Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo
- Kaori Sakai†1,
- Ludivine Taconnat†2, 3,
- Nero Borrega1,
- Jennifer Yansouni2, 3,
- Véronique Brunaud2, 3,
- Christine Paysant-Le Roux2, 3,
- Etienne Delannoy2, 3,
- Marie-Laure Martin Magniette2, 3, 4,
- Loïc Lepiniec1,
- Jean Denis Faure1,
- Sandrine Balzergue2, 3, 5Email author and
- Bertrand Dubreucq1Email authorView ORCID ID profile
© The Author(s) 2018
Received: 14 June 2017
Accepted: 15 January 2018
Published: 3 February 2018
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.
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.
Multicellular (higher) organisms like plants are characterized by cell-specific differentiation and tissue formation during development. Over the last decades, many studies have addressed the question of gene expression during plant growth, under stress conditions, at precise stages or in specific genetic backgrounds. One aim of modern biology is to provide more quantitative and tissue- or cell-specific analyses with regard to the regulatory networks controlling biological processes. Nevertheless, the characterization and isolation of cell types organised in a three-dimensional space requires specific and tricky physical manipulations (for review see ). Different approaches have been used including, the expression of reporter genes coupled to affinity purification system (e.g. INTACT, , fluorescent-activated cell sorting [2, 13, 35, 44], or manual dissection based on morphological characters). These techniques are limited in their application due to the need for protoplasting or genetic transformations with cell type-specific markers.
Laser Assisted Microdissection (LAM) allows precise recovery of specific tissues or cell types, based on their morphology or fluorescence staining when cell specific markers have been introduced. If based on paraffin embedded sections, the identification of specific zones can be facilitated by the use of a microscope, although decreasing the size of the section lowers the amount of RNA extracted. Similarly, very small areas of interest will have poor total RNA content, hampering comprehensive transcriptional analysis. The size of the zone of interest and its subsequent RNA content are therefore a crucial issue in the production of good-quality data. Coupling LAM to quantitative RT-PCR and then to DNA chips has increased the number of genes detected and thus, these approaches have been widely and successfully used on seed [4, 5, 37]. However, the development of NGS (Next Generation Sequencing) technologies offers many other advantages: sensitivity, ability to quantify expression in species for which no genome sequences are available (i.e. new species of interest), access to differentially spliced forms or to non-coding RNAs. Moreover, in a specific case of plant-pathogen interaction studies, the LAM provides also a cellular-level resolution to reveal the often low coverage of pathogen transcripts . The NGS has an additional advantage compared to traditional chip hybridization, as it requires a smaller amount of total RNA for analysis. Some studies combined LAM, RNA ribosomal depletion and RNA-seq experiment in order to reveal the entire diversity of transcripts [16, 30]. In these cases, the amounts of total RNA coming from LAM is in the range of ng, requiring spending a lot of time to microdissection step, often at the expense of RNA quality or/and biological repetition number. Therefore, we combined ultra-low-RNA-seq sequencing with laser microdissection for undertaking precise and comprehensive analyses of the epidermal versus mesophyll cell transcriptomes of the cotyledons, at the torpedo-shaped stage of the small Arabidopsis embryo.
The development of Arabidopsis embryo has been extensively described [17, 41]. Briefly, after fertilization, the zygotic cell gains polarity and develops following a precise pattern of divisions to give rise to specific cell types (epidermis, vascular bundles, cortex, and mesophyll). The epidermis, which originates from the differentiation of the protoderm at the dermatogen stage, marks the junction between the embryo and the external environment . This cell layer forms a hydrophobic barrier over the aerial surfaces of the plant . Specific secondary metabolites accumulate in the epidermis in the form of cuticular fatty acids. The cutin and flavonoids then form protective compounds. Previous analyses have shown that several genes are specifically expressed in the epidermis during aerial organ development  or during embryo development [15, 27, 40].
In this paper, we investigated the effect of decreasing the total amount of tissue and RNA template using ultra-low RNA-seq methods and the technical limit of this approach. After validation, the optimized method was used to characterize genes that are differentially expressed in the epidermis versus mesophyll cells at the torpedo stage of Arabidopsis embryo development.
Plant material and growth conditions
Arabidopsis thaliana plants, accession Columbia (Col-0), were grown in a greenhouse under the following conditions: 13 h of light, 25 °C/17 °C day/night, and irrigated three times per week with mineral nutrient solution. To harvest seeds at defined developmental stages, individual flowers were tagged on the day of opening, and then opened flowers and developing siliques were counted daily. Siliques at 8 days after fertilization, corresponding to seeds containing embryos at linear stage were harvested under RNase free conditions: all materials and working surfaces were treated with RNase Zap (Ambion) and immediately fixed in 3:1 (vol/vol) ethanol:acetic acid at 4 °C on ice. Siliques were cut at the edge into ∼ 1 cm segments before fixation to allow better penetration of the fixator. The seeds were fixed under vacuum for 1 h and left O/N in the fixator at 4 °C.
The plant material was dehydrated in a graded ethanol series (70% 1 h, 85% 1 h, 95% 1 h, 100% 1 h two times, 100% ethanol O/N), and infiltrated with histoclear (1:3 1 h, 1:1 1 h, 3:1 2h30 histoclear: ethanol). This was followed by 100% histoclear for 30 min three times. Samples were then incubated with 1:1 paraffin/histoclear for 1 h and paraffin 100% at 60 °C O/N. The paraffin was replaced twice over 1–2 days. Seeds were sectioned at 8 µm thickness using an automatic microtome (Microm HM 355S) and mounted on polyethylene napthalate (PEN)-membrane slides (Zeiss) in RNase-free conditions. Slides were dried with a hot plate set at 24 °C and deparaffinized twice in 100% histoclear for 1 min and dehydrated in 100% ethanol for 1 min. Laser capture microdissection was performed using a PALM MicroBeam system (Zeiss). For pilot experiment, 20 whole cotyledons sections coming from 6 siliques on 3 individual plants were microdissected and captured. For the second experiment, each tissue type (i.e. Epidermis and Mesophyll) of each seed was separately microdissected to minimize contamination from adjacent cell and tissue types (Fig. 2a). Four biological replicates harvested at four different dates were captured for each tissue type. Each biological replicate consisted of around 25 microdissected tissue sections from at least 5 siliques coming from one individual plant. All tissues were captured in a collection tube with adhesive cap (Zeiss) within 10 min to maximize the quality of total RNA extraction.
Microdissected samples were harvested and incubated directly into RNA extraction buffer. Total RNA was extracted using the Arcturus Pico RNA extraction kit (Thermo Fisher Scientific, Inc) and then treated with RNase-free DNase (1:8 dilution of DNase I in RDD buffer; Qiagen). RNA quantity and quality were checked by microcapillary electrophoresis RNA 6000 Pico Chip (Agilent 2100 BioAnalyzer; Agilent Technologies Waldbroon, Germany). RIN (RNA Integrity Number, Agilent) obtained were around 6.3 (6.0 for epiderm and 6.5 mesophyll: range between 5.7 and 6.7).
For the pilot experiment, in order to focus on optimising the quality of extracted RNA in relation to the time spent on microdissection, 6 dilutions of a same RNA sample of whole cotyledons were used (5 ng, 100 pg, 75 pg, 50 pg, 25 pg, and 10 pg). For the second experiment, the objective of which was to compare epidermis and mesophyll tissues, a dilution of 100 pg of total RNA was used. cDNA syntheses were performed using the SMARTer Ultra Low Input RNA Kit for Sequencing-v4 (Clontech Laboratories, Inc.) and libraries were prepared according to DNA Sample Preparation Illumina kit instructions with a different bar code for each sample (Illumina, Cat. Nos. FC-131-1024). Ultra-low RNA-seq libraries were checked for their quality on High-sensitivity DNA chip using Agilent 2100 bioanalyzer (Waldbroon, Germany) before Illumina sequencing (Illumina®, California, U.S.A.). The UltraLowRNA-seq samples were sequenced in Paired-End (PE) with a read length of 100 bases. For the pilot experiment, the six libraries were sequenced on Hiseq 2000 machine. Samples with a dilution of 100 pg and 5 ng were first sequenced to obtain around 50 million reads/sample. Samples with a dilution of 100 pg, 75 pg, 50 pg, 25 pg, and 10 pg were then sequenced at a second date to obtained around 30 million reads/sample. The 100 pg sample was thus sequenced twice and used to correct a sequencing bias effect. The value of Spearman’s correlation coefficient between the two 100 pg samples was 0.61 before correction and 0.86 after. After correction, the results of the two sequencing for the 100 pg sample were similar and only the second one was kept for the analyses. For the second experiment, the eight libraries were sequenced on Hiseq 2000 machine to obtained around 35 million reads/sample with a multiplexing rate of 4 libraries/lane.
Bioinformatics and statistical analyses
The raw data (fastq) were trimmed for Phred Quality Score > 20, read length > 30 bases, and the ribosome sequences were removed with tool sortMeRNA . The mapper Bowtie version 2  was used to align reads against the A. thaliana transcriptome (with ‘local’ option and other default parameters). The 33602 annotated genes were extracted from TAIR (v10) database corresponding to the representative gene model (longest CDS) given by TAIR. The abundance of each gene was calculated by a home-made script which counts only paired-end reads for which both reads map unambiguously one gene, removing multi-hits.
All the statistical analyses were done with the R software using also EdgeR package version 3.8.6 . For the pilot experiment, to compare the gene expression, the raw counts were normalized to take the difference of the library sizes into account with TMM method and a sequencing date effect. It was done with a negative binomial generalized linear model with one factor (sequencing date). Normalized counts equal to the raw counts divided by the scaling factor minus a date sequencing effect. The second experiment concerned the tissue comparison between epidermis and mesophyll. First, genes which did not have at least 1 read after a count per million normalization in at least one half of the samples, were discarded. Then, raw counts were normalized using TMM method and count distribution was modelled with a negative binomial generalized linear model where the tissue type and the harvest date were taken into account and where the dispersion is estimated by the edgeR method. A likelihood ratio test was performed to evaluate a tissue effect. Raw p-values were adjusted with the Benjamini–Hochberg procedure to control the False Discovery Rate (FDR). A gene was declared differentially expressed if it’s adjusted p value ≤ 0.05.
RNA-Seq projects were deposited in the international repository GEO (Gene Expression Omnibus, Edgard et al. ): http://www.ncbi.nlm.nih.gov/geo/; accession no. GSEGSE NGS2014_01_MicroD GSE98176 163 and NGS2012_02_MicroD GSE98313, according to the MINSEQE ‘minimum information about a high-throughput sequencing experiment’. All steps of the experiment, from growth conditions to bioinformatics analyses, were detailed in CATdb : http://tools.ips2.u-psud.fr.fr/CATdb/; Project: NGS2012_02_microD and NGS2014_01.
Read mapping and gene detection statistics based on starting RNA quantity
Librairy size (million)
Mapped reads (million) (percentage)
Detected genes (level 0)
Common reads with 5 ng reference sample (percentage)
We then performed a microdissection experiment using a 100 pg RNA template and 4 biological replicates, with the aim to identify differentially expressed genes of epidermis versus mesophyll cells. Here, we used cotyledons at the torpedo stage of embryo development (see Methods section). To assess data quality, we performed a principal component analysis (Additional file 3: Fig. S3). The latter clearly showed no replicate bias and the two first axes, which represent 41.46% of the explained variance discriminate the two types of tissue. It is to be noted that epidermis samples are much more variable than mesophyll samples that may reflect a contamination by endosperm-specific expressed genes during the dissection process.
Number of differentially expressed genes detected based on number of biological replicates
Number of repeats
Log ratio for DE genes
Number of DE genes
Transcription factors differentially expressed in the epidermis in the developing embryo.
Expressed in the embryo
myb domain protein 56
NAC domain containing protein 2
WUSCHEL-related homeobox 8
Transcriptional factor B3 family protein
myb domain protein 118
Suh et al. 
SALT TOLERANCE ZINC FINGER
C-REPEAT/DRE BINDING FACTOR 1
myb domain protein 96
myb domain protein 17
ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5
RESPONSIVE TO HIGH LIGHT 41
ASYMMETRIC LEAVES 2
myb domain protein 94
Nakamura et al. 
myb domain protein 111
myb domain protein 38
myb domain protein 4
Abe et al. 
MERISTEM LAYER 1
Sessions et al. 
Ryu et al. 
JASMONATE INSENSITIVE 1
myb domain protein 11
Stracke et al. 2007
ENHANCER OF GLABRA3
Bernhardt et al. 
GROWTH-REGULATING FACTOR 4
WRKY DNA-binding protein 22
LEAFY COTYLEDON 1
Lotan et al. 
Homeobox-leucine zipper protein HAT4
FREEZING TOLERANCE QTL 4
ABNORMAL FLORAL ORGANS
Tsuchiya et al. 
ARABIDOPSIS RESPONSE REGULATOR
In this paper we have undertaken a microdissection followed by RNA-Seq in developing Arabidopsis embryos. We showed that robust and comprehensive RNA-Seq experiment can be performed starting with very small amount of starting material and optimized microdissection time. Embedded sections usually produce lower quality RNA than fresh tissue. However, the quality of the section remains much higher in embedded tissues compared to cryo-sections, which shrivel quickly at room temperature under the LAM. A key advantage of embedded sections is to reduce damages to organ structure before and during micro-dissection. Thus, the combination of LAM and RNA-seq provide new powerful approach for investigating the transcriptome of specific tissues or cell types. First, based on mapped reads and detected genes, we concluded that a low amount of total RNA (i.e. 10 pg), is sufficient to generate libraries and produce RNA-seq data. The number of genes detected was comprised between 15936 and 17446, which is fully comparable with embryo data obtained in previous studies based on microarrays technologies [7, 37]. Surprisingly, the number of genes detected in the 5 ng sample is lower than expected when compared to other samples (i.e. 100–50 pg), nevertheless we can observe a decrease of the number of genes detected according to the RNA quantity. The overlap between the genes detected in the reference sample (5 ng) and the other samples was good (≥ 90% with 75 and 100 pg), regardless of the variations of the library size. Based on these results and PCA/distribution profiles, we therefore recommend a minimum of 50 pg of total RNA to perform a comprehensive analysis of the expressed genome. Lower quantity of total RNA (up to 10 pg) can be used to raise libraries and sequence successfully, essentially in order to check for the presence of a given gene, but is not fully quantitative. Last, as expected, increasing the number of biological repeats positively impacted the number of DEGs detected (e.g. an additional repeat increases by around 10% the number of DEG detected).
Dissecting the epidermis for RNA-seq profiling at the torpedo stage of embryo development was a real challenge to tackle with Arabidopsis. Thus, we evaluated the approach on two well-known primary and secondary metabolic pathways, namely wax and flavonoids, both occuring preferentially in epidermal cells. Only 4 differentially genes that are supposed to be directly involved in wax and cutin biosynthesis were found: one MAH-like (At4g39150), WAX2 (At5g57800), CUT1 (At1g68530) and HOTHEAD (At1g72970). The well-characterized genes involved in epicuticular waxes and cutin like CER1, CER3 or CER4 are however not found in the dissected RNA dataset. It has to be noted that the precise biochemical functions of these 4 genes are still unknown. On the contrary, the function of acyl-CoA biosynthetic is better characterized. As expected, ACC1 gene, which is involved in malonyl-CoA synthesis was found to be preferentially expressed in epidermal cells that is consistent with previous data . Acyl-CoA is associated with epicuticular wax synthesis in developing stems . Several of them were found to have also a differentially enhanced expression in the embryo epidermis. For instance, out of the 21 KCS genes coding the first enzyme of the acyl-CoA elongation complex), eleven showed an induced expression in stem epidermis, among which six were also induced in embryo epidermis . Two out of the three remaining genes of the elongase complex, KCR and ECR were also found to be induced in the embryo epidermis. The remaining member HACD coded by PASTICCINO gene was strongly expressed in seedling epidermis and was found as the most-differentially expressed gene in the apical part of the globular embryo [8, 26]. Other epicuticular waxes and cutin genes like CER1, CER3 or CER4 were not isolated, suggesting that they are either not differentially expressed between the two tissues studied or expressed at low levels, below the detection threshold of the methods used. In conclusion, the embryo epidermis showed specific expression of fatty acid genes involved in the epicuticular waxes and cutin biosynthesis leading most probably to an increase in acyl-CoA elongation and in wax or cutin export.
For the flavonoid pathway, most of the EBGs involved in the biosynthesis of flavonoid precursors (i.e. dihydroflavonols) were found to be preferentially expressed in the epidermal cells. This is fully consistent with the simultaneous characterization of MYB111, which is involved in the regulation of these EBGs  and ENHANCER OF GLABRA 3 (EGL3, At1g63650) acting both as a regulator of epidermal cell fate (see below) and flavonoid biosynthesis . Moreover, this is also consistent with the usual epidermal localization of flavonoids, which can participate to the cuticle layer, providing some protective barrier against biotic or abiotic stresses [23, 24].
Last, we focused on DE transcription factors (TF). Among the most differentially expressed genes in the epidermal cells compared to the mesophyll cell, we identified PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (AtML1), two homologous HD-Zip expressed in epidermal cells [1, 27, 32], as well as WEREWOLF (WER) and EGL3 that interact to control epidermal cell fate in root [33, 36]. Our results are fully consistent with the epidermal localisation and function of these TF and again, validate the approach. It has to be noticed that MYB118 was identified as DE expressed with a high log ratio (> 5, Table 3). This transcription factor was functionally analysed and mRNA accumulation as well as promoter analysis showed that its expression is restricted to the endosperm . This clearly suggests a probable contamination of the epidermis by endosperm tissues, probably when the microdissection itself was performed, a little bit outside the epidermis to protect the tissue. Thus the data we provide here are not strictly epidermis specific but represent an epidermis-enriched fraction of the genes expressed during embryogenesis. We could have narrow the dissection beam to the epidermis’s cell wall but would probably have decreased the quality of the mRNA during the process. Nevertheless, our results also provide new putative epidermal-specific or preferentially-expressed TFs (Table 3). They pave the way for new functional analyses of these interesting candidates for the regulation of epidermal cell differentiation and metabolic pathways. Many other factors have been identified as DE without preliminary data about their possible localisation, some of them being highly expressed in the epidermis, suggesting major roles in the specificity or in the differentiation of the epidermis in the growing embryo. It is to be noticed that, for many of the TF, the count number was relatively high in both cell layers, suggesting a difference of expression but no cell specificity. For some TF the count number was very low, or even not detected at all, outside the epidermis, suggesting these genes are not only DE in the epidermis compared to mesophyll, but specific for the epidermis. To go further in the characterization of these DE genes, it would be also very interesting to study the promoter sequences of all these epidermal specific or preferentially expressed genes in order to characterize putative conserved epidermis specific cis-elements and then isolate new regulatory genes and build the regulatory networks involved. More broadly, identifying co-expressed genes in specific tissues or cell layers will allow a much more detailed analysis of these regulatory networks.
We have set up a robust protocol for RNA seq analysis of ultra low quantity of template RNA obtained after laser-assisted-microdissection. We have shown that the protocol is still working below 50 pg of total RNA despite lower number of detected genes. This could be useful for some applications necessarily requiring a very small amount of starting material although not fully quantitative. We have successfully applied this method to the analysis of the epidermis in developing embryos, allowing detection of many differentially expressed transcription factors in this cell layer. This analysis will help for very precise transcriptomic analysis of specific tissues that could be of great use for a better understanding of gene regulation in specific organ or cell types.
BD and SB conceived the project, and designed experiments. KS and ND performed LAM experiment. LT and YJ performed RNA-seq libraries experiment. VB, CPL, ED and MLMM performed RNA-seq analysis. BD, LL and JDF analysed the transcriptomic data from Arabidopsis embryo. BD, SB, LL, JDF, VB and MLMM wrote the manuscript. All authors read and approved the final manuscript.
The authors also acknowledge the IG-CNS partner for the libraries sequencing. The IJPB and IPS2 institutes benefit from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).
The authors declare that they have no competing interests.
Availability of data and materials
The transcriptomic data have been submitted to GEO data depository NGS2014_01_MicroD GSE98176 and NGS2012_02_MicroD GSE98313 accessible through https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=obetguoslnifniz&acc=GSE98176 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ktszcacybtmzhwf&acc=GSE98313.
Consent for publication
Ethics approval and consent to participate
This work has benefited from a French State grant (LabEx Saclay Plant Sciences-SPS, ref. ANR-10-LABX-0040-SPS), managed by the French National Research Agency under an “Investments for the Future” program (ref. ANR-11-IDEX-0003-02).
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