Tree Genetics & Genomes
DOI 10.1007/s11295-013-0691-z
ORIGINAL PAPER
Temperature-dependent differential transcriptomes
during formation of an epigenetic memory in Norway
spruce embryogenesis
Igor A. Yakovlev & YeonKyeong Lee & Björn Rotter &
Jorunn E. Olsen & Tore Skrøppa & Øystein Johnsen &
Carl Gunnar Fossdal
Received: 3 January 2013 / Revised: 4 September 2013 / Accepted: 2 December 2013
# Springer-Verlag Berlin Heidelberg 2014
Abstract Embryogenesis is the initial stage of plant life,
when the basics of body plan and the post-embryonic devel-
opment are laid down. Epigenetic memory formed in the
Norway spruce embryos permanently affect the timing of
bud burst and bud set in progenies, vitally important adaptive
traits in this long-lived forest species. The epigenetic memory
marks are established in response to the temperature condi-
tions prevailing during zygotic and somatic embryogenesis;
the epitype is fixed by the time the embryo is fully developed
and is mitotically propagated throughout the tree’s life span.
Somatic embryogenesis closely mimics the natural zygotic
embryo formation and results in epigenetically different plants
in a predictable temperature-dependent manner with respect to
altered phenology. Using Illumina-based Massive Analysis of
cDNA Ends, the transcriptome changes were monitored in
somatic embryos during morphogenesis stage under two dif-
ferent temperatures (18 vs. 30 °C). We found distinct differ-
ences in transcriptomes between the genetically identical em-
bryogenic tissues grown under the two epitype-inducing tem-
peratures suggesting temperature-dependent canalizing of
Communicated by J. Dean
Electronic supplementary material The online version of this article
(doi:10.1007/s11295-013-0691-z) contains supplementary material,
which is available to authorized users.
I. A. Yakovlev (*) : T. Skrøppa : C. G. Fossdal
Norwegian Forest and Landscape Institute, P.O. Box 115, 1431 Ås,
Norway
e-mail: yai@skogoglandskap.no
Y. Lee : J. E. Olsen : Ø. Johnsen
Department of Plant and Environmental Sciences, Norwegian
University of Life Sciences, 1432 Ås, Norway
B. Rotter
GenXPro GmbH, Frankfurter Innovationszentrum (FIZ),
Altenhöferallee 3, 060438 Frankfurt am Main, Germany
gene expression during embryo formation, putatively based
on chromatin modifications. From 448 transcripts of genes
coding for proteins involved in epigenetic machinery, we
found 35 of these to be differentially expressed at high level
under the epitype-inducing conditions. Therefore, temperature
conditions during embryogenesis significantly alter transcrip-
tional profiles including numerous orthologs of transcriptional
regulators, epigenetic-related genes, and large sets of un-
known and uncharacterized transcripts.
Keywords Conifers . Picea abies . Epigenetic memory .
Transcriptome . Next-generation (high-throughput)
sequencing . Embryogenesis
Abbreviations
MACE Massive Analysis of cDNA Ends
DE Differentially expressed
CE “Cold” embryogenesis environment (18 °C
or C in libraries definitions)
WE “Warm” embryogenesis environment (30 °C
or W in libraries definitions)
RT-PCR Real-time reverse transcription polymerase
chain reaction
Introduction
Long-lived trees in the temperate regions of the world need to
adjust their growth cycle to maximize growth during the
growth season and cease growth and form buds as well as
attain cold hardiness prior to onset of the winter (references
in(Olsen 2010). Thus, it came as a great surprise to forest
geneticists that these adaptive traits believed to have high
heritability is greatly influenced by epigenetic changes, which
could explain part of the clinal variation seen in nature for this

species (Yakovlev et al. 2012). It has been firmly established
that the timing of bud burst and bud set in Picea abies
(Norway spruce) is regulated by an epigenetic memory set
by the temperature conditions during zygotic and somatic
embryogenesis (Kvaalen and Johnsen 2008; Johnsen et al.
2005a, b, 2009; Skrøppa et al. 2007; Yakovlev et al. 2012).
The environmental conditions during embryogenesis result in
the formation of epitypes, and the epigenetic memory is
mitotically propagated resulting in significant and life-lasting
phenotypic changes in seasonal timing of bud phenology.
Similar epigenetic effects have been observed in progeny of
white spruce (Picea glauca), P. glauca×Picea engelmannii
crosses (Webber et al. 2005; Stoehr et al. 1998), Scots pine
(Pinus sylvestris) (Dormling and Johnsen 1992), Larix spp.
(Greenwood and Hutchison 1996), and longleaf pine (Pinus
palustris) (Schmidtling and Hipkins 2004). However, there is
a lack of research on this phenomenon in angiosperm trees.
To study the epigenetic aspects and to rule out classical
genetic effects, somatic embryogenesis provides an excellent
way to explore the lasting impact of environmental influence
on phenotypic performance. Significant phenotypic variability
has been observed in genetically uniform populations of
cloned plants. Some of this so-called somaclonal variation
may have a strong epigenetic basis and potentially be useful
for plant breeding and adaptation (Kaeppler et al. 2000).
Previously, zygotic and somatic propagation of Norway
spruce embryos at different temperatures were successfully
used to study the epigenetic memory effects (Kvaalen and
Johnsen 2008). By using somatic embryos, it is possible to
mimic the natural zygotic embryogenic processes during seed
formation to generate genetically identical but epigenetically
different plants. Thus, somatic embryos provide an ideal mod-
el to study epigenetic memory establishment in spruce. Re-
cently, transcriptional changes during embryogenesis had
been studied in spruce (Vestman et al. 2011) and pine (Vega-
Bartol et al. 2013). It is established that angiosperms and
gymnosperms show strong similarities in the sequences of
their DE genes during embryogenesis, so most of the differ-
ences between gymnosperm and angiosperm morphology and
development could be explained by the modest differences in
the activity or expression (Cairney and Pullman 2007).
The underlying molecular mechanisms behind the epige-
netic memory are currently under active investigation, mostly
based on model organisms with short generation times
(Thellier and Lüttge 2013). Multiple molecular mechanisms
are likely involved in epigenetic memory establishment and its
maintenance, including transmittable DNA methylation, mul-
tiple protein-based chromatin modifications as well as by non-
coding RNA-based mechanisms (Heo and Sung 2011; Satake
and Iwasa 2012; Boyko and Kovalchuk 2010; Saze 2008;
Angel et al. 2011). Currently, the investigation started in
Norway spruce, including transcriptional and microRNA
changes between epitypes (Yakovlev et al. 2010, 2011). All
Tree Genetics & Genomes
evidence strongly suggests that epigenetic memory is
established exclusively during the initial stages of embryo
development in Norway spruce and involves no change in
the primary DNA sequence (Kvaalen and Johnsen 2008).
Therefore, we hypothesize that epigenetic memory mecha-
nisms influence phenotype through altered regulation of gene
expression impacted by mitotically propagated temperature-
dependent chromatin modifications and non-coding RNA.
The epigenetic memory regulation may have evolutionary
implications and the fact that there are family differences, in
this epigenetically caused shift in the growth cycle, suggests
that there are allelic differences in the underlying epigenetic
molecular machinery. This reflects that both epigenetics and
classical genetics are integral parts of adaptive evolution.
Thus, formation of different epitypes helps a species to cope
with a rapidly changing environment and is potentially an
effective adaptive mechanism for organisms with very long
generation times. Furthermore, such a mechanism can also
counteract negative effects caused by rapid gene flow between
latitudinal and altitudinal populations (Yakovlev et al. 2012).
Next-generation sequencing (NGS) or high-throughput
technologies have revolutionized genomics studies (Lister
et al. 2009; Metzker 2010) and have been further developed
for highly accurate digital transcriptome analysis (Matsumura
et al. 2010) and epigenomics studies (Schmitz and Zhang
2011; He et al. 2011). NGS of transcriptomes is particularly
attractive for species with extremely large genomes (Kelly and
Leitch 2011), like the conifer Norway spruce. There are nu-
merous methods for sequencing-based transcriptome analysis,
like Digital Gene Expression TAG (DGE-TAG), DeepSAGE,
high-throughput SuperSAGE, and RNA-Seq (Matsumura
et al. 2010). Here, for high-resolution gene expression analy-
sis, we used a novel method called Massive Analysis of cDNA
Ends (MACE) (http://www.genxpro.info/products_and_
services/Transcriptomics/RNAseq_MACE_SuperSAGE_
digital_gene_Expression/) developed by GenXPro GmbH.
The method is a derivate of a technique first used by Torres
et al. (2008) or Eveland et al. (2008), but uses Illumina
sequencing and avoids PCR-introduced quantification bias
by applying a PCR bias proof technology “TrueQuant”. The
method provides very good cost/output ratio, higher coverage,
and better quantification of the transcriptome when compared
to regular RNA-seq.
To approach the molecular basis of the epigenetic memory
formation affected by conditions during embryogenesis, we
aimed at the identification of transcripts differentially
expressed during the particular stages of somatic embryogen-
esis, when the epigenetic memory in Norway spruce is
established, using Illumina-based MACE analysis. We studied
in vitro propagated somatic embryos from two previously
phenotypically well-characterized full-sib genotypes during
morphogenesis stage using established epitype-inducing tem-
perature conditions (18 vs. 30 °C). We found striking
Tree Genetics & Genomes
temperature-induced differences in transcriptomes for both of
the embryogenic genotypes studied. Thus, formation of the
epigenetic memory induced by warm and cold conditions
during somatic embryogenesis is accompanied by differential
expression of different sets of genes, including epigenetic
machinery-related genes.
Materials and methods
Plant material and sample collection
For transcriptome analyses, somatic embryos were generated
in vitro according to experiment 4 by Kvaalen and Johnsen
(2008). The embryogenic samples were started from two
zygotic seeds (genotypes) originating from the controlled
crosses of the same grafted Picea abies (L.) Karst. parents
(♀#2650 × ♂#2707). One of these two zygotic seeds
(genotype) was produced under native outdoor conditions
[genotype denoted as “outdoors” (O)] while the second seed
was produced in heated greenhouse conditions [genotype
denoted as “greenhouse” (G)]. The somatic callus tissue gen-
erated from these two zygotic embryos (genotypes) gave upon
in vitro embryogenic culturing, at the two (18 vs. 30 °C)
epitype-inducing conditions, rise to somatic embryos with a
high epigenetic memory response growing into somatic plants
(epitypes) with significant phenotypic differences in adaptive
bud phenology (Johnsen et al. 2005b).
For induction and proliferation (morphogenesis stage) of
the two embryonic callus, the AL medium (Kvaalen et al.
2005) was supplemented with inositol (10 %, w/v) and revised
vitamin mixture used for Douglas-fir cotyledon (Gupta and
Durzan 1985). This medium was supplemented with 2,4-
dichlorophenoxyacetic acid (2,4-D; 10 μM),
benzylaminopurine (BA; 5 μM), and sucrose (1 %, w/v),
and solidified with Phytagel (P-8169; Sigma, 0.3 % w/v).
For maturation of the embryogenic callus cultures, growth
regulators 2,4-D and BA were omitted and 20 μM abscisic
acid (ABA; A8451, Sigma) was added. The carbon source
was changed from sucrose to maltose and the medium was
supplemented with polyethylene glycol (PEG4000; Fluka,
Switzerland) (5 %, w/v). During the whole duration of the
morphogenesis and maturation stages, the embryogenic cul-
tures of the two genotypes O and G were subjected to a
temperature treatment at either 18 °C [“cold” epitype-
inducing embryogenesis environment (CE)] or 30 °C (“warm”
epitype-inducing embryogenesis environment (WE)]. Sam-
ples were collected from callus cultures at morphogenesis
stage 1 (earliest stage of embryo formation). Thus, four dif-
ferent samples were denoted OC and OW (stage 1 embryos
from O genotype generated at 18 °C cold and 30 °C warm
epitype-inducing temperatures correspondingly), and GC and
GW (stage 1 embryos from G genotype generated at 18 °C
cold and 30 °C warm epitype-inducing temperatures corre-
spondingly) were used for transcriptome sequencing (libraries
L1–L4). In each case, two biological replicates, each contain-
ing 100 mg of stage 1 embryo tissue, were sampled and
immediately frozen in liquid nitrogen before storage at
−80 °C.
RNA extraction
Total RNA extracted from stage I embryogenic tissue was
grinded in liquid nitrogen, using an Epicentre MasterPure™
RNA Purification Kit (Epicentre, Madison, WI, USA,
#MCR85102), according to the manufacturer’s instruction.
Total RNA preparations were stored at −80 °C. The integrity
and quantity of total RNA was assessed by Agilent 2100
Bioanalyzer with RNA 6000 Nano Kit (Agilent, Santa Clara,
CA, USA #5067-1511).
Construction of MACE libraries and sequencing
Massive Analysis of 3′-cDNA Ends (MACE) libraries were
constructed by GenXPro GmbH from ≈10 μg of total RNA
using the “MACE-Kit” (GenXPro GmbH, Frankfurt am
Main, Germany). The technique analyzes 3′-biotinylated
cDNA ends of fragmented cDNA, whose 5′-end is sequenced
essentially, as described by Torres et al. (2008), but here an
Illumina GAII sequencer (Illumina, Inc., San Diego, CA,
USA) was used for sequencing. In order to avoid bias during
the PCR amplification steps, the GenXPro’s PCR-bias-proof
technology “TrueQuant” was employed allowing
distinguishing PCR copies from original transcripts. The ra-
tionale behind the TrueQuant method is to barcode each
template molecule with a unique barcode prior to PCR. Copies
of the DNA fragments can then be identified by analyzing the
barcode and the first 20 base pairs of the sequence, which
must appear in a unique combination and otherwise eliminat-
ed from the dataset after sequencing. For each library, 89-bp
tags, originating from fragments of around 100–300 bp of the
3′-end of the transcript molecules, were sequenced, starting
from the 5′-end of the fragments. GXP-Tag sorter software
provided by GenXPro GmbH was used to quantify the
transcripts.
Treatment of raw data
Tag sequences were annotated using MegaBLAST against
public databases in the respective order:
NCBI_Entrez_Picea_allEST, DFCI (TIGR) Spruce Gene In-
dex (v. 5) “all_TIGR_Plant” (plant entries of the TIGR data-
base), and contigs obtained after assembly of 454 sequenced
normalized cDNA (unpublished). A MACE read was consid-
ered as “no hit” when there were more than six mismatches. In
addition, “no hit” reads were assembled also. Obtained

MACE tag generated contigs consisted of transcripts that
extended the combined sequenced length of the tags. The
obtained contigs were again annotated to the reference data-
bases to which also the SwissProt protein database of all plant
species was added. For the Gene Ontology (GO) category
annotations, we used primarily the TIGR-gene indices. If there
was no annotation with TIGR, we performed a BLASTX to
the SwissProt database (plant entries only) and used the GO
annotation. Additionally, for comparative study, from the total
amount of annotated genes we constructed the list genes
encoding proteins involved into epigenetic regulation, based
on extensive review of the literature (Chen et al. 2010; Matzke
and Mittelsten Scheid 2006).
After pairwise comparisons of transcript levels, GO enrich-
ment analysis and visualization of data were done using
STDGE2GO, a web-based toolkit, provided by GenXPro
GmbH (http://genxpro.ath.cx). Statistical analysis of
differentially expressed (DE) tags was conducted using the
probability (p) value according to the description of Audic and
Claverie (1997). The pairwise expression ratios of the tags
were calculated as follows: ratio R=log2(Library1/Library2),
after normalizing to 1 million tags (tags per million—tpm).
Tags absent in any one of the libraries (with zero quantities)
were replaced by 0.05 to allow calculation of the ratio. En-
richment was then calculated by Fisher’s exact test. All tran-
scripts below a p value threshold (threshold defined in the
tool) were used for the enrichment calculation.
Comparison of MACE expression profiles to RT-PCR
For cDNA synthesis and RT-PCR validation of the MACE
transcriptome sequencing results, we used RNA extracted
from a biological replicate of the same stage I embryogenic
tissues (genotypes) used for the MACE library constructions.
We selected 13 unique transcripts differentially expressed
between MACE library pairs for both genotypes. Selection
was based on at least two criteria: strong changes in fold
change (FC) values (either up- or down-regulated in libraries
pairwise comparisons, FC >2, p<e−10) and average abundance
of more than 100 copies per million. When possible, we
selected candidates putatively involved in epigenetic regula-
tion in other species, but also unknown or uncharacterized
candidates. Primers were designed based on the sequences of
EST homologs from the DFCI Spruce Gene Index (v.5),
NCBI databases, and from sequence contigs obtained after
the MACE sequencing.
Gene-specific primers were designed using Primer3
(Rozen and Skaletsky 2000) with default parameters and
amendments in the following criteria: melting temperature
around 70 °C and product size between 90 and 120 bp. The
list of the studied gene homologs and their primer sequences
are shown in Table S1. Total RNA was reverse transcribed
(800 ng per reaction) using the TaqMan Reverse Transcription
Tree Genetics & Genomes
kit (Applied Biosystems, Carlsbad, CA, USA, #8080234) in a
50-μl-reaction volume. RT-PCR amplification was performed
in a 25-μl-reaction volume, using 2 μl of a threefold diluted
cDNA solution as template, 12.5 μl of 1× SYBR Green master
mix, and 200 nM of each primer. RT-PCRs were performed
using the 7500 Real-time PCR System (Applied Biosystems)
with standard cycling parameters. All reactions were done in
triplicate and a no-template control was run for each primer
pair. For data analysis, the arithmetic mean of two biological
replicates was calculated. Target gene expression was normal-
ized to the average of transcripts of the spruce ACTIN
(PaACT) and TRANSLATION ELONGATION FACTOR-1-
ALPHA (PaEF1α) (Table S1 and Fig. S1).
Absolute quantification was performed using the 7500-
system SDS software. Data were further processed in MS
Excel and additionally analyzed using RT2 Profiler PCR Ar-
ray Data Analysis web portal from SABiosiences/Qiagen
(Frederick, MD, USA) (http://pcrdataanalysis.sabiosciences.
com/pcr/arrayanalysis.php) using portal defaults.
Results
Sequencing of MACE libraries
A total of more than 8.5 million reads with 4,937,676 tags was
sequenced from the four MACE libraries (OC, OW, GC, GW)
produced from the stage I embryo tissues of Norway spruce
originating from greenhouse originating genotype G and out-
door originating genotype O generated at both 18 °C cold (C)
or 30 °C warm (W) epitype-inducing conditions. The number
of reads was unevenly distributed between the four libraries.
However, common libraries’ structures allowed their mutual
comparisons (Figs. S1 and S2). The largest amount of reads
was obtained in OC library (more than 5.4 million). In each of
the other libraries, close to 1 million reads were obtained.
After grouping and annotation of the sequence tags, a total
of 53,000–120,000 unique transcripts were identified in each
of the four libraries (Table 1). Unique transcripts were se-
quence tags that were annotated to separate accessions in the
public databases allowing a maximum of six mismatches or
gave unique contigs after velvet assembly (Zerbino and
Birney 2008). In total, only around 60–70 % of all tags
showed matches to available ESTs and were used for further
analysis.
Pairwise comparison and assignment of unique transcripts
to Gene Ontology categories
For pairwise comparison and identification of unique differ-
entially expressed (DE) transcripts in the four MACE librar-
ies, we used the STDGE2GO-Tool developed by GenXPro. It
allows also defining the gene ontology (GO) categories of
Tree Genetics & Genomes

Table 1 Characteristics of
MACE libraries made from em- Tags Reads Libraries
bryogenic tissues of two sibling
genotypes (outdoor—O and OC GC OW GW
greenhouse—G) of Norway
spruce grown under warm Hits, S+AS 2,645,680 5,678,042 3,706,088 472,487 597,250 902,217
(30 °C—W) or cold (18 °C—C) No hit 2,291,996 2,855,900 1,715,947 299,977 368,742 471,234
epitype-inducing conditions
Total 4,937,676 8,533,942 5,422,035 772,464 965,992 1,373,451
Tags per million reads
Hits, S+AS 683,522.7 611,663.1 618,275.8 656,898.6
No hit 316,476.6 388,337.8 381,723.7 343,102.2
Hits, ‰ 68.4 61.2 61.8 65.7
S sense direction, AS antisense Unique transcripts 143,723 119,501 53,223 61,188 70,264
direction

unique transcripts and selecting the unique DE transcripts
within particular GO terms of the different hierarchical GO
levels based on the calculated P levels stating the probability
of a GO term to be enriched.
GOs provide a standardized set of terms to describe
genes and gene products consistently in different species
and databases (Consortium GO 2004). Almost 40,000
unique transcripts were functionally categorized and
assigned to be involved in three principal GO categories
at an identity threshold ≤1E−10: (1) molecular function
(nearly 25,000–35,000 unique transcripts), (2) cellular com-
ponents (nearly 31,000–41,000 unique transcripts), and (3)
biological processes (nearly 25,000–35,000 unique tran-
scripts). As expected with this level of deep sequencing,
no distinct differences were observed among library pairs in
terms of the number of different GO categories present
(Table S2.1).
A large number of various GO term groups were
enriched in DE transcripts between temperature treat-
ments (Fig. 1) and genotypes (Tables S2.2 and S2.3).
Moreover, 2,667 tags, common for two genotypes, were
DE at different culturing temperatures. The highest num-
ber of DE tags assigned to GO terms related to cytoplas-
mic part, response to stimulus and stress, reproduction,
embryonic development, and regulation of growth and
development. Within most of the DE GO terms, unique
transcript number was dependent on temperature condi-
tions. Amount of DE upregulated tags was generally
higher in 18 °C cold epitype-inducing condition (CE),
except the circadian rhythm, rhythmic processes, and
multiple translation initiation factors (eIF) complex
tags.
Unique transcript abundance and distribution were plotted
for pairwise comparisons of different libraries (Fig. S3).
Scatterplots showed quite disperse distribution of unique tran-
scripts with assigned and not-assigned GO terms between
transcriptomes at different temperature conditions within the
same genotypes. They also showed DE unique transcripts as
the outliers of the “main” dot cloud.
Global transcriptome differences in embryogenic tissue
under different temperatures
Pairwise comparisons of cold and warm transcriptomes for
both genotypes revealed significant transcriptomic distinc-
tions between the temperature treatments (Table 2). Setting a
minimum threshold of 2-fold DE, within the genotype G,
1,408 transcripts were up-regulated in CE and 2,421 tran-
scripts in 30 °C warm epitype-inducing condition (WE), com-
paring to each other. Similarly, there were 2,746 transcripts
upregulated in outdoor genotype O in CE and 933 in WE.
Although by raising the minimum threshold to 4- and 8-fold
change, we decreased the number of DE transcript tags; it was
still kept high. Generally, in O genotype, higher amounts of
upregulated transcripts were found in CE and in G genotype in
WE. Fifty of the most upregulated transcripts in two family-
related genotypes developed in CE and WE are listed in
Tables S3.1a, b and S3.2a, b.
Combining both genotypes G and O into one group and
comparing between the two epitype-inducing temperature
treatments allowed identifying common transcripts with sim-
ilar differential expression patterns dependent on temper-
ature (Table 2). Between CE and WE, there were 1,608
common for both genotypes DE genes with FC >2, 1,001
were upregulated in CE compared to WE, and 607 in
WE compared to CE. Generally, CE exhibited a higher
number of upregulated transcripts (e.g., 111 with FC >8)
compared to WE (24 with FC >8).
In CE for both genotypes, more abundant were tran-
scripts of late embryogenesis abundant proteins (LEAs),
as well as several methyltransferases, class IV
chitinases, transcription factors, and uncharacterized or
predicted proteins. In WE, there were higher transcript
levels of genes encoding unknown or uncharacterized
proteins, ribonucleases, cell-wall-associated hydrolases,
heat-shock proteins, glycine-rich RNA-binding proteins,
and some transcription factors. Twenty-five most upreg-
ulated differentially expressed transcripts are shown in
Tables S3.3 and S3.4.
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Fig. 1 List of the gene ontology

(GO) categories most enriched
(p<0.01) in DE unique transcripts
during embryogenesis in Norway
spruce grown under 18 °C cold
(CE) or 30 °C warm (WE)
epitype-inducing embryogenesis
conditions. The assignments were
based on significant (<e−10) plant
species hits against the Database.
The x-axis indicates the number of
unique transcripts

Analysis of transcript abundance of spruce homologs of genes

Table 2 Amounts of differentially expressed (DE) transcript (>10 rpm,
involved in epigenetic regulation
upregulated in cold/warm conditions) with different fold change differ-
ences (≥8, ≥4, and >2) (for samples description, see Table 1) A large number of genes and transcription factors described to
be involved in different pathways of epigenetic machinery
Filtering conditions Within genotypes Number of DE genes
(>100 tpm) number of DE genes related to treatment were identified, including (but not limited to) DNA
methylation/demethylation, chromatin and histones modifica-
Outdoor, Greenhouse, Cold/warm tion, and small RNAs-regulated gene silencing. Totally, in the
C/W C/G four libraries we defined 448 such unique transcripts. Among
FC ≥8 397/70 308/360 111/24 them, we found 24 transcripts of DNA cytosine-specific meth-
FC ≥4 979/265 580/921 311/117
yltransferases, three of adenosine-specific methyltransferase,
FC >2 2,746/933 1,408/2,421 1,001/607
and three transcripts involved in demethylation. Also, we
identified a large set of transcripts involved in histone
Tree Genetics & Genomes
Table 3 Differentially expressed Norway spruce transcripts of molecular
components of epigenetic regulation shared between genotypes in the
four sequenced libraries (OC, OW, GC, GW). The libraries were made
from stage I embryogenic tissues of the O and G genotypes of Norway
modifications: 28 transcripts of histone methyltransferases,
12 of polycomb group proteins, 27 of PHD domain protein,
nine of chromo domain-containing proteins, five of histone
demethylases, 12 of histone acetyltransferases, 13 of
bromodomain-containing protein, and 31 of histone
deacetylases. Additionally, we defined 71 transcripts of
spruce obtained under 18 °C cold (C) or 30 °C warm (W) epitype-
inducing embryogenesis conditions (tpm≥10, p≤e−10). The total list of
identified Norway spruce homologs of genes described to be involved in
epigenetic regulation is shown in Table S4
chromatin formation/remodeling proteins and 119 transcripts
related to small RNA biogenesis, including 11 transcripts of
Argonaut/Zwille-like proteins (Table S4). Of all analyzed
transcripts, we found 35 DE transcripts between WE and CE
(tpm≈≥10, p≤0.01) common for both family-related geno-
types (Table 3). We found that different sets of genes were

Table 3 (continued)
FC ratios for genes upregulated in 18 °C cold epitype-inducing conditions (CE) shown in red, for genes upregulated in 30 °C warm epitype-inducing
conditions (WE) in green
activated at different temperatures during early stage of em-
bryogenesis. In CE, there was higher expression of genes
coding for different chromatin remodeling factors and small
RNA pathway proteins. In WE, there was higher presence of
transcripts of histone acetyltransferases, deacetylases, and
polycomb group proteins. Different DNA and histone meth-
yltransferases were generally higher expressed in both CE and
WE, but chromo-domain-containing histone methyltransfer-
ases and histone lysine-specific demethylases showed higher
transcript abundance in CE.
Confirmation of MACE data in embryogenic tissues
by qRT-PCR
A subset of 13 unique DE transcripts (Table S1) was selected
for analysis by qRT-PCR on the basis of strong up- or down-
regulation in pairwise comparisons of MACE libraries and
transcript abundance. For comparison between MACE tran-
script counts and qRT-PCR data, we used ratios of transcript
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counts/quantities between pairs of compared libraries
(Table S5). For all of the studied genes, there were similar
trends in transcript levels for compared library pairs. However,
the amplitude of differences in qRT-PCR data was generally
lower than in the MACE direct transcript counts. For qRT-
PCR, the highest determined FC ratio of 16.03 was found for
DNA-CYTOSINE METHYLTRANSFERASE (PaDCM1). In
comparison, the FC value was 200.91 for this transcript in the
MACE direct transcript count. For the MACE data, the highest
FC differences reached several thousand such as for a putative
uncharacterized protein (PaPUP4) and an uncharacterized pro-
tein, related to heat response processes (PaRTH1).
Discussion
Early stages of embryogenesis are of great importance for the
development of future plants. We used the MACE technique
to study the transcriptomic differences in somatic embryos
Tree Genetics & Genomes
during morphogenesis stage 1 between two epitype-inducing
temperatures, providing candidate genes for further studies of
the epigenetic memory establishment in plants. In the four
studied libraries, we identified 53,000–119,000 unique tran-
scripts. Around 40 % of tags found in developing spruce
embryos have “no hit” among the available ESTs at the
available Databases. Previous studies show that the conifer
embryo markedly differs from other gymnosperm tissues in
terms of the range of genes transcribed (Cairney and Pullman
2007). These transcripts, putatively, are embryo specific and
unique for a particular stage of embryogenesis. They were not
used in further analysis but could be of great importance to
decipher the specific molecular changes during
embryogenesis.
By the latest estimate, the nuclear genome of P. abies
contains up to 28,354 well-supported genes (Nystedt et al.
2013). Thus, even taking into consideration embryo-specific
genes, allelic variants, and naturally occurring antisense tran-
scripts, our libraries cover well the transcript diversity in the
embryos. Long, 3′-UTR-based MACE sequence reads facili-
tate unambiguous gene assignment. However, as gene assign-
ment and annotation in huge genomes like spruce necessarily
must still depend on available EST collections, unambiguous
distinguishing of closely related gene family members re-
mains a challenge. Future and deeper sequencing or sequenc-
ing of a larger number of embryonic samples will provide
enhanced statistical support for the rare transcripts.
Normal embryonic growth is accompanied by a defined
pattern of transcribed genes resulting in the formation of
embryos (Stasolla et al. 2004; Vestman et al. 2011). Pairwise
comparisons of combined transcriptomes revealed a large
number of DE genes within each of the two sibling genotypes
O and G in response to different epitype-inducing tempera-
tures during somatic embryogenesis and allowed identifying
DE transcripts and transcripts with similar expression patterns.
There was nearly no overlap in content of most highly DE
genes between the two epitype-inducing temperature condi-
tions. Among the most highly DE genes, there was a large set
of genes typically involved in embryo development, such as
heat-shock proteins, chitinases, etc. (Wiweger et al. 2003;
Stasolla et al. 2004; Cairney and Pullman 2007). Particularly
high level of transcripts for heat-shock proteins was found in
WE, which act to protect embryos from unfavorable heat
stress conditions (Su and Li 2008) and involved in epigenetic
regulation of heat signaling (Kim et al. 2010). In CE, we
detected a number of LEA proteins genes and chitinases, as
well as several methyltransferases putatively involved in epi-
genetic processes. Additionally, a large set of unknown and
uncharacterized genes with no predicted homology to any
characterized plant mRNA sequence were obtained (e.g.,
Table S3). This could indicate the existence of novel genes
or non-coding transcripts giving rise to small regulatory RNAs
involved in epigenetic regulation.
We consider that the defined transcriptome changes during
somatic embryogenesis caused by the epitype-inducing tem-
perature conditions are (at least in part) involved in establish-
ing epigenetic memory marks. Transcripts showing similar
expression patterns for both genotypes and DE in response
to temperature are candidate genes involved in formation of
epigenetic memory or directly regulated by the induced mem-
ory marks. Transcriptome differences during embryogenesis
could conceivably be caused by somaclonal variation such as
somatic recombination (Wang and Wang 2012; Kaeppler et al.
2000). However, the directed and predictable change in bud
phenology and the observed similarities in gene expression
changes caused by the epitype-inducing temperature condi-
tions could not be caused by random genomic rearrangements
and is most likely regulated by epigenetic factors (Yakovlev
et al. 2012).
The obtained transcript profiling data allow identifying and
quantitatively analyzing genes encoding proteins involved in
specific cellular processes and epigenetic memory formation
in particular. The magnitude of the transcriptome differences
during WE and CE, enveloping several hundreds of genes, is
comparable with the gene expression differences observed
during important developmental shifts, like bud burst, bud
set, or induction of flowering (Rohde et al. 2007; Ruttink
et al. 2007; Huang et al. 2012). It is tempting to speculate
the existence of some kind of “multi-positional thermal
switch” canalizing the genes transcription in response to the
surrounding temperature during embryo formation. This
switching mechanism involve multiple concurrent processes
in developing embryo tissues resulting in activation or silenc-
ing of different genes or gene variants and maybe whole
pathways. Such epigenetic memory gene switching circuits
might be based on histone modification/substitution affecting
chromatin structure, currently described for plants and animals
and resembling predominant chromatin states in Arabidopsis
(Nicol-Benoît et al. 2012; Grimanelli and Roudier 2013;
Seffer et al. 2013; Feil and Fraga 2012). A possible link
b e t w ee n c h r o m a t i n s t r u ct u r e an d e x p r es s i o n o f
embryogenesis-related genes in conifers has already been
proposed (Uddenberg et al. 2011). A histone modifica-
tion mechanism might be directly affected by tempera-
ture or indirectly through some unknown temperature-
sensing mechanisms involving Polycomb Group (PcG)
proteins (Butenko and Ohad 2011) and/or by a small
RNA-based regulating mechanism initially transferred
from parents (or mother cell in case of mitotical propa-
gation) and putatively re-established/facilitated by the
surrounding temperature (Huff and Zilberman 2012;
Ahmad et al. 2010). Such a chromatin modification-
based memory might activate specific sets of transcrip-
tion factors and/or microRNA genes, which will keep the
epitype identity through subsequent cell divisions
(Thellier and Lüttge 2013).

We identified and analyzed 448 unique transcripts
encoding proteins described to be involved in epigenetic
machinery (Chen et al. 2010; Matzke and Mittelsten Scheid
2006). Most of them were DE between temperature treatments
during the somatic embryogenesis, however, either with a
very low expression levels (low-frequency tags) or without
consistent pattern in their expression. Among these, several
groups of transcripts were detected that matched to the same
EST. As we currently could not distinguish well between
paralogous genes and gene alleles, we considered these as
alleles of the same gene. In most cases, there were one or two
alleles with high frequency and others with very low number
of reads. Sometimes such low-expressed allelic variants
showed opposite expression pattern between temperature
treatments, so the role and importance of such low-
frequency alleles in genes related to epigenetic regulation
(parental effects, imprinting) should be further studied.
Thirty-five highly expressed transcripts orthologous to
epigenetic-related genes showed consistent transcript patterns
differing between CE and WE for both genotypes O and G.
Being common for both sibling genotypes (i.e., genotype
independent), it is conceivable that these 35 candidate genes
could be involved in the establishment of temperature-induced
epigenetic marks. These DE candidates include members
from all the main groups of genes known to be involved in
epigenetic regulation. DNA methylation is considered a high-
ly flexible and inheritable epigenetic means to control gene
expression (Vanyushin and Ashapkin 2011; Chen et al. 2010).
DNA-CYTOSINE METHYLTRANSFERASE (DCM2) and
VARIANT IN METHYLATION (VIM1), which were upregulat-
ed in CE and WE, respectively, encode proteins catalyzing
methylation at CG context. In addition, in CE adenine-specific
DNA METHYLASE transcripts were upregulated. Large sets of
transcripts encoding histone modification proteins were up-
regulated both in CE and in WE. HISTONE METHYLTRAN
SFERASES were upregulated in both conditions; a lysine-
specific DEMETHYLASE JMJ14-like transcript was upregu-
lated only in CE. Similarly, in CE the amount of a transcript
encoding five chromatin-remodeling proteins was consider-
ably higher than in WE. Transcripts of two PcG proteins
(CYSTEINE-RICH POLYCOMB-LIKE PROTEIN 1 and
VERNALIZATION 1) were highly upregulated in WE com-
pared to CE. PcG proteins are shown to be directly involved in
epigenetic memory regulation (Angel et al. 2011; Butenko and
Ohad 2011) through chromatin modification-based
(H3K27me3) gene silencing. Furthermore, in WE there were
higher number of transcripts of bromodomain proteins, his-
tone acetyltransferases, and histone deacetylases. Thus, it
appears that both acetylation and deacetylation of histones
are more actively ongoing in WE compared to CE.
Comparing transcript counts by MACE and RT-PCRs in
most cases confirmed differential gene expression between the
four libraries. Observed discrepancies in the expression level
Tree Genetics & Genomes
change could be explained by the primers’ non-specificity
during RT-PCR (i.e., amplifies several paralogs) or that the
endogenous controls are not as stably expressed during em-
bryogenesis as in mature tissues. Additionally, due to the
limited amount of tissues, we used RNA from a different
biological replicate than the one used for sequencing. This
being said, we consider that the MACE data provide a better
estimate of transcriptome differences during formation of the
epigenetic memory affected than RT-PCR due to the greater
depth of transcripts and allelic variants obtained by
sequencing.
The time scale for establishment of epigenetic memory
marks during embryogenesis may be weeks rather than days;
studies on zygotic seeds indicate that the epigenetic response
accumulates until the seed is fully mature (Besnard et al. 2008;
Skrøppa et al. 2007). In order to have adaptive value, it may be
laid down over the entire differentiation into a mature embryo,
thus long enough to reflect general weather trends and not
day-to-day stochastic local weather variation. We are going to
test this in future studies comparing the effect of WE and CE
on the transcriptomes during all stages of embryo develop-
ment in vitro from morphogenesis state (current study) until
the stage when we will get a fully formed embryo.
In conclusion, we revealed that different epitype-inducing
temperatures during somatic embryogenesis lead to changes
in the transcriptomes of formed epitypes. Similarities of tran-
scriptional response in sibling genotypes support the notion
that the temperature during embryogenesis somehow morphs
“the epigenetic landscape”, by long-term altering gene regu-
latory networks, which then impacts on the individual’s phe-
notype (i.e., phenology impacting on fitness) and capacity to
respond to changes in the environment such as seasonal
changes needed for survival. Genes involved in establishing
epigenetic marks as well as those whose expression is affected
by such marks are likely to be found among the DE genes
described here. This study provides a basis for further eluci-
dation of the fine-tuned gene regulation during the formation
of the epigenetic memory at the early stages of embryogene-
sis. The identification of epigenetic changes such as the his-
tone modification and/or DNA-methylation changes of spe-
cific gene locations in the genome and how this affects
shifting the plants growth rhythm phenology is a major chal-
lenge for the near future.
Acknowledgments The authors would like to thank Tone I. Melby
(Norwegian University of Life Sciences) for assistance in RNA extraction
and Anne E. Nilsen (Norwegian Forest and Landscape Institute) for
valuable help during in vitro culturing. In addition, we would like to
thank Ruth Jüngling and Nico Krezdorn (GenXPro GmbH) for
conducting the sequencing and the initial bioinformatics processing of
data. We express additional gratitude to Damien Vaisettes (Institut Na-
tional Des Sciences Appliquees, France) for valuable technical help with
primer testing and running qRT-PCRs. This work was supported by the
Norwegian Research Council (FRIBIO Grant #191455/V40) and the EU
FP7 project ProCoGen.
Tree Genetics & Genomes
Data Archiving Statement Unique transcripts from four libraries
using Illumina-based MACE analysis were deposited to the SRA (Short
Read Archive, NCBI) and got the following accession: PRJNA184229
and ID: 184229.
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