associated with low-temperature treatment, isolation and transfer to in vitro culture has been shown to disturb redox homeostasis and generate relatively high levels of reactive oxygen species (ROS), affecting microspore vitality. The aim of this study was to investigate whether controlled plant growth, specific tiller pre-treatment and culture conditions could improve the potential of microspores to cope with stress and effectively induce ME. To understand the mechanism of the stress response, hydrogen peroxide levels, total activity and the content of the most important low-molecular-weight antioxidants (glutathione and ascorbate), as well as the content of selected macro- (Mg, Ca, NA, K) and micronutrients (Mn, Zn, Fe, Cu, Mo) were determined. These analyses, combined with the cytological characteristics of the microspore suspensions, allowed us to demonstrate that an increased microspore vitality and stronger response to ME induction were associated with higher stress resistance based on more efficient ROS scavenging and nutrient management. It was shown that a modified procedure, combining a low temperature with mannitol and sodium selenate tiller pre-treatment, reduced oxidative stress and improved the effectiveness of ME in winter wheat lines.
| S-EPMC10857491 | biostudies-literature
Project description:Plant microspores can be reprogrammed from their normal pollen development to an embryogenic route in a process termed microspore embryogenesis or androgenesis. Stress treatment has a critical role in this process, inducing the dedifferentiation of microspores and conditioning the following androgenic response. In this study, we have used three barley doubled haploid lines with similar genetic background but different androgenic response. The Barley1 GeneChip was used for transcriptome comparison of these lines after mannitol stress treatment, allowing the identification of 213 differentially expressed genes. Most of these genes belong to the functional categories "cell rescue, defense, and virulence"; "metabolism"; "transcription"; and "transport". These genes were grouped into clusters according to their expression profiles among lines. A principal component analysis allowed us to associate specific gene expression clusters to phenotypic variables. Genes associated with the ability of microspores to divide and form embryos were mainly involved in changes in the structure and function of membranes, efficient use of available energy sources, and cell fate. Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration.
| S-EPMC2700865 | biostudies-literature
Project description:Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7 d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10 642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered.
| S-EPMC2486481 | biostudies-literature
Project description:Microspore embryogenesis is a biotechnological process that allows to rapidly obtain doubled haploid plants for breeding programs. The process is initiated by the application of stress treatment which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), increase in MCA proteolytic activity, and activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programmes.
| S-EPMC7861468 | biostudies-literature
Project description:BackgroundA mannitol stress treatment and a subsequent application of n-butanol, known as a microtubule-disrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat. To characterize changes in cortical (CMT) and endoplasmic (EMT) microtubules organization and dynamics, associated with ME induction treatments, immunocytochemistry studies complemented by confocal laser scanning microscopy (CLSM) were accomplished. This technique has allowed us to perform advanced 3- and 4D studies of MT architecture. The degree of MT fragmentation was examined by the relative fluorescence intensity quantification.ResultsIn uni-nucleated mannitol-treated microspores, severe CMT and EMT fragmentation occurs, although a complex network of short EMT bundles protected the nucleus. Additional treatment with n-butanol resulted in further depolymerization of both CMT and EMT, simultaneously with the formation of MT aggregates in the perinuclear region. Some aggregates resembled a preprophase band. In addition, a portion of the microspores progressed to the first mitotic division during the treatments. Bi-nucleate pollen-like structures showed a high MT depolymerization after mannitol treatment and numerous EMT bundles around the vegetative and generative nuclei after n-butanol. Interestingly, bi-nucleate symmetric structures showed prominent stabilization of EMT.ConclusionsFragmentation and stabilization of microtubules induced by mannitol- and n-butanol lead to new configurations essential for the induction of microspore embryogenesis in bread wheat. These results provide robust insight into MT dynamics during EM induction and open avenues to address newly targeted treatments to induce ME in recalcitrant species.
| S-EPMC8656030 | biostudies-literature