Opposite Auxin Dynamics Determine the Gametophytic and Embryogenic Fates of the Microspore.

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us 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 the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the 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 programs.

Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement.

Under stress, isolated microspores are reprogrammed in vitro towards embryogenesis, producing doubled haploid plants that are useful biotechnological tools in plant breeding as a source of new genetic variability, fixed in homozygous plants in only one generation. Stress-induced cell death and low rates of cell reprogramming are major factors that reduce yield. Knowledge gained in recent years has revealed that initiation and progression of microspore embryogenesis involve a complex network of factors, whose roles are not yet well understood. Here, I review recent findings on the determinant factors underlying stress-induced microspore embryogenesis, focusing on the role of autophagy, cell death, auxin, chromatin modifications, and the cell wall. Autophagy and cell death proteases are crucial players in the response to stress, while cell reprogramming and acquisition of totipotency are regulated by hormonal and epigenetic mechanisms. Auxin biosynthesis, transport, and action are required for microspore embryogenesis. Initial stages involve DNA hypomethylation, H3K9 demethylation, and H3/H4 acetylation. Cell wall remodelling, with pectin de-methylesterification and arabinogalactan protein expression, is necessary for embryo development. Recent reports show that treatments with small modulators of autophagy, proteases, and epigenetic marks reduce cell death and enhance embryogenesis initiation in several crops, opening up new possibilities for improving in vitro embryo production in breeding programmes.

Development of the ovule and seed of Habanero chili pepper (Capsicum chinense Jacq.): Anatomical characterization and immunocytochemical patterns of pectin methyl-esterification.

Ovule and seed development in plants has long fascinated the scientific community given the complex cell coordination implicated in these processes. These cell events are highly conserved but are not necessarily representative of all plants. In this study, with the aim of obtaining information regarding the cellular patterns that follow the usual development of the ovule and the zygotic embryo, we carried out an integral anatomical study of the Capsicum chinense Jacq., floral buds and seeds at various days during maturation. This study allowed us to identify the main histo-morphological stages accompanying the transition of somatic cells into the macrospore, female gamete, and the zygotic embryogenesis. This knowledge is fundamental for future biotechnological research focused on solving the morphological recalcitrance observed during the in vitro induction of somatic or microspore embryogenesis in Capsicum. For the first time in C. chinense, we have described the hypostases, a putative source of plant growth regulators, and "the corrosion cavity", a space around the embryo. Additionally, the cell wall pectin-esterification status was investigated by immunohistology. At early stages of morphogenesis, the pectin is highly methyl-esterified; however, methyl-esterification decreases gradually throughout the process. A comparison of the results obtained here, together with the histo- and immunological changes occurring during the somatic and microspore embryogenesis, should help to elucidate the biochemical mechanisms that trigger the morphogenic events in Capsicum spp.

The effects of organophosphorus insecticides and heavy metals on DNA damage and programmed cell death in two plant models.

The ubiquity of pollutants, such as agrochemicals and heavy metals, constitute a serious risk to human health. To evaluate the induction of DNA damage and programmed cell death (PCD), root cells of Allium cepa and Vicia faba were treated with two organophosphate insecticides (OI), fenthion and malathion, and with two heavy metal (HM) salts, nickel nitrate and potassium dichromate. An alkaline variant of the comet assay was performed to identify DNA breaks; the results showed comets in a dose-dependent manner, while higher concentrations induced clouds following exposure to OIs and HMs. Similarly, treatments with higher concentrations of OIs and HMs were analyzed by immunocytochemistry, and several structural characteristics of PCD were observed, including chromatin condensation, cytoplasmic vacuolization, nuclear shrinkage, condensation of the protoplast away from the cell wall, and nuclei fragmentation with apoptotic-like corpse formation. Abiotic stress also caused other features associated with PCD, such as an increase of active caspase-3-like protein, changes in the location of cytochrome C (Cyt C) toward the cytoplasm, and decreases in extracellular signal-regulated protein kinase (ERK) expression. Genotoxicity results setting out an oxidative via of DNA damage and evidence the role of the high affinity of HM and OI by DNA molecule as underlying cause of genotoxic effect. The PCD features observed in root cells of A. cepa and V. faba suggest that PCD takes place through a process that involves ERK inactivation, culminating in Cyt C release and caspase-3-like activation. The sensitivity of both plant models to abiotic stress was clearly demonstrated, validating their role as good biosensors of DNA breakage and PCD induced by environmental stressors.

Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley.

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.

Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba.

Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated. In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation. These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction.

BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus.

BACKGROUND: Pectins are one of the main components of plant cell walls. They are secreted to the wall as highly methylesterified forms that can be de-esterified by pectin methylesterases (PMEs). The degree of methylesterification of pectins changes during development, PMEs are involved in the cell wall remodeling that occurs during diverse plant developmental processes. Nevertheless, the functional meaning of pectin-related wall remodeling in different cell types and processes remains unclear. In vivo, the microspore follows the gametophytic pathway and differentiates to form the pollen grain. In vitro, the microspore can be reprogrammed by stress treatments becoming a totipotent cell that starts to proliferate and follows the embryogenic pathway, a process known as microspore embryogenesis. RESULTS: To investigate if the change of developmental programme of the microspore towards embryogenesis involves changes in pectin esterification levels, which would cause the cell wall remodeling during the process, in the present study, dynamics of PME expression and degrees of pectin esterification have been analysed during microspore embryogenesis and compared with the gametophytic development, in Brassica napus. A multidisciplinary approach has been adopted including BnPME gene expression analysis by quantitative RT-PCR, fluorescence in situ hybridization, immuno-dot-blot and immunofluorescence with JIM5 and JIM7 antibodies to reveal low and highly-methylesterified pectins. The results showed that cell differentiation at advanced developmental stages involved induction of BnPME expression and pectin de-esterification, processes that were also detected in zygotic embryos, providing additional evidence that microspore embryogenesis mimics zygotic embryogenesis. By contrast, early microspore embryogenesis, totipotency and proliferation were associated with low expression of BnPME and high levels of esterified pectins. CONCLUSIONS: The results show that the change of developmental programme of the microspore involves changes in pectin esterification associated with proliferation and differentiation events, which may cause the cell wall remodeling during the process. The findings indicate pectin-related modifications in the cell wall during microspore embryogenesis, providing new insights into the role of pectin esterification and cell wall configuration in microspore totipotency, embryogenesis induction and progression.

Detection of Epigenetic Modifications During Microspore Embryogenesis: Analysis of DNA Methylation Patterns Dynamics.

Methylation of 5-deoxy-cytidines of DNA constitutes a prominent epigenetic modification of the chromatin fiber which is locked in a transcriptionally inactive conformation. Changes in global DNA methylation are involved in many plant developmental processes during proliferation and differentiation events. The analysis of the changes of global DNA methylation distribution patterns during microspore embryogenesis induction and progression will inform on the regulatory mechanisms of the process, helping in the design of protocols to improve its efficiency in different species. To investigate the DNA methylation dynamics during microspore embryogenesis in the different cell types present in the cultures, the analysis of spatial and temporal pattern of nuclear distribution of 5-methyl-deoxy-cytidine (5mdC) constitutes a potent approach. The immunolocalization of 5mdC on sections and subsequent confocal laser microscopy analysis have been developed for in situ cellular analysis of a variety of plant samples, including embryogenic microspore and anther cultures. Quantification of 5mdC immunofluorescence intensity by image analysis software also permits to estimate differences in global DNA methylation levels among different cell types during development.

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L.

BACKGROUND: In Quercus suber, cork oak, a Mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. Despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. The search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. In this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. Rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach. RESULTS: Results showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. DNA methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers. CONCLUSIONS: These findings suggest that DNA hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species.

Changes in histone methylation and acetylation during microspore reprogramming to embryogenesis occur concomitantly with Bn HKMT and Bn HAT expression and are associated with cell totipotency, proliferation, and differentiation in Brassica napus.

In response to stress treatments, microspores can be reprogrammed to become totipotent cells that follow an embryogenic pathway producing haploid and double-haploid embryos which are important biotechnological tools in plant breeding. Recent studies have revealed the involvement of DNA methylation in regulating this process, but no information is available on the role of histone modifications in microspore embryogenesis. Histone modifications are major epigenetic marks controlling gene expression during plant development and in response to environmental changes. Lysine methylation of histones, accomplished by histone lysine methyltransferases (HKMTs), can occur on different lysine residues, with histone H3K9 methylation being mainly associated with transcriptionally silenced regions. In contrast, histone H3 and H4 acetylation is carried out by histone acetyltransferases (HATs) and is associated with actively transcribed genes. In this work, we analyzed 3 different histone epigenetic marks: dimethylation of H3K9 (H3K9me2) and acetylation of H3 and H4 (H3Ac and H4Ac) during microspore embryogenesis in Brassica napus by Western blot and immunofluorescence assays. The expression patterns of histone methyltransferase BnHKMT and histone acetyltransferase BnHAT genes have also been analyzed by qPCR. Our results revealed different spatial and temporal distribution patterns for methylated and acetylated histone variants during microspore embryogenesis and their similarity with the expression profiles of BnHKMT and BnHAT, respectively. The data presented suggest the participation of H3K9me2 and HKMT in embryo cell differentiation and heterochromatinization events, whereas H3Ac, H4Ac, and HAT would be involved in transcriptional activation, totipotency, and proliferation events during cell reprogramming and embryo development.

Changes in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley.

Under specific stress treatments, the microspore can be induced in vitro to deviate from its gametophytic development and to reprogram towards embryogenesis, becoming a totipotent cell and forming haploid embryos. These can further regenerate homozygous plants for production of new isogenic lines, an important biotechnological tool for crop breeding. DNA methylation constitutes a prominent epigenetic modification of the chromatin fiber which regulates gene expression. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation; however, the relationship between global DNA methylation and genome-wide expression patterns is still poorly understood. In this work, the dynamics of global DNA methylation levels and distribution patterns were analyzed during microspore reprogramming to embryogenesis and during pollen development in Hordeum vulgare. Quantification of global DNA methylation levels and 5-methyl-deoxycytidine (5mdC) immunofluorescence were conducted at specific stages of pollen development and after reprogramming to embryogenesis to analyze the epigenetic changes that accompany the change of developmental program and cell fate. The results showed low DNA methylation levels in microspores and a high increase along pollen development and maturation; an intense 5mdC signal was concentrated in the generative and sperm nuclei whereas the vegetative nucleus exhibited a weaker DNA methylation signal. After inductive stress treatment, low methylation levels and faint 5mdC signals were observed in nuclei of reprogrammed microspores and 2-4-cell proembryos. This data revealed a global DNA hypomethylation during the change of the developmental program and first embryogenic divisions. This is in contrast with the hypermethylation of generative and sperm cells of the male germline during pollen maturation, suggesting an epigenetic regulation after induction of microspore embryogenesis. At later embryogenesis stages, global DNA methylation progressively increased, accompanying embryo development and differentiation events like in zygotic embryos, corroborating that DNA methylation is critical for the regulation of gene expression in microspore embryogenesis.

Epigenetic changes accompany developmental programmed cell death in tapetum cells.

The tapetum, the nursing tissue inside anthers, undergoes cellular degradation by programmed cell death (PCD) during late stages of microspore-early pollen development. Despite the key function of tapetum, little is known about the molecular mechanisms regulating this cell death process in which profound nuclear and chromatin changes occur. Epigenetic features (DNA methylation and histone modifications) have been revealed as hallmarks that establish the functional status of chromatin domains, but no evidence on the epigenetic regulation of PCD has been reported. DNA methylation is accomplished by DNA methyltransferases, among which DNA methyl transferase 1 (MET1) constitutes one of the CG maintenance methyltransferase in plants, also showing de novo methyltransferase activity. In this work, the changes in epigenetic marks during the PCD of tapetal cells have been investigated by a multidisciplinary approach to reveal the dynamics of DNA methylation and the pattern of expression of MET1 in relation to the main cellular changes of this PCD process which have also been characterized in two species, Brassica napus and Nicotiana tabacum. The results showed that tapetum PCD progresses with the increase in global DNA methylation and MET1 expression, epigenetic changes that accompanied the reorganization of the nuclear architecture and a high chromatin condensation, activity of caspase 3-like proteases and Cyt c release. The reported data indicate a relationship between the PCD process and the DNA methylation dynamics and MET1 expression in tapetal cells, suggesting a possible new role for the epigenetic marks in the nuclear events occurring during this cell death process and providing new insights into the epigenetic control of plant PCD.

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus.

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39-4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39-4 gene, JIM13 and JIM14 epitopes found specifically in 2-4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.

The 5-methyl-deoxy-cytidine (5mdC) localization to reveal in situ the dynamics of DNA methylation chromatin pattern in a variety of plant organ and tissue cells during development.

DNA methylation of cytosine residues constitutes a prominent epigenetic modification of the chromatin fiber which is locked in a transcriptionally inactive conformation leading to gene silencing. Plant developmental processes, as differentiation and proliferation, are accompanied by chromatin remodeling and epigenetic reprogramming. Despite the increasing knowledge gained on the epigenetic mechanisms controlling plant developmental processes, the knowledge of the DNA methylation regulation during relevant developmental programs in flowering plants, such as gametogenesis or embryogenesis, is very limited. The analysis of global DNA methylation levels has been frequently conducted by high performance capillary electrophoresis, and more recently also by ELISA-based assays, which provided quantitative data of whole organs and tissues. Nevertheless, to investigate the DNA methylation dynamics during plant development in different cell types of the same organ, the analysis of spatial and temporal pattern of nuclear distribution of 5-methyl-deoxy-cytidine (5mdC) constitutes a potent approach. In this work, immunolocalization of 5mdC on sections and subsequent confocal laser microscopy analysis have been applied for in situ cellular analysis of a variety of plant cells, tissues and organs with different characteristics, e.g. hardness, heterogeneity, cell accessibility, tissue compactness, etc.; the results demonstrated the versatility and feasibility of the approach for different plant samples, and revealed defined DNA methylation nuclear patterns associated with differentiation and proliferation events of various plant cell types and developmental programs. Quantification of 5mdC immunofluorescence intensity by image analysis software also permitted to estimate differences in global DNA methylation levels among different cells types of the same organ during development.

DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis.

Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes.

A new microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials, expresses auxin and efficiently regenerates doubled-haploid plants in Brassica napus.

BACKGROUND: Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction. RESULTS: We have developed a new in vitro system at lower temperature (18°C) to efficiently induce microspore embryogenesis throughout two different developmental pathways: one involving the formation of suspensor-like structures (52.4%) and another producing multicellular embryos without suspensor (13.1%); additionally, a small proportion of non-responsive microspores followed a gametophytic-like development (34.4%) leading to mature pollen. The suspensor-like pathway followed at 18°C involved the establishment of asymmetric identities from the first microspore division and an early polarity leading to different cell fates, suspensor and embryo development, which were formed by cells with different organizations and endogenous auxin distribution, similar to zygotic embryogenesis. In addition, a new strategy for germination of microspore derived embryos was developed for achieving more than 90% conversion of embryos to plantlets, with a predominance of spontaneous doubled haploids plants. CONCLUSION: The present work reveals a novel mechanism for efficient microspore embryogenesis induction in B. napus using continuous low temperature treatment. Results indicated that low temperature applied for longer periods favours an embryogenesis pathway whose first division originates asymmetric cell identities, early polarity establishment and the formation of suspensor-like structures, mimicking zygotic embryogenesis. This new in vitro system provides a convenient tool to analyze in situ the mechanisms underlying different developmental pathways during the microspore reprogramming, breaking or not the cellular symmetry, the establishment of polarity and the developmental embryo patterning, which further produce mature embryos and plants.

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley.

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.