Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

KEY MESSAGE: In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways. In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

Light induces gene expression to enhance the synthesis of storage reserves in Brassica napus L. embryos.

KEY MESSAGE: In this manuscript, we disclosed the influence of light on the accumulation of storage reserves in B. napus embryos.1.Light induced the gene expression in the developing embryos of B. napus.2.Light promoted the starch synthesis in chloroplasts of B. napus embryos.3.Light enhanced the metabolic activity of storage reserve synthesis in B. napus embryos. Light influences the accumulation of storage reserves in embryos, but the molecular mechanism was not fully understood. Here, we monitored the effects of light on reserve biosynthesis in Brassica napus by comparing embryos from siliques grown in normal light conditions to those that were shaded or masked (i.e., darkened completely). Masked embryos developed more slowly, weighed less, and contained fewer proteins and lipids than control embryos. They also had fewer and smaller oil bodies than control embryos and lacked chloroplasts, where starch grains are usually synthesized. The levels of most amino acids, carbohydrates, and fatty acids were higher in masked embryos than in control or shaded embryos, whereas the levels of these metabolites in the masked endosperms were lower than those in control and shaded endosperm. Transcriptome analysis indicated that genes involved in photosynthesis (42 genes), amino acid biosynthesis (51 genes), lipid metabolism (61 genes), and sugar transport (13 genes) were significantly repressed in masked embryos. Our results suggest that light contributes to reserve accumulation in embryos by inducing the expression of metabolic genes, thereby enhancing the biosynthesis of storage reserves.

Genetic Mapping Combined with a Transcriptome Analysis to Screen for Candidate Genes Responsive to Abscisic Acid Treatment in Brassica napus Embryos During Seed Germination.

Brassica napus embryos contain precursor tissues for the leaves, stem, and root, as well as the cotyledons, and these precursor tissues play key roles in seed germination, seedling survival, and subsequent seedling growth. Abscisic acid (ABA) plays a prominent role in the inhibition of seed germination. The underlying molecular mechanisms of the embryo responses to ABA stress followed by inhibited seed germination have not been reported in B. napus to date. In this study, we conducted quantitative trait locus (QTL) analysis of B. napus seed in response to ABA stress using 170 recombinant inbred lines. Furthermore, we performed transcriptome sequencing (RNA-seq) analyses by using B. napus ZS11 embryos under sterile deionized water (control) and 10 mg/L (10A), 20 mg/L (20A), and 30 mg/L (30A) ABA treatment conditions. In total, 10 QTLs were screened for explaining 2.70-6.73% of the phenotypic variation under ABA stress. In addition, 1495, 3332, and 3868 differentially expressed genes (DEGs) were identified in the "control vs 10A," "control vs 20A," and "control vs 30A" comparisons, respectively. Gene Ontology (GO) enrichment analysis indicated that DEG functions are mainly related to response to stimuli, response to oxygen-containing compounds, response to lipids, and the transport and seed dormancy processes. These DEGs mainly participated in the response to plant hormone signal transduction, starch and sucrose metabolism, cutin, suberine, and wax biosynthesis, and phenylpropanoid biosynthesis processes pathways. Our results provide a foundation for further explorations of the molecular regulatory mechanisms of B. napus embryos in response to abiotic stress during the seed germination stage.

Isolated Microspore Culture in Brassica napus.

Isolated microspore culture is the most efficient technique among those used to induce microspore embryogenesis. In the particular case of Brassica napus, it is also the most widely used and optimized. In this chapter, we describe a protocol for microspore culture in B. napus which includes the steps necessary to isolate and culture microspores, to induce microspore-derived embryos, to produce doubled haploid plants from them, as well as to check for the developmental stage of the microspores isolated, their viability, and the ploidy level of regenerated plantlets.

Two young genes reshape a novel interaction network in Brassica napus.

New genes often drive the evolution of gene interaction networks. In Brassica napus, the widely used genic male sterile breeding system 7365ABC is controlled by two young genes, Bnams4b and BnaMs3. However, the interaction mechanism of these two young genes remains unclear. Here, we confirmed that Bnams4b interacts with the nuclear localised E3 ligase BRUTUS (BTS). Ectopic expression of AtBRUTUS (AtBTS) and comparison between Bnams4b -transgenic Arabidopsis and bts mutants suggested that Bnams4b may drive translocation of BTS to cause various toxic defects. BnaMs3 gained an exclusive interaction with the plastid outer-membrane translocon Toc33 compared with Bnams3 and AtTic40, and specifically compensated for the toxic effects of Bnams4b . Heat shock treatment also rescued the sterile phenotype, and high temperature suppressed the interaction between Bnams4b and BTS in yeast. Furthermore, the ubiquitin system and TOC (translocon at the outer envelope membrane of chloroplasts) component accumulation were affected in Bnams4b -transgenic Arabidopsis plants. Taken together, these results indicate that new chimeric Bnams4b carries BTS from nucleus to chloroplast, which may disrupt the normal ubiquitin-proteasome system to cause toxic effects, and these defects can be compensated by BnaMs3-Toc33 interaction or environmental heat shock. It reveals a scenario in which two population-specific coevolved young genes reshape a novel interaction network in plants.

Comparative Transcriptomics Analysis of Brassica napus L. during Seed Maturation Reveals Dynamic Changes in Gene Expression between Embryos and Seed Coats and Distinct Expression Profiles of Acyl-CoA-Binding Proteins for Lipid Accumulation.

Production of vegetable oils is a vital agricultural resource and oilseed rape (Brassica napus) is the third most important oil crop globally. Although the regulation of lipid biosynthesis in oilseeds is still not fully defined, the acyl-CoA-binding proteins (ACBPs) have been reported to be involved in such metabolism, including oil accumulation, in several plant species. In this study, progressive changes in gene expression in embryos and seed coats at different stages of seed development were comprehensively investigated by transcriptomic analyses in B. napus, revealing dynamic changes in the expression of genes involved in lipid biosynthesis. We show that genes encoding BnACBP proteins show distinct changes in expression at different developmental stages of seed development and show markedly different expression between embryos and seed coats. Both isoforms of the ankyrin-repeat BnACBP2 increased during the oil accumulation period of embryo development. By contrast, the expression of the three most abundant isoforms of the small molecular mass BnACBP6 in embryos showed progressive reduction, despite having the highest overall expression level. In seed coats, BnACBP3, BnACBP4 and BnACBP5 expression remained constant during development, whereas the two major isoforms of BnACBP6 increased, contrasting with the data from embryos. We conclude that genes related to fatty acid and triacylglycerol biosynthesis showing dynamic expression changes may regulate the lipid distribution in embryos and seed coats of B. napus and that BnACBP2 and BnACBP6 are potentially important for oil accumulation.

Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer.

Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.

Correlation analysis of the transcriptome and metabolome reveals the regulatory network for lipid synthesis in developing Brassica napus embryos.

KEY MESSAGE: In this manuscript, we explored the key molecular networks for oil biosynthesis with the transcriptome and metabolome of B. napus embryo at different developmental stages. Brassica napus (B. napus) is an important oil crop worldwide, yet the molecular pathways involved in oil biosynthesis in seeds are not fully understood. In this study, we performed a combined investigation of the gene expression profiles and metabolite content in B. napus seeds at 21, 28 and 35 days after flowering (DAF), when seed oil biosynthesis takes place. The total triacylglycerol (TAG) content in seed embryos increased over the course of seed maturation, and was accompanied by changes in the fatty acid profile, an increase in lipid droplets, and a reduction in starch grains. Metabolome analysis showed that the total amino acid, free fatty acid and organic acid contents in seed embryos decreased during seed maturation. In total, the abundance of 76 metabolites was significantly different between 21 and 28 DAF, and 68 metabolites changed in abundance between 28 and 35 DAF. Transcriptome analysis showed that the set of genes differentially expressed between stages was significantly enriched in those related to lipid metabolism, transport, protein and RNA metabolism, development and signaling, covering most steps of plant lipid biosynthesis and metabolism. Importantly, the metabolite and gene expression profiles were closely correlated during seed development, especially those associated with TAG and fatty acid biosynthesis. Further, the expression of major carbohydrate metabolism-regulating genes was closely correlated with carbohydrate content during seed maturation. Our results provide novel insights into the regulation of oil biosynthesis in B. napus seeds and highlights the coordination of gene expression and metabolism in this process.

Genome-wide screening and analysis of imprinted genes in rapeseed (Brassica napus L.) endosperm.

Species-specific genomic imprinting is an epigenetic phenomenon leading to parent-of-origin-specific differential expression of maternally and paternally inherited alleles. To date, no studies of imprinting have been reported in rapeseed, a tetraploid species. Here, we analysed global patterns of allelic gene expression in developing rapeseed endosperms from reciprocal crosses between inbred lines YN171 and 93275. A total of 183 imprinted genes, consisting of 167 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs), were identified from 14,394 genes found to harbour diagnostic SNPs between the parental lines. Some imprinted genes were validated in different endosperm stages and other parental combinations by RT-PCR analysis. A clear clustering of imprinted genes throughout the rapeseed genome was identified, which was different from most other plants. Methylation analysis of 104 out of the 183 imprinted genes showed that 11 genes (7 MEGs and 4 PEGs) harboured differentially methylated regions (DMRs). Unexpectedly, only 1 MEG out of these 11 genes had a DMR that exhibited high CG methylation rate in paternal allele and had big difference between parent alleles. These results extend our understanding of gene imprinting in plants and provide potential avenues for further research in imprinted genes.

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.

Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding.

Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.

Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture.

Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 3',5'-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism.

Dynamic Metabolic Profiles and Tissue-Specific Source Effects on the Metabolome of Developing Seeds of Brassica napus.

Canola (Brassica napus) is one of several important oil-producing crops, and the physiological processes, enzymes, and genes involved in oil synthesis in canola seeds have been well characterized. However, relatively little is known about the dynamic metabolic changes that occur during oil accumulation in seeds, as well as the mechanistic origins of metabolic changes. To explore the metabolic changes that occur during oil accumulation, we isolated metabolites from both seed and silique wall and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 443 metabolites were identified from four developmental stages. Dozens of these metabolites were differentially expressed during seed ripening, including 20 known to be involved in seed development. To investigate the contribution of tissue-specific carbon sources to the biosynthesis of these metabolites, we examined the metabolic changes of silique walls and seeds under three treatments: leaf-detachment (Ld), phloem-peeling (Pe), and selective silique darkening (Sd). Our study demonstrated that the oil content was independent of leaf photosynthesis and phloem transport during oil accumulation, but required the metabolic influx from the silique wall. Notably, Sd treatment resulted in seed senescence, which eventually led to a severe reduction of the oil content. Sd treatment also caused a significant accumulation of fatty acids (FA), organic acids and amino acids. Furthermore, an unexpected accumulation of sugar derivatives and organic acid was observed in the Pe- and Sd-treated seeds. Consistent with this, the expression of a subset of genes involved in FA metabolism, sugar and oil storage was significantly altered in Pe and Sd treated seeds. Taken together, our studies suggest the metabolite profiles of canola seeds dynamically varied during the course of oil accumulation, which may provide a new insight into the mechanisms of the oil accumulation at the metabolite level.

Regulation of FATTY ACID ELONGATION1 expression in embryonic and vascular tissues of Brassica napus.

The expression of the FATTY ACID ELONGATION1 genes was characterised to provide insight into the regulation of very long chain fatty acid (VLCFA) biosynthesis in Brassica napus embryos. Each of the two rapeseed homoeologous genes (Bn-FAE1.1 and Bn-FAE1.2) encoding isozymes of 3-keto-acylCoA synthase, a subunit of the cytoplasmic acyl-CoA elongase complex that controls the production of elongated fatty acids, are expressed predominantly in developing seeds. The proximal regions of the Bn-FAE1.1 and Bn-FAE1.2 promoters possess strong sequence identity suggesting that transcriptional control of expression is mediated by this region which contains putative cis-elements characteristic of those found in the promoters of genes expressed in embryo and endosperm. Histochemical staining of rapeseed lines expressing Bn-FAE1.1 promoter:reporter gene fusions revealed a strong expression in the embryo cotyledon and axis throughout the maturation phase. Quantitative analyses revealed the region, -331 to -149, exerts a major control on cotyledon specific expression and the level of expression. A second region, -640 to -475, acts positively to enhance expression levels and extends expression of Bn-FAE1.1 into the axis and hypocotyl but also acts negatively to repress expression in the root meristem. The expression of the Bn-FAE1.1 gene was not restricted to the seed but was also detected in the vascular tissues of germinating seedlings and mature plants in the fascicular cambium tissue present in roots, stem and leaf petiole. We propose that Bn-FAE1.1 expression in vascular tissue may contribute VLCFA for barrier lipid synthesis and reflects the ancestral function of FAE1 encoded 3-keto-acylCoA synthase.

Comprehensive selection of reference genes for quantitative gene expression analysis during seed development in Brassica napus.

KEY MESSAGE: MicroRNAs have higher expression stability than protein-coding genes in B. napus seeds and are therefore good reference genes for miRNA and mRNA RT-qPCR analysis. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has become the "gold standard" to gain insight into function of genes. However, the accuracy of the technique depends on appropriate reference genes for quantification analysis in different experimental conditions. Accumulation of microRNAs (miRNAs) has also been studied by RT-qPCR, but there are no reference genes currently validated for normalization of Brassica napus miRNA expression data. In this study, we selected 43 B. napus miRNAs and 18 previously validated mRNA reference genes. The expression stability of the candidate reference genes was evaluated in different tissue samples (stages of seed development, flowers, and leaves) using geNorm, NormFinder, and RefFinder analysis. The best-ranked reference genes for expression studies during seed development (miR167-1_2, miR11-1, miR159-1 and miR168-1) were used to asses the expression of miR03-1. Since candidate miRNAs showed higher expression stability than protein-coding genes in most of the tested conditions, the expression profile of DGAT1 gene was compared when normalized by the four most stable miRNAs reference genes and by the four most stable mRNA reference genes. The expected expression pattern of DGAT1 during seed development was achieved with the use of miRNA as reference genes. In conclusion, the most stable miRNA reference genes can be employed in the normalization of RT-qPCR quantification of miRNAs and protein-coding genes. This work is the first to perform a comprehensive survey of the stability of miRNA reference genes in B. napus and provides guidelines to obtain more accurate RT-qPCR results in B. napus seeds studies.

Identification and genetic characterization by high-throughput SNP analysis of intervarietal substitution lines of rapeseed (Brassica napus L.) with enhanced embryogenic potential.

KEY MESSAGE: Seven intervarietal substitution lines were identified with embryogenic potentials up to 40.4 times that of the recurrent parent, providing an ideal material for further in depth studies of this trait. To identify genomic regions that carry genetic factors controlling embryogenic potential of isolated microspores of rapeseed, marker segregations were analysed in a segregating population of haploid microspore-derived embryos and a BC1 population from a cross between 'Express 617' and 'RS239'. After map construction 15 intervarietal substitution lines from the same cross with 'Express 617' as recurrent parent were selected with donor segments covering five genomic regions that had shown skewed segregations in the population of microspore-derived embryos but not in the BC1 population. By comparing the embryogenic potential of microspores of the 15 substitution lines and 'Express 617', seven lines were identified with significantly enhanced embryogenic potential ranging from 4.1 to 40.4 times that of 'Express 617'. To improve the genetic characterization of the selected lines, they were subjected to a high-throughput SNP analysis using the Illumina Infinium 60K chip for rapeseed. Based on 7,960 mapped SNP markers, one to eight donor segments per line, which cover 0.64-6.79% of the 2,126.1 cM of the SNP map, were found. The SNP analysis also gave evidence that homoeologous exchanges had occurred during the development of the substitution line population, increasing the genetic diversity within this population. By comparing donor segments between lines with significantly enhanced embryogenic potential and non-significant lines, 12 genomic regions were identified that may contain genetic factors controlling embryogenic potential in rapeseed. These regions range in size from 0 (represented by just one marker) to 26.8 cM and cover together just 5.42% of the SNP map.

Plant embryogenesis requires AUX/LAX-mediated auxin influx.

The plant hormone auxin and its directional transport are known to play a crucial role in defining the embryonic axis and subsequent development of the body plan. Although the role of PIN auxin efflux transporters has been clearly assigned during embryonic shoot and root specification, the role of the auxin influx carriers AUX1 and LIKE-AUX1 (LAX) proteins is not well established. Here, we used chemical and genetic tools on Brassica napus microspore-derived embryos and Arabidopsis thaliana zygotic embryos, and demonstrate that AUX1, LAX1 and LAX2 are required for both shoot and root pole formation, in concert with PIN efflux carriers. Furthermore, we uncovered a positive-feedback loop between MONOPTEROS (ARF5)-dependent auxin signalling and auxin transport. This MONOPTEROS-dependent transcriptional regulation of auxin influx (AUX1, LAX1 and LAX2) and auxin efflux (PIN1 and PIN4) carriers by MONOPTEROS helps to maintain proper auxin transport to the root tip. These results indicate that auxin-dependent cell specification during embryo development requires balanced auxin transport involving both influx and efflux mechanisms, and that this transport is maintained by a positive transcriptional feedback on auxin signalling.

The influence of heat stress on auxin distribution in transgenic B. napus microspores and microspore-derived embryos.

Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro-cultured microspores and on microspore-derived embryo development. In the "mild" heat-treated (1 day at 32 °C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 µm, endogenous auxin content in a single cell corresponded to concentration of 1.01 µM. In mcs subjected to a prolonged heat (5 days at 32 °C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning.

Induction of embryogenesis from isolated microspore cultures is a complex experimental system where microspores undergo dramatic changes in developmental fate. After ~40 years of application of electron microscopy to the study of the ultrastructural changes undergone by the induced microspore, there is still room for new discoveries. In this work, high pressure freezing and freeze substitution (HPF/FS), the best procedures known to date for ultrastructural preservation, were used to process Brassica napus microspore cultures covering all the stages of microspore embryogenesis. Analysis of these cultures by electron microscopy revealed massive processes of autophagy exclusively in embryogenic microspores, but not in other microspore-derived structures also present in cultures. However, a significant part of the autophagosomal cargo was not recycled. Instead, it was transported out of the cell, producing numerous deposits of extracytoplasmic fibrillar and membranous material. It was shown that commitment of microspores to embryogenesis is associated with both massive autophagy and excretion of the removed material. It is hypothesized that autophagy would be related to the need for a profound cytoplasmic cleaning, and excretion would be a mechanism to avoid excessive growth of the vacuolar system. Together, the results also demonstrate that the application of HPF/FS to the study of the androgenic switch is the best option currently available to identify the complex and dramatic ultrastructural changes undergone by the induced microspore. In addition, they provide significant insights to understand the cellular basis of induction of microspore embryogenesis, and open a new door for the investigation of this intriguing developmental pathway.