Tracing 100 million years of grass genome evolutionary plasticity.

Grasses derive from a family of monocotyledonous plants that includes crops of major economic importance such as wheat, rice, sorghum and barley, sharing a common ancestor some 100 million years ago. The genomic attributes of plant adaptation remain obscure and the consequences of recurrent whole genome duplications (WGD) or polyploidization events, a major force in plant evolution, remain largely speculative. We conducted a comparative analysis of omics data from ten grass species to unveil structural (inversions, fusions, fissions, duplications, substitutions) and regulatory (expression and methylation) basis of genome plasticity, as possible attributes of plant long lasting evolution and adaptation. The present study demonstrates that diverged polyploid lineages sharing a common WGD event often present the same patterns of structural changes and evolutionary dynamics, but these patterns are difficult to generalize across independent WGD events as a result of non-WGD factors such as selection and domestication of crops. Polyploidy is unequivocally linked to the evolutionary success of grasses during the past 100 million years, although it remains difficult to attribute this success to particular genomic consequences of polyploidization, suggesting that polyploids harness the potential of genome duplication, at least partially, in lineage-specific ways. Overall, the present study clearly demonstrates that post-polyploidization reprogramming is more complex than traditionally reported in investigating single species and calls for a critical and comprehensive comparison across independently polyploidized lineages.

Custom methods to identify conserved genetic modules applied to novel transcriptomic data from Amborella trichopoda.

We have devised a procedure for the inter-species comparison of transcriptomic data and used this procedure to reconstruct the expression dynamics of major genetic modules that were present at least 149 million years ago in the most recent common ancestor of living angiosperms. We began by using laser-assisted microdissection to generate novel transcriptomic data from female flower tissues of Amborella trichopoda, the likely sister to all other living angiosperms. We then employed a gene-expression clustering method, followed by a custom procedure to compare genetic modules on the basis of gene orthology between Amborella and the molecular-genetic model angiosperm Arabidopsis thaliana. Using this protocol, we succeeded in identifying nine major genetic modules that appear to have conserved their expression dynamics from an early stage in angiosperm evolution. The genes of these modules, representing over 5000 orthogroups, include around one third of those known to control female reproductive development in Arabidopsis. Our study constitutes a proof of concept for the comparison of transcriptomic data between widely diverged plant species and represents a first step in the large-scale analysis of gene expression dynamics in a macro-evolutionary context.

Variation in Expression of the HECT E3 Ligase UPL3 Modulates LEC2 Levels, Seed Size, and Crop Yields in Brassica napus.

Identifying genetic variation that increases crop yields is a primary objective in plant breeding. We used association analyses of oilseed rape/canola (Brassica napus) accessions to identify genetic variation that influences seed size, lipid content, and final crop yield. Variation in the promoter region of the HECT E3 ligase gene BnaUPL3 C03 made a major contribution to variation in seed weight per pod, with accessions exhibiting high seed weight per pod having lower levels of BnaUPL3 C03 expression. We defined a mechanism in which UPL3 mediated the proteasomal degradation of LEC2, a master transcriptional regulator of seed maturation. Accessions with reduced UPL3 expression had increased LEC2 protein levels, larger seeds, and prolonged expression of lipid biosynthetic genes during seed maturation. Natural variation in BnaUPL3 C03 expression appears not to have been exploited in current B napus breeding lines and could therefore be used as a new approach to maximize future yields in this important oil crop.

Regulation of FUSCA3 Expression During Seed Development in Arabidopsis.

FUSCA3 (FUS3) is a master regulator of seed development important in establishing and maintaining embryonic identity whose expression is tightly regulated at genetic and epigenetic levels. Despite this prominent role, the control of FUS3 expression remains poorly understood. Promoter and functional complementation analyses provided insight into the regulation of FUS3. W-boxes present in the promoter proximal to the start of transcription are recognized by WRKY type-1 factors which are necessary for the activation of FUS3 expression. The RY motif, the binding site of B3 factors, is important for the activation of FUS3 in the embryo proper but not in the suspensor. The loss of a negative regulatory sequence (NRS) leads to preferential expression of FUS3 in the vasculature of vegetative tissues. Since the NRS includes the RY motif, mechanisms of activation and repression target adjacent or overlapping regions. These findings discriminate the regulation of FUS3 from that of LEAFY COTYLEDON2 by the control exerted by WRKY factors and by the presence of the RY motif, yet also confirm conservation of certain regulatory elements, thereby implicating potential regulation by BASIC PENTACYSTEINE (BPC) factors and POLYCOMB REPRESSIVE COMPLEX2 (PRC2).

Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1.

In Arabidopsis seeds, embryo growth is coordinated with endosperm breakdown. Mutants in the endosperm-specific gene ZHOUPI (ZOU), which encodes a unique basic helix-loop-helix (bHLH) transcription factor, have an abnormal endosperm that persists throughout seed development, significantly impeding embryo growth. Here we show that loss of function of the bHLH-encoding gene INDUCER OF CBP EXPRESSION 1 (ICE1) causes an identical endosperm persistence phenotype. We show that ZOU and ICE1 are co-expressed in the endosperm and interact in yeast via their bHLH domains. We show both genetically and in a heterologous plant system that, despite the fact that both ZOU and ICE1 can form homodimers in yeast, their role in endosperm breakdown requires their heterodimerization. Consistent with this conclusion, we confirm that ZOU and ICE1 regulate the expression of common target genes in the developing endosperm. Finally, we show that heterodimerization of ZOU and ICE1 is likely to be necessary for their binding to specific targets, rather than for their nuclear localization in the endosperm. By comparing our results with paradigms of bHLH function and evolution in animal systems we propose that the ZOU/ICE1 complex might have ancient origins, acquiring novel megagametophyte-specific functions in heterosporous land plants that were conserved in the angiosperm endosperm.

Deciphering the Molecular Mechanisms Underpinning the Transcriptional Control of Gene Expression by Master Transcriptional Regulators in Arabidopsis Seed.

In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors.

MYB118 represses endosperm maturation in seeds of Arabidopsis.

In the exalbuminous species Arabidopsis thaliana, seed maturation is accompanied by the deposition of oil and storage proteins and the reduction of the endosperm to one cell layer. Here, we consider reserve partitioning between embryo and endosperm compartments. The pattern of deposition, final amount, and composition of these reserves differ between the two compartments, with the embryo representing the principal storage tissue in mature seeds. Complex regulatory mechanisms are known to prevent activation of maturation-related programs during embryo morphogenesis and, later, during vegetative growth. Here, we describe a regulator that represses the expression of maturation-related genes during maturation within the endosperm. MYB118 is transcriptionally induced in the maturing endosperm, and seeds of myb118 mutants exhibit an endosperm-specific derepression of maturation-related genes associated with a partial relocation of storage compounds from the embryo to the endosperm. Moreover, MYB118 activates endosperm-induced genes through the recognition of TAACGG elements. These results demonstrate that the differential partitioning of reserves between the embryo and endosperm in exalbuminous Arabidopsis seeds does not only result from developmental programs that establish the embryo as the preponderant tissue within seeds. This differential partitioning is also regulated by MYB118, which regulates the biosynthesis of reserves at the spatial level during maturation.

ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development.

In angiosperm seeds the embryo is embedded within the endosperm, which is in turn enveloped by the seed coat, making inter-compartmental communication essential for coordinated seed growth. In this context the basic helix-loop-helix domain transcription factor AtZHOUPI (AtZOU) fulfils a key role in both the lysis of the transient endosperm and in embryo cuticle formation in Arabidopsis thaliana. In maize (Zea mays), a cereal with a persistent endosperm, a single gene, ZmZOU, falls into the same phylogenetic clade as AtZOU. Its expression is limited to the endosperm where it peaks during the filling stage. In ZmZOU-RNA interference knock-down lines embryo size is slightly reduced and the embryonic suspensor and the adjacent embryo surrounding region show retarded breakdown. Ectopic expression of ZmZOU reduces stomatal number, possibly due to inappropriate protein interactions. ZmZOU forms functional heterodimers with AtICE/AtSCREAM and the closely related maize proteins ZmICEb and ZmICEc, but its interaction is more efficient with the ZmICEa protein, which shows sequence divergence and only has close homologues in other monocotyledonous species. Consistent with the observation that these complexes can trans-activate target gene promoters from Arabidopsis, ZmZOU partially complements the Atzou-4 mutant. However, structural, trans-activation and gene expression data support the hypothesis that ZmZOU and ZmICEa may have coevolved to form a functional complex unique to monocot seeds. This divergence may explain the reduced functionality of ZmZOU in Arabidopsis, and reflect functional specificities which are unique to the monocotyledon lineage.

The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes.

MAIN CONCLUSION: The protein, phospholipid and sterol composition of the oil body surface from the seeds of two rapeseed genotypes was compared in order to explain their contrasted oil extractability. In the mature seeds of oleaginous plants, storage lipids accumulate in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids surrounded by a phospholipid monolayer in which structural proteins are embedded. The physical stability of OBs is a consequence of the interactions between proteins and phospholipids. A detailed study of OB characteristics in mature seeds as well as throughout seed development was carried out on two contrasting rapeseed genotypes Amber and Warzanwski. These two accessions were chosen because they differ dramatically in (1) crushing ability, (2) oil extraction yield and, (3) the stability of purified OBs. Warzanwski has higher crushing ability, better oil extraction yield and less stable purified OBs than Amber. OB morphology was investigated in situ using fluorescence microscopy, transmission electron microscopy and pulsed field gradient NMR. During seed development, OB diameter first increased and then decreased 30 days after pollination in both Amber and Warzanwski embryos. In mature seeds, Amber OBs were significantly smaller. The protein, phospholipid and sterol composition of the hemi-membrane was compared between the two accessions. Amber OBs were enriched with H-oleosins and steroleosins, suggesting increased coverage of the OB surface consistent with their higher stability. The nature and composition of phospholipids and sterols in Amber OBs suggest that the hemi-membrane would have a more rigid structure than that of Warzanwski OBs.

Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds.

Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. OBs are covered with specific proteins embedded in a single layer of phospholipids. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development. Analyses were carried out with different genotypes: the wild type and three mutants affected in the accumulation of various oleosins (OLE1, OLE2, and OLE4), three major OB proteins. Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t). Our results indicate that OB size increases sharply during seed maturation, in part by OB fusion, and then decreases until the end of the maturation process. In single, double, and triple mutant backgrounds, the size and spatial distribution of OBs are modified, affecting in turn the total lipid content, which suggests that the oleosins studied have specific functions in the dynamics of lipid accumulation.

Transcriptional regulation of Arabidopsis LEAFY COTYLEDON2 involves RLE, a cis-element that regulates trimethylation of histone H3 at lysine-27.

LEAFY COTYLEDON2 (LEC2) is a master regulator of seed development in Arabidopsis thaliana. In vegetative organs, LEC2 expression is negatively regulated by Polycomb Repressive Complex2 (PRC2) that catalyzes histone H3 Lys 27 trimethylation (H3K27me3) and plays a crucial role in developmental phase transitions. To characterize the cis-regulatory elements involved in the transcriptional regulation of LEC2, molecular dissections and functional analyses of the promoter region were performed in vitro, both in yeast and in planta. Two cis-activating elements and a cis-repressing element (RLE) that is required for H3K27me3 marking were characterized. Remarkably, insertion of the RLE cis-element into pF3H, an unrelated promoter, is sufficient for repressing its transcriptional activity in different tissues. Besides improving our understanding of LEC2 regulation, this study provides important new insights into the mechanisms underlying H3K27me3 deposition and PRC2 recruitment at a specific locus in plants.

Evolution goes GAGA: GAGA binding proteins across kingdoms.

Chromatin-associated proteins (CAP) play a crucial role in the regulation of gene expression and development in higher organisms. They are involved in the control of chromatin structure and dynamics. CAP have been extensively studied over the past years and are classified into two major groups: enzymes that modify histone stability and organization by post-translational modification of histone N-Terminal tails; and proteins that use ATP hydrolysis to modify chromatin structure. All of these proteins show a relatively high degree of sequence conservation across the animal and plant kingdoms. The essential Drosophila melanogaster GAGA factor (dGAF) interacts with these two types of CAP to regulate homeobox genes and thus contributes to a wide range of developmental events. Surprisingly, however, it is not conserved in plants. In this review, following an overview of fly GAF functions, we discuss the role of plant BBR/BPC proteins. These appear to functionally converge with dGAF despite a completely divergent amino acid sequence. Some suggestions are given for further investigation into the function of BPC proteins in plants.

Recent progress in molecular genetics and omics-driven research in seed biology.

Elucidating the mechanisms that control seed development, metabolism, and physiology is a fundamental issue in biology. Michel Caboche had long been a catalyst for seed biology research in France up until his untimely passing away last year. To honour his memory, we have updated a review written under his coordination in 2010 entitled "Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research". This review encompassed different molecular aspects of seed development, reserve accumulation, dormancy and germination, that are studied in the lab created by M. Caboche. We have extended the scope of this review to highlight original experimental approaches implemented in the field over the past decade such as omics approaches aimed at investigating the control of gene expression, protein modifications, primary and specialized metabolites at the tissue or even cellular level, as well as seed biodiversity and the impact of the environment on seed quality.

L'élucidation des mécanismes qui contrôlent le développement, le métabolisme et la physiologie des graines est une question fondamentale en biologie. Michel Caboche a longtemps été un catalyseur de la recherche en biologie des graines en France jusqu'à son décès prématuré l'année dernière. Pour honorer sa mémoire, nous avons mis à jour une revue écrite sous sa coordination en 2010 intitulée « Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research ¼. Cette revue englobait différents aspects moléculaires du développement des graines, de l'accumulation des réserves, de la dormance et de la germination, qui sont étudiés dans le laboratoire créé par M. Caboche. Nous avons étendu la portée de cette revue pour mettre en évidence des approches expérimentales originales mises en œuvre dans le domaine au cours de la dernière décennie, telles que les approches omiques visant à étudier le contrôle de l'expression des gènes, les modifications des protéines, les métabolites primaires et spécialisés au niveau des tissus ou même des cellules, tout en tenant compte de la biodiversité des graines et de l'impact de l'environnement sur leur qualité.

Expression variation in connected recombinant populations of Arabidopsis thaliana highlights distinct transcriptome architectures.

BACKGROUND: Expression traits can vary quantitatively between individuals and have a complex inheritance. Identification of the genetics underlying transcript variation can help in the understanding of phenotypic variation due to genetic factors regulating transcript abundance and shed light into divergence patterns. So far, only a limited number of studies have addressed this subject in Arabidopsis, with contrasting results due to dissimilar statistical power. Here, we present the transcriptome architecture in leaf tissue of two RIL sets obtained from a connected-cross design involving 3 commonly used accessions. We also present the transcriptome architecture observed in developing seeds of a third independent cross. RESULTS: The utilisation of the novel R/eqtl package (which goal is to automatize and extend functions from the R/qtl package) allowed us to map 4,290 and 6,534 eQTLs in the Cvi-0 × Col-0 and Bur-0 × Col-0 recombinant populations respectively. In agreement with previous studies, we observed a larger phenotypic variance explained by eQTLs in linkage with the controlled gene (potentially cis-acting), compared to distant loci (acting necessarily indirectly or in trans). Distant eQTLs hotspots were essentially not conserved between crosses, but instead, cross-specific. Accounting for confounding factors using a probabilistic approach (VBQTL) increased the mapping resolution and the number of significant associations. Moreover, using local eQTLs obtained from this approach, we detected evidence for a directional allelic effect in genes with related function, where significantly more eQTLs than expected by chance were up-regulated from one of the accessions. Primary experimental data, analysis parameters, eQTL results and visualisation of LOD score curves presented here are stored and accessible through the QTLstore service database http://qtlstore.versailles.inra.fr/. CONCLUSIONS: Our results demonstrate the extensive diversity and moderately conserved eQTL landscape between crosses and validate the utilisation of expression traits to explore for candidates behind phenotypic variation among accessions. Furthermore, this stresses the need for a wider spectrum of diversity to fully understand expression trait variation within a species.

Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research.

Seeds play a fundamental role in colonization of the environment by spermatophytes, and seeds harvested from crops are the main food source for human beings. Knowledge of seed biology is therefore important for both fundamental and applied issues. This review on seed biology illustrates the important progress made in the field of Arabidopsis seed research over the last decade. Access to 'omics' tools, including the inventory of genes deduced from sequencing of the Arabidopsis genome, has speeded up the analysis of biological functions operating in seeds. This review covers the following processes: seed and seed coat development, seed reserve accumulation, seed dormancy and seed germination. We present new insights in these various fields and describe ongoing biotechnology approaches to improve seed characteristics in crops.

Genetic and Molecular Control of Somatic Embryogenesis.

Somatic embryogenesis is a method of asexual reproduction that can occur naturally in various plant species and is widely used for clonal propagation, transformation and regeneration of different crops. Somatic embryogenesis shares some developmental and physiological similarities with zygotic embryogenesis as it involves common actors of hormonal, transcriptional, developmental and epigenetic controls. Here, we provide an overview of the main signaling pathways involved in the induction and regulation of somatic embryogenesis with a focus on the master regulators of seed development, LEAFY COTYLEDON 1 and 2, ABSCISIC ACID INSENSITIVE 3 and FUSCA 3 transcription factors whose precise role during both zygotic and somatic embryogenesis remains to be fully elucidated.

The Seed Development Factors TT2 and MYB5 Regulate Heat Stress Response in Arabidopsis.

HEAT SHOCK FACTOR A2 (HSFA2) is a regulator of multiple environmental stress responses required for stress acclimation. We analyzed HSFA2 co-regulated genes and identified 43 genes strongly co-regulated with HSFA2 during multiple stresses. Motif enrichment analysis revealed an over-representation of the site II element (SIIE) in the promoters of these genes. In a yeast 1-hybrid screen with the SIIE, we identified the closely related R2R3-MYB transcription factors TT2 and MYB5. We found overexpression of MYB5 or TT2 rendered plants heat stress tolerant. In contrast, tt2, myb5, and tt2/myb5 loss of function mutants showed heat stress hypersensitivity. Transient expression assays confirmed that MYB5 and TT2 can regulate the HSFA2 promoter together with the other members of the MBW complex, TT8 and TRANSPARENT TESTA GLABRA 1 (TTG1) and that the SIIE was involved in this regulation. Transcriptomic analysis revealed that TT2/MYB5 target promoters were enriched in SIIE. Overall, we report a new function of TT2 and MYB5 in stress resistance and a role in SIIE-mediated HSFA2 regulation.

Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis.

Seeds represent the main source of nutrients for animals and humans, and knowledge of their biology provides tools for improving agricultural practices and managing genetic resources. There is also tremendous interest in using seeds as a sustainable alternative to fossil reserves for green chemistry. Seeds accumulate large amounts of storage compounds such as carbohydrates, proteins and oils. It would be useful for agro-industrial purposes to produce seeds that accumulate these storage compounds more specifically and at higher levels. The main metabolic pathways necessary for oil, starch or protein accumulation are well characterized. However, the overall regulation of partitioning between the various pathways remains unclear. Such knowledge could provide new molecular tools for improving the qualities of crop seeds (Focks and Benning, 1998, Plant Physiol. 118, 91). Studies to improve understanding of the genetic controls of seed development and metabolism therefore remain a key area of research. In the model plant Arabidopsis, genetic analyses have demonstrated that LEAFY COTYLEDON genes, namely LEC1, LEC2 and FUSCA3 (FUS3), are key transcriptional regulators of seed maturation, together with ABSCISIC ACID INSENSITIVE 3 (ABI3). Interestingly, LEC2, FUS3 and ABI3 are related proteins that all contain a 'B3' DNA-binding domain. In recent years, genetic and molecular studies have shed new light on the intricate regulatory network involving these regulators and their interactions with other factors such as LEC1, PICKLE, ABI5 or WRI1, as well as with sugar and hormonal signaling. Here, we summarize the most recent advances in our understanding of this complex regulatory network and its role in the control of seed maturation.

Regulation of HSD1 in seeds of Arabidopsis thaliana.

The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition faithfully reflected mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines overexpressing HSD1 were then obtained to test the importance of proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate and mobilize storage lipids. Interestingly, overexpressors of HSD1 over-accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

BACKGROUND: Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. METHODS AND RESULTS: We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. CONCLUSION: This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.