Research

The regulation of cambial activity in Chinese fir (Cunninghamia

lanceolata) involves extensive transcriptome remodeling
Zongbo Qiu1,2,3, Lichuan Wan3, Tong Chen3, Yinglang Wan1, Xinqiang He4, Shanfa Lu5, Yanwei Wang1 and
Jinxing Lin1
1
College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China; 2College of Life Sciences, Henan Normal University, Xinxiang 453007 China;
3
Key Laboratory of Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China; 4College of Life Sciences, Peking University, Beijing 100871, China;
5
Medicinal Plant Cultivation Research Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China

Summary
Author for correspondence:  Chinese fir (Cunninghamia lanceolata), a commercially important tree for the timber and
Jin Xing Lin pulp industry, is widely distributed in southern China and northern Vietnam, but its large and
Tel: +86 10 62836211
complex genome has hindered the development of genomic resources. Few efforts have
Email: linjx@ibcas.ac.cn
focused on analysis of the modulation of transcriptional networks in vascular cambium during
Received: 31 January 2013 the transition from active growth to dormancy in conifers.
Accepted: 27 March 2013
 Here, we used Illumina sequencing to analyze the global transcriptome alterations at the
different stages of vascular cambium development in Chinese fir.
New Phytologist (2013) 199: 708–719  By analyzing dynamic changes in the transcriptome of vascular cambium based on our RNA
doi: 10.1111/nph.12301 sequencing (RNA-Seq) data at the dormant, reactivating and active stages, many potentially
interesting genes were identified that encoded putative regulators of cambial activity, cell
Key words: Chinese fir (Cunninghamia
division, cell expansion and cell wall biosynthesis and modification. In particular, the genes
lanceolata), RNA-Seq, transcriptome, involved in transcriptional regulation and hormone signaling were highlighted to reveal their
vascular cambium, wood formation. biological importance in the cambium development and wood formation.
 Our results reveal the dynamics of transcriptional networks and identify potential key
components in the regulation of vascular cambium development in Chinese fir, which will
contribute to the in-depth study of cambial differentiation and wood-forming candidate
genes in conifers.

expression (DGE)) have provided an opportunity to address

Introduction
Wood represents one of the most important sources of energy on
earth and is an environmentally acceptable future alternative to
fossil fuel resources. The perennial stem growth habit of most
tree species is characterized by secondary growth that results in a
cumulative increase in girth during each growth cycle. This is
achieved by the cell division activity of the vascular cambium,
with subsequent differentiation of secondary xylem toward the
inside of the cambium and secondary phloem toward the outside
(Schrader et al., 2004; Wang et al., 2007). Recent advances in the
understanding of these processes have revealed that wood forma-
tion is under highly regulated genetic control, notably at the tran-
scriptional level (Hertzberg et al., 2001; Wang et al., 2007).
Therefore, a better understanding of the regulation of cambial
activity and wood formation is essential for improving the quality
of wood using molecular biological techniques.
The recent development of novel high-throughput sequencing
technologies (i.e. next-generation sequencing (NGS), such as
Solexa/Illumina RNA-Seq (RNA sequencing) and digital gene
wood formation-associated genes in tree species by de novo assem-
bly or mapping and quantification of transcriptomes. Transcrip-
tome sequencing is an efficient means to generate functional
genomic data for nonmodel organisms or those with genome
characteristics prohibitive to whole-genome sequencing
(Raherison et al., 2012). Production and analysis of expressed
sequence tags (ESTs) from wood-forming tissues have increased
our understanding of the gene regulation involved in wood for-
mation (Allona et al., 1998; Kirst et al., 2003; Pavy et al., 2005;
Foucart et al., 2006; Wang et al., 2010a). In particular, cloning
and identification of genes from the cambium of woody plants
are important strategies in the investigation of wood formation
(Hertzberg et al., 2001; Schrader et al., 2004; Wang et al.,
2010a), because the secondary growth in woody plants is initiated
from meristematic cambium cells that retain a perpetual cell divi-
sion ability. In recent years, genomic approaches (e.g. microarray
analyses) have been used to elucidate transcriptional networks
associated with various stages of activity–dormancy transitions in
the model plant poplar (Schrader et al., 2004; Druart et al., 2007;

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Ruttink et al., 2007; Larisch et al., 2012). Schrader et al. (2004)
and Druart et al. (2007) reported a comprehensive analysis to
investigate cambial dormancy utilizing cryosection-isolated cam-
bial cells from the woody plant Populus tremula during active
growth and dormancy using microarray experiments. However,
few transcriptome-level studies have been carried out to date on
the transition from active growth to dormancy in the vascular
cambium in coniferous species. Such studies would bridge the
physiological and anatomical changes during wood formation
with the molecular data.
Chinese fir (Cunninghamia lanceolata) is an evergreen conifer in
the family of Taxoidiaceae, which is the most commercially
important coniferous species widely distributed in southern China
and northern Vietnam. Furthermore, Chinese fir is the most
important fast-growing timber tree of the warm regions south of
the Yangtze River. Because of the limited number of EST
sequences and the total absence of genomic sequences available,
our knowledge, at the molecular level, of regulation of wood for-
mation in Chinese fir remains poorly understood. In particular,
genes expressed only during the transition between stages are likely
to control the entire process of wood formation. This prompted us
to analyze the transcriptome of isolated cambial tissues during
three sequential developmental stages in the nonmodel tree Chi-
nese fir growing under natural conditions. This approach has
allowed us to reveal the dynamic changes in the key transcriptional
and development networks during the distinct stages of the
dormancy–activity cycle. These results provide an insight into the
molecular basis underlying the physiological and anatomical
changes in dormant, reactivating, and active cambium cells. Fur-
thermore, using cryosectioning to isolate cambial meristem for
analysis has allowed a much higher cellular resolution in defining
the transcriptional and development profiles in wood formation in
gymnosperm. To the best of our knowledge, this study is the first
to characterize the transcriptome dynamics of Chinese fir using
RNA-Seq, which may serve as a gene expression profile blueprint
for cambium development and wood formation in the conifer.
Materials and Methods
Plant material and total RNA isolation
Chinese fir (Cunninghamia lanceolata (Lamb.) Hook, the wide
type) trees were grown under standard glasshouse conditions
(70% relative humidity and 25 : 18°C, day : night). Fresh leaves,
stems and roots of 2-month-old young seedlings were harvested
and stored at 80°C until use. Dry seeds were stored at 4°C.
Total RNA was extracted from seeds and roots using RNAiso-
mate for plant tissue and RNAiso plus (Takara, Dalian, Liaoning,
China) according to the manufacturer’s instructions. Total RNA
from leaves and stems was isolated using Concert Plant
RNA Reagent (Invitrogen), following the supplied protocol.
RNA quality was verified using a 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA, USA), and all four samples
had an RNA integrity number (RIN) value of > 8.0. A total of
20 lg RNA was pooled from the four tissues equally for cDNA
preparation.
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cDNA library preparation and transcriptome sequencing
Illumina sequencing using the GAII platform was performed at
the Beijing Genomics Institute (BGI)-Shenzhen, Shenzhen,
China (http://www.genomics.cn/index.php) according to the
manufacturer’s instructions (Illumina, San Diego, CA, USA).
Briefly, magnetic beads with oligo(dT) were utilized to isolate
poly(A) mRNA following collection of total RNA from Chinese
fir tissues. A fragmentation buffer was added to break the mRNA
into short fragments. Using these short fragments as templates, a
random hexamer primer was used to synthesize first-strand
cDNA. Second-strand cDNA was synthesized using buffers,
dNTPs, RNase H, and DNA polymerase I. Short fragments were
purified with a QiaQuick PCR extraction kit (Qiagen) and
resolved with an elution buffer for end repair and by addition of
poly(A). Thereafter, the short fragments were connected with
sequencing adapters. For PCR amplification, we selected suitable
fragments as templates based on the results of agarose gel electro-
phoresis. Finally, the library was sequenced using an Illumina
HiSeqTM 2000. The sequencing data were deposited in the US
National Center for Biotechnology Information (NCBI)
Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/
Traces/sra; Wheeler et al., 2008) under accession number
SRA053525.
Analysis of Illumina transcriptome sequencing results
De novo assembly was carried out using SOAPdenovo-Trans
(version 1.04; http://soap.genomics.org.cn/soapdenovo.html)
with the default parameters, except for the K-mer value (Li
et al., 2010). After assessing different K-mer sizes, 29-mer
yielded the best assembly for the desired application and was
chosen to construct the de Bruijn graph. Although this higher
value reduced the number of assembled contigs, it increased the
reliability and resulted in longer contigs. The contigs without N
were obtained by conjoining the K-mers in an unambiguous
path. The reads were then mapped back to contigs to construct
scaffolds using the paired end information. SOAPdenovo con-
nected the contigs using N to represent unknown sequences
between each pair of contigs, and thus scaffolds were made.
Paired-end reads were again used for gap-filling of scaffolds to
obtain sequences with the least Ns (unknown sequences) and
that could not be extended at either end. Such sequences were
defined as unigenes used for blast search and annotation against
an NCBI nonredundant protein (Nr) database (http://www.
ncbi.nlm.nih.gov) and Swiss-Prot protein database (http://www.
expasy.ch/sprot) using an E-value cutoff of 10 5. Functional
annotation by gene ontology terms (GO, http://www.geneontol-
ogy.org) was analyzed using the Blast2GO program (Conesa
et al., 2005). Unigene sequences were also aligned to the Cluster
of Orthologous Groups (COG) database (http://www.ncbi.nlm.
nih.gov/COG) to predict and classify possible functions. The
Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG;
http://www.genome.jp/kegg) annotation were carried out
according to the KEGG database (Ogata et al., 1999) using
BLASTx with an E-value threshold of 10 5.
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Differential gene expression library preparation
Dormant, reactivating, and active cambium samples were
obtained from the stems of Chinese fir trees (the wide type,
c. 16 yr old, 15 m tall, 12 cm diameter) growing under natural
conditions in the Forestry Station at Fuzhou, China (45.44′N,
126.36′E). Samples were collected on 25 December 2010, 20
March 2011, and 1 June 2011, to cover the major stages of the
dormancy–activity cycle. Small blocks (4 9 2 9 2 cm3) contain-
ing secondary phloem, vascular cambium, and secondary xylem
were sampled c. 1.5 m above ground from 20 independent trees
at each time point. Samples were flash-frozen in liquid nitrogen
and stored at 80°C. Frozen stem segments were trimmed to
blocks of c. 30 mm length (axial) and 3.0 mm width (tangential).
Tangential sections through the cambial region of the stem were
obtained using a cryomicrotome and frozen in liquid nitrogen, as
described by Uggla et al. (1996) with modification. Briefly,
30 lm sections were isolated by tangential cryosectioning at
24°C with a Leica CM1850 Cryostat (Leica Microsystems
Nussloch GmbH, Nussloch, Germany) equipped with a steel
knife. Transverse sections taken from both ends of the specimen
and stained with aniline blue were used to locate the position of
tangential sections. For each time point, 90 sections were used to
prepare total RNA for the construction of cDNA libraries and
sequencing using Illumina GA II. Total RNAs from dormant,
reactivating, and active cambial meristems, respectively were
isolated using Concert Plant RNA Reagent (Invitrogen) follow-
ing the supplied protocol. RNA integrity was confirmed using a
2100 Bioanalyzer (Agilent Technologies). The short-read data
sets are available at the NCBI SRA with the accession number
GSE37152.
Anatomical observations of the vascular cambium
Blocks of c. 5 mm3 including secondary phloem, vascular cam-
bium, and secondary xylem cut from the stems of Chinese fir
were collected and fixed in 2.5% glutaraldehyde in 100 mM
phosphate buffer (pH 7.2). After dehydration through an ethanol
series, the samples were embedded into Spurr’s resin. Sectioning
was performed using a Leica microtome. Sections (1 lm) were
mounted on slides, stained with 0.25% (w/v) toluidine blue O
(Sigma), and observed under a Zeiss Axioskop 2 Plus microscope
equipped with a computer-assisted digital camera. Images were
processed using Photoshop (Adobe, San Jose, CA, USA).
Analysis and mapping of Illumina short reads
The RNA-Seq bioinformatic analysis pipeline is shown in the
Supporting Information (Fig. S1). Before mapping reads to the
reference database, all reads were filtered to remove adaptors,
low-quality reads, and reads with unknown bases. The clean read
expression distribution was used to evaluate the normality of the
whole data. All clean reads were mapped to our transcriptome
reference sequences using SOAPaligner/soap2, and mismatches
of only 1 bp were considered. The number of unambiguous clean
reads for each gene was calculated and then normalized to the
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number of reads per kilobase per million clean reads (RPKM;
Mortazavi et al., 2008), uniquely aligning within each sample.
Bioinformatics for functional annotation of differential
gene expression
A rigorous algorithm to identify differentially expressed genes
was developed based on the method of Audic & Claverie (1997).
The false discovery rate (FDR) was used to determine the thresh-
old of P-value in multiple test and analysis. We used
FDR < 0.001 and the absolute value of log2(ratio)  2 as thresh-
olds to determine the significance of gene expression difference
(Benjamini & Yekutieli, 2001). Furthermore, an additional crite-
rion, which involved using only differentially expressed genes
(DEGs) with a minimum of fourfold change, was adopted for
further analysis.
Real-time quantitative reverse transcription polymerase
chain reaction (qRT-PCR) validation and expression
analysis
Total RNAs were isolated from dormant, reactivating, and active
cambium, as described earlier. First-strand cDNA synthesis was
performed using Superscript II reverse transcriptase (Invitrogen)
according to the manufacturer’s instructions, using 1 lg of total
RNA and oligo(dT) primers. qRT-PCR was performed using a
Rotor-Gene 3000 real-time PCR detection system (Qiagen)
using SYBR® qPCR Mix (Toyobo, Tokyo, Japan) according to
the manufacturer’s protocol. Gene-specific primers were designed
using the Primer Express software version 3.0 (Applied Biosys-
tems, Foster City, CA, USA; Table S1). Quantitative PCR reac-
tions were conducted in 20 ll volumes containing 2 ll diluted
cDNA, 300 nM of each primer, and 10 ll of the Thunderbird
SYBR Green PCR Master Mix with the following cycling condi-
tions: 95°C for 2 min, 40 cycles at 95°C for 15 s, 60°C for 15 s,
and 72°C for 15 s. After amplification, a thermal denaturing
cycle at 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s was car-
ried out to determine the dissociation curves and verify the speci-
ficity of the amplifications. All reactions were performed in
biological triplicates, and the results were expressed relative to the
expression levels of an internal reference gene, glyceraldehyde 3-
phosphate dehydrogenase (GAPDH, CV170251), in each sample
using the 2 DDCt method (Livak & Schmittgen, 2001).
Results
High-throughput transcriptome sequencing and read
assembly
To maximize the number of genes included in the transcriptome,
a cDNA sample was prepared from an equal mixture of total
RNA isolated from seeds, roots, stems, and leaves, and sequenced
using the Illumina high-throughput sequencing platform. After
stringent quality checks and data cleaning, we obtained
27 666 672 reads containing a total of 2490 000 480 nucleotides
(2.49 Gb). The average read size, Q20 percentage (sequencing
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error rate < 1%), and GC (guanine + cytosine) percentage were classification. Among the 25 COG categories, the cluster for
90 bp, 94.51%, and 45.04%, respectively. Based on the high- ‘general function prediction only’ (3138, 16.77%) represented
quality reads, 417 105 contigs were assembled with an average the largest group, followed by ‘transcription’ (1487, 7.95%) and
length of 144 bp. With paired-end joining and gap-filling, the ‘replication, recombination, and repair’ (1432, 7.92%). The fol-
contigs were further assembled into 84 980 scaffolds with an lowing categories, ‘nuclear structure’ (2, 0.01%), ‘extracellular
average length of 385 bp, including 6548 scaffolds larger than structures’ (4, 0.02%), and ‘cell motility’ (79, 0.42%), repre-
1000 bp. After employed local assembly with the unmapped end sented the smallest groups (Fig. 1).
to fill in the small gaps within the scaffolds, the de novo assembly Gene ontology (GO) assignments were also used to classify the
yielded 59 669 unigenes with an average length of 497 bp functions of the predicted Chinese fir genes. Based on sequence
(Table 1). The size distribution of these scaffolds and unigenes is homology, 12 365 sequences can be categorized into 44 func-
shown in Fig. S2 and Table S2. To demonstrate the quality of tional groups (Fig. S3). In each of the three main GO classifica-
sequencing data, we randomly selected six unigenes and designed tions (biological process, cellular component, and molecular
six pairs of primers for RT-PCR, products that were confirmed function), the ‘metabolic process’, ‘cell part’, and ‘catalytic activ-
by Sanger sequencing. ity’ terms, respectively, were dominant. We also noticed a high
percentage of genes from the ‘cellular process’, ‘organelle’, and
‘binding’ categories, but few from ‘biological adhesion’, ‘extracel-
Gene annotation and functional classification
lular region part’, and ‘electron carrier activity’ (Fig. S3). The GO
For validation and annotation of assembled unigenes, sequence analysis indicated that the identified genes are associated with vari-
similarity searches were conducted against the NCBI Nr database ous biological processes. Five thousand five hundred and eighty
and the Swiss-Prot protein database using the BLASTx algorithm sequences were annotated as belonging to the ‘metabolic process’
with an E-value threshold of 10 5. The results indicated that of category, which may allow for the identification of novel genes
59 669 unigenes, 34 749 (58.23%) had a significant similarity to involved in the secondary metabolism pathways in wood formation.
known proteins in the Nr database (Table 2). Owing to the lack The KEGG Pathway database records the networks of molecu-
of Chinese fir genome and EST information, 41.77% of unigenes lar interactions in the cells, and variants of them specific to partic-
could not be matched to known genes. Similarly, up to 35 782 ular organisms. Based on a comparison against the KEGG
unigenes (59.97% of the total) had no Swiss-Prot annotation database using BLASTx with an E-value cutoff of < 10 5, of the
(Table 2). 59 669 unigenes, 16 517 (27.68%) had significant matches in the
To further evaluate the completeness of our transcriptome database and were assigned to 119 KEGG pathways (Table 2).
library and the effectiveness of our annotation process, we The most represented pathways were ‘metabolism pathways’
searched the annotated sequences for genes involved in COG (4213 members), ‘biosynthesis of secondary metabolites’ (2484
classifications. Of 34 749 Nr hits, 18 713 sequences had a COG members), ‘plant–pathogen interaction’ (1283 members),
‘spliceosome’ (893 members), and ‘phenylpropanoid biosynthe-
Table 1 Summary for the Chinese fir (Cunninghamia lanceolata)
transcriptome sis’ (663 members). These annotations provide a valuable
resource for investigating the processes, functions, and pathways
Total number of reads 27 666 672 involved in wood formation.
Total nucleotide length 2490 000 480 bp
Average read length 90 bp
Total number of contigs 417 105 Cambial activity of Chinese fir at three developmental
Mean length of contigs 144 bp
stages
Total number of scaffolds 84 980
Mean length of scaffolds 385 bp The cambium shows seasonal variation in cell division, termed
Total number of unigenes 59 669
the dormant, reactivating, and active seasons. We initially per-
Mean length of unigenes 497 bp
formed anatomical observations of the cambial zone to identify
the timing of the key events of the activity–dormancy cycle,
Table 2 Annotation of unigene sequences in Chinese fir (Cunninghamia
induction, and the cessation of cambial cell division. The vascular
lanceolata)
cambium is defined as the actively dividing layer of cells that lies
Sequence Number of annotated Percentage of annotated between secondary xylem and secondary phloem cells. As shown
database unigene sequences unigene sequences in Fig. 2, it is obvious that dormant cambium cells were arranged
very compactly and were easily distinguished from terminal late-
Total unigenes 59 669 100
Nr 34 749 58.23
wood tracheids and differentiated secondary phloem cells in the
Swiss-Prot 23 887 40.03 samples collected on 25 December 2010 (Fig. 2a,b). In samples
KEGG 16 517 27.68 collected on 20 March 2011, cambial reactivation was evident,
GO 12 365 20.72 with thin-walled cells displaying some cell expansion but remain-
COG 18 713 31.36 ing at two to three cells (Fig. 2c,d), and on 1 June 2011, a
Nr, NCBI nonredundant protein database; Swiss-Prot, Swiss-Prot protein dramatic increase in the width of the cambial zone reflected a
database; KEGG, Kyoto Encyclopedia of Genes and Genomes Pathway; fully activated cambium (Fig. 2e,f). Based on the anatomical
GO, gene ontology; COG, clusters of orthologous groups. observation, we used cryosectioning to isolate cambial meristem

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Fig. 1 Clusters of orthologous groups (COG)

classifications in Chinese fir (Cunninghamia
lanceolata). These 18 713 sequences have a
COG classification within the 25 categories.

(a) (b)

(c) (d)

(e) (f)

Fig. 2 Transverse sections through dormant

(a, b), reactivating (c, d), and active (e, f)
Chinese fir (Cunninghamia lanceolata)
cambial zones. Asterisks indicate the
approximate location of the tangential
cryosections used for library generation and
Illumina sequencing. Bars, 50 lm (a, f); same
magnification in panels (a, c, e) and (b, d, f).

cells, which allowed a higher cellular resolution for defining tran-

scriptional and development profiles, and overcame the limita-
tions arising from the mixed samples in many previous
investigations. Importantly, use of this technique provides new
information about the genetic regulation involved in wood
formation and facilitates the dynamic analysis by separating
samples.
A snapshot of Chinese fir mRNA profiling in dormant,
reactivating, and active vascular cambium
To investigate the annotated transcriptome assembly served as a
reference for RNA-Seq profiling of stage-specific expression, we
conducted a small RNA-Seq experiment using tangential cryosec-
tions of dormant, reactivating, and actively growing Chinese fir

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vascular cambium and mapped the resulting reads to our refer-
ence transcriptome. Using Illumina high-throughput sequencing,
11.6–12.1 million clean reads were generated in the analyzed
samples and a mean of 5.8 million unique reads in each cambium
sample were mapped uniquely to the transcriptome of Chinese
fir, corresponding to 49.3% of the high-quality reads (Table S3).
Filtered with an FDR  0.001 and |log2(ratio)|  2, the
expression of 4415 DEGs was found to be significantly changed
between the dormant and active cambium libraries. The 883
DEGs in both the reactivating and active cambium libraries
showed quantitative differences. In addition, we compared the
dormant and reactivating cambium libraries, and 4018 variant
genes were found, of which 1548 were up-regulated and 2470
were down-regulated (Fig. 3). Larger numbers of genes were up-
or down-regulated at the active and dormant stages than at the
reactivating stage, indicating that the transcript abundance
changed dramatically at these key switches among developmental
stages of vascular cambium.
At the active and dormant stages, genes whose transcript abun-
dance exhibited highly dynamic changes (|log2(ratio)|  4,
Fig. 4) included genes encoding transcription regulators (auxin-
induced protein, PIN-like auxin efflux carrier, thaumatin-like
protein, putative nodulin like-protein, receptor-like protein
kinase, somatic embryogenesis receptor kinase), transcription fac-
tors (R2R3-MYB transcription factor, class III homeodomain
leucine zipper protein, transcription factor AP2-EREBP, zinc fin-
ger protein; |log2(ratio)|  4; Fig. 4), cell cycle-associated pro-
teins (mitotic cyclin A1-like protein, putative A-like cyclin,
minichromosome maintenance protein, plant mitotic spindle
assembly checkpoint protein mad2), cell wall-associated proteins
(beta-glucosidase, endo-beta-1,3-glucanase, celullose synthase,
xyloglucan endotransglucosylase, pectin methylesterase), and
4000
3000 2886
Number of DGEs
2470
2000
1529 1548
1000
731
152
0 division, and cell expansion during vascular cambium develop-
Active vs dormant Active vs reactivating Reactivating vs
dormant
Fig. 3 Changes in gene expression profile during the different stages of
cambium development in Chinese fir (Cunninghamia lanceolata). The
number of up- (blue bars) and down-regulated (pink bars) genes between
active and dormant, active and reactivating, and reactivating and dormant
stages are summarized. For example, ‘1529’ indicates that the expression
of 1529 genes was significantly higher at the dormant stage than at the
active stage; and ‘2886’ means that the expression of 2886 genes was
significantly lower at the dormant than at the active stage. DGE, digital
gene expression.
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stress-associated proteins (late embryogenesis abundant protein,
disease resistance associated protein, peroxidase, low temperature
induced-like protein; Fig. 4, Table S4). Therefore, the change in
expression patterns of distinct transcripts suggests the require-
ment of different development events from dormant to active
growth. For example, 12 preferentially expressed transcription
factor-related transcripts accumulated to a higher level in the
active phase than in the reactivating and dormant phases, which
indicates that the active phase may need more transcription
factors than the dormant and reactivating phases (Fig. 4, Table
S4). In addition, seven genes that are involved in cell cycle and
expansion and 15 genes that are involved in cell wall remodeling
showed the highest accumulation in the reactivating and active
stages (Fig. 4, Table S4), as expected for actively dividing cells.
Interestingly, > 85% of the DEGs were classified as either ‘no
hits’ or ‘proteins of unknown function’ (Table S5), suggesting
that these genes are only present in trees and might play impor-
tant roles in cambium development and wood formation.
Verification of the gene expression profiles by qRT-PCR
To further verify the expression profiles of genes in our Illumina
sequencing analyses, we have selected 12 DEGs by qRT-PCR
using the same samples originally used for RNA-Seq, including
genes encoding class III homeodomain leucine zipper protein,
zinc finger protein, R2R3-MYB transcription factor, ARF-L1
protein, PIN-like auxin efflux carrier, auxin-induced protein
5NG4, putative beta-1,3-glucanase, endo-beta-1,4-glucanase,
putative A-like cyclin, xyloglucan endotransglycosylase hydrolase,
pectate lyase and histone H4, respectively. These genes were
selected for their key roles in regulating cambial activity, cell divi-
sion, and cell expansion. The results presented in Fig. 5 showed
that the expression levels of 10 genes were higher in active and
reactivating cambium than in dormant cambium, including
genes encoding class III homeodomain leucine zipper protein,
ARF-L1 protein, PIN-like auxin efflux carrier, auxin-induced
protein 5NG4, putative beta-1,3-glucanase, endo-beta-1,4-glu-
canase, putative A-like cyclin, xyloglucan endotransglycosylase
hydrolase, pectate lyase and histone H4, whereas two genes
encoding zinc finger protein and R2R3-MYB transcription factor
were more highly expressed in active cambium than in reactivat-
ing and dormant cambium. These results indicated that there was
a close correlation between the expression changes (fold differ-
ence) measured by RNA-Seq and those by qRT-PCR (Fig. 5),
further indicating the reliability of our sequencing data as well as
confirming the differences in regulation of cambial activity, cell
ment.
Discussion
Illumina paired-end sequencing, assembly, and functional
annotation
The transcriptome is the complete set and quantity of transcripts
in a cell at a specific developmental stage or under a physiological
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condition. Therefore, transcriptome analysis is of importance in
interpreting the functional elements of the genome and elucidat-
ing the molecular constituents of cells and tissues. In the present
study, we sampled the pooled transcriptomes of roots, stems,
leaves, and seeds of Chinese fir using Illumina paired-end
sequencing technology to generate a large-scale EST database.
Approximately 2.5 Gb of data was generated and assembled into
59 669 unigenes. This large number of reads with paired-end
information produced much longer unigenes (mean, 497 bp)
than those in previous studies (Novaes et al., 2008; Meyer et al.,
2009; Wang et al., 2010b). This increased coverage depth of
transcriptome facilitated de novo assembly, enhanced sequencing
accuracy, and avoided possible contamination.
Of the Chinese fir unigenes, 58.23% (34 749 of 59 669) had
homologs in the Nr databases, whereas only 38.50, 16.20, and
46.21% unigenes had homologs in the Nr database in
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Fig. 4 Expression profile of 122 differentially
expressed genes involved in transcription
regulation, phytohormone signaling, cell
division and cell expansion, cell wall
remodeling, cell wall biosynthesis, defense/
stress response, transport, kinase,
cytoskeleton and others in the dormant,
reactivating, and active cambia of Chinese fir
(Cunninghamia lanceolata). The bar
represents the scale of the expression levels
for each gene (log10RPKM (number of reads
per kilobase per million clean reads)) in the
dormant, reactivating, and active cambia as
indicated by red/green rectangles. Red
indicates up-regulation of genes and green
indicates down-regulation. All genes in this
list have a P-value for differential expression
< 10 5. AC, dormant cambium; RC, reactiv-
ating cambium; DC, dormant cambium.
Complete information for each gene list can
be found in Table S4.
Epimedium sagittatum (Zeng et al., 2010), whitefly (Wang et al.,
2010b), and sweet potato (Wang et al., 2010c), respectively. The
higher percentage of hits found in our paired-end sequencing was
largely the result of the greater number of long sequences in the
unigene database of Chinese fir, in agreement with the earlier
findings in Taxus (Hao et al., 2011) and Siraitia grosvenorii (Tang
et al., 2011). Importantly, we can assign a number of these unig-
enes to a wide range of GO categories and COG classifications
(Figs 1, S3), indicating that a wide diversity of transcripts
involved in wood formation are represented in the sequence data
of this species, reflecting the complexity of differential develop-
mental stages in woody plants. Furthermore, most representative
unigenes were annotated to specific pathways, such as metabolic
pathways, biosynthesis of secondary metabolites, plant–pathogen
interactions, spliceosome, and phenylpropanoid biosynthesis
using the KEGG database (Table 2), leading us to conclude that
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Fig. 4 Continued.
most of the genes we identified are involved in cambial differenti-
ation and wood formation.
Global changes of gene expression during the transition
from active growth to dormancy
The vascular cambium forms a continuous cylinder of meriste-
matic cells in the stem, producing secondary phloem on the
outside and secondary xylem or wood on the inside. Druart et al.
(2007) demonstrated that extensive changes in the transcriptome
took place in the cambial zone of the model tree aspen during the
course of their activity–dormancy cycle. By utilizing microarray
technology, Larisch et al. (2012) uncovered significant differences
between gene profiles in cambial and ray cells from wood samples
of poplar (Populus 9 canescens) during early spring (reactivation)
and active growth. Other studies have observed similar large
shifts in gene expression by utilizing microarray technology on
large-scale transcript profiling to examine gene expression
changes in cambial tissues of forest trees (Schrader et al., 2004;
Holliday et al., 2008; Ko et al., 2011; Leonardo et al., 2012), but
little attention has been paid to gene expression of cambial cells
from the woody plant Chinese fir during dormancy, reactivation
and active growth. In the present study, using RNA-Seq to exam-
ine global gene expression profiles over a time course of cambium
development in Chinese fir, we found that the expression of 4415
DEGs was up- or down-regulated (|log2(ratio)|  2) between
the dormant and active cambium libraries, indicating consider-
able changes of gene expression in vascular cambium during the
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Research 715
–3.0 0.0 3.0
transition from active growth to dormancy. Although the genes
identified here showed similar functions to those reported previ-
ously (Hertzberg et al., 2001; Zhang et al., 2011), it is of interest
to note that > 5% of the genes have not been reported in previous
studies. Since we analyzed the transcriptional profiles following
the dynamic process of cambium development from active
growth to dormancy, we obtained more genes with dynamic
changes in transcript abundance or those that are only strongly
transcribed during the transitions.
Genes involved in cell division and cell expansion during
vascular cambium development
Cell division is one of the key processes taking place in the cam-
bial zone and the majority of cell cycle genes were up-regulated
in Chinese fir vascular cambium during the active stage (Fig. 4),
as expected for actively dividing cells. These genes include cyclin
A1 (Unigene57815 and Unigene56058) and cyclin (Uni-
gene4080), which are similar to several genes involved in cell
cycle control, like cyclins A and B on maximum gene expression
in poplar cambium zone using microarray approach by Hertzberg
et al. (2001). The qRT-PCR analysis of one cell cycle gene,
cyclins A, indicated that the abundance of this mRNA was higher
in active and reactivating cambium than in dormant cambium
(Fig. 5). The high abundance of cyclin homolog transcripts in
active and reactivating cambium also reflected a positive correla-
tion between cambium cell division and key cell cycle gene
expression. In contrast to the active stage, the decline in the
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(a) (b) (c)

Expression level (fold difference)

Fig. 5 Confirmation of the expression profiles of 12 transcripts in Chinese fir (Cunninghamia lanceolata) by quantitative reverse transcription polymerase
chain reaction (qRT-PCR). Correlation between the expression profiles of the 12 transcripts determined by RNA-Seq (red bars) and qRT-PCR (blue bars).
The 12 points (A–L) from left to right on the x-axis represent genes encoding class III homeodomain leucine zipper protein, zinc finger protein, R2R3-MYB
transcription factor, ARF-L1 protein, PIN-like auxin efflux carrier, auxin-induced protein 5NG4, putative beta-1,3-glucanase, endo-beta-1,4-glucanase,
putative A-like cyclin, xyloglucan endotransglycosylase hydrolase, pectate lyase and histone H4, respectively. The y-axes show expression levels (fold
difference) determined by qRT-PCR and RNA-Seq.

transcript abundance of the core cell cycle genes in vascular cam-
bium during the dormant stage correlates well with the cessation
of cambial cell division (Li et al., 2009). As many of those genes
involved in cell division are down-regulated in the transition
from the active to the dormant stage (Brown et al., 2005; Druart
et al., 2007; Leonardo et al., 2012), we can conclude that the key
cell cycle protein transcripts expressed preferentially at the active
stage may be essential for cambial cell division, as expected
changes during the growth cessation and reactivation cycles.
The expression of histone H4, as a marker for cell division, is
associated with DNA replication (Hertzberg et al., 2001). In our
experiment, the expression of the histone H4 gene was maximal
during the active and reactivating stages and declined at the dor-
mant stage, in support of the conclusions drawn from the cell
cycle genes that cell proliferation is at its highest in active cam-
bium. After cell division, cells in the cambial zone expand in the
axial and radial directions. Cell wall expansion plays a crucial role
in shaping the morphology of plants. During cell extension,
modifications in the structure and composition of the cross-
linked pectin xyloglucans occur. Xyloglucan endotransglycosylas-
es (XETs) are responsible for cell wall remodeling during primary
cell wall biosynthesis by cutting and rejoining the xyloglucan
chains (Wang et al., 2007). Previous studies have shown that sev-
eral genes encoding xyloglucan endotransglycosylase (XTH), pec-
tin methylesterase and expansin are involved in the cell wall
remodeling and expansion processes in coniferous species (Wang
et al., 2007; Maurice et al., 2011). In this study, we found that
the mRNA levels of several genes were more than fourfold higher
in reactivating and active vascular cambium than in dormant vas-
cular cambium, including those encoding XTH (Unigene28011
and Unigene47961), pectin methylesterase (Unigene38919,
Unigene35308 and Unigene227), pectin esterase (Uni-
gene34411, Unigene57279, Unigene52430), pectate lyase (Uni-
gene42642 and Unigene52045) and expansin (Unigene7691,
Unigene57087 and Unigene25630), suggesting that the
expression of these genes is responsible for the cell wall loosening
and cell elongation during the process. Furthermore, three
putative genes encoding beta-1,3-glucanase and seven putative
genes encoding endo-1,4-beta-glucanase, which are involved in
cell wall loosening and elongation, displayed higher expression
levels (more than fourfold differential expression) in active cam-
bium than in dormant cambium. These results suggest that these
highly differentially expressed genes are involved in a broad range
of physiological functions, especially in cell division and cell
expansion during vascular cambium development.
Genes involved in the auxin response pathway during
vascular cambium development
Auxin plays a pivotal role in various aspects of plant growth and
developmental processes, such as cambial cell division, cell wall
loosening and cell elongation, vascular tissue differentiation, and
secondary xylem development in trees, through regulation of the
expression of early auxin response factors (ARFs; Schrader et al.,
2004; Nilsson et al., 2008; Hayashi, 2012). It was demonstrated
that auxin can be transported between cells in a directional man-
ner, which is mediated by several families of auxin transporters.
AUX1/LAX symporters function as auxin influx carriers,
whereas the PIN-formed protein (PIN) family of auxin efflux
facilitators export auxin from the cell (Hayashi, 2012). By com-
paring the changes in the components of the auxin signaling and
transport machinery in dormant, reactivating, and active cambia,
we found that genes encoding the PIN-like auxin efflux carrier
(Unigene48697 and Unigene35220) and auxin-induced protein
(Unigene54524, Unigene45986 and Unigene11714) were
highly expressed (more than fourfold differential expression) in
vascular cambium at the reactivating and active stages, when
more cambial cells are actively expanding and elongating. These
results suggest that the expression changes of auxin transport
genes may contribute to the setting up of auxin concentration

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gradients once again during vascular cambium development and
wood formation.
Alongside polar auxin transport, auxin signaling also plays an
essential role in several developmental processes, from root and
shoot development to flower and fruit development in plants. It
was found that one IAA-induced transcription factor is up-regu-
lated during poplar secondary vascular tissue regeneration after
bark girdling (Zhang et al., 2011). Wang et al. (2007) reported
several members of the auxin signaling pathway in Chinese fir. In
the present investigation, we identified homologs of genes
involved in auxin response, such as ARF in active cambium,
using RNA-Seq and qRT-PCR, suggesting that the ARF genes
are involved in the regulation of cambium formation and wood
formation. Further studies on the roles of additional auxin polar
transport carriers and auxin response factors will help us to
understand the roles of auxin in cambial cell development and
wood formation.
Genes involved in transcriptional regulation during vascular
cambium development
Transcription factors that are expressed predominantly during
vascular development and secondary growth are of considerable
interest because of the economic importance of wood and wood
fibers. Ingraham et al. (1988) found that the homeodomain-con-
taining superfamily of transcription factors participated in a
wide variety of plant developmental processes. Increasing evi-
dence indicates that the leucine zipper-associated HD-Zip, knot-
ted-related KNOX, and zinc finger-associated ZF-HD
homeodomain-containing transcription factors are associated
with processes related to meristem functions in both shoot and
root apical meristems, polarity of lateral organs, and develop-
ment of primary vascular tissues in several species (Federico
et al., 2007; Ilegems et al., 2010). Previous studies indicated that
the poplar homolog to Arabidopsis, ATHB-8, was highly
expressed in the cambial meristem (Hertzberg et al., 2001).
ATHB-8 is a member of the HD-Zip III class of transcription
factors that is expressed in provascular cells of Arabidopsis, where
it has been proposed to regulate vascular development (Kim
et al., 2008). Our present results, which were obtained from the
RNA-Seq and qRT-PCR, showed that these genes encoding
class III homeodomain-leucine zipper protein and zinc finger
transcription factor were specifically expressed in active cam-
bium from Chinese fir, suggesting that these genes are required
to balance cell proliferation and the maintenance and specifica-
tion of undifferentiated cells during active growth, but are not
necessary for dormancy.
Although most attention has been focused on the homeodo-
main-containing transcription factors, transcription factors
belonging to other families are involved in regulating cambium
development and wood formation. For instance, it has been
shown that some members of the R2R3-MYB family are essential
for controlling lignin deposition and secondary wall formation in
tree species including pine and spruce by interacting with other
R2R3-MYB genes, activated by NAC (NAM/ATAF/CUC) tran-
scription factor master switches and binding to AC (ACCTACC)
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Research 717
elements (Bedon et al., 2007; Zhong et al., 2008; Zhong & Ye,
2009). The genes MYB1 and MYB8 from Pinus taeda are poten-
tially important players in conifers in the regulation of secondary
cell wall biosynthesis, including lignin deposition (Bomal et al.,
2008). Moreover, it has recently been shown that transcriptional
regulators, including genes encoding zinc finger and AP2-EREBP
transcription factors, were more highly expressed during cell wall
thickening in cotton fiber development than during the earlier
elongation stage (Al-Ghazi et al., 2009). A regulatory role for
AP2-EREBPs in poplar secondary cell wall metabolism has also
been suggested (van Raemdonck et al., 2005). Our RNA-Seq and
qRT-PCR data indicated that these genes encoding R2R3-MYB
and AP2-EREBP transcription factors were up-regulated in active
cambium from Chinese fir. Given that these transcription factors
may regulate, directly or indirectly, secondary wall biosynthesis
and vascular development, characterization of the DEGs encod-
ing transcription factors might shed light on the regulation of
wood formation in conifer.
Conclusions
Vascular cambium is the lateral meristem producing xylem cells
inwards and phloem cells outwards in plant stems. The genome-
wide transcriptome and RNA-Seq analysis presented in this study
has expanded our knowledge of this process by identifying dra-
matically expressed genes involved in crucial biological processes
such as cell division, cell expansion, secondary cell wall biosyn-
thesis and hormone pathways. The approach of combining tan-
gential cryosectioning and transcriptome analysis is a valuable
tool for the investigation of tissue-specific expression in Chinese
fir. Importantly, the high-resolution expression patterns pre-
sented here highlight our understanding of the molecular mecha-
nisms involved in vascular cambium development and wood
formation in the conifers.
Acknowledgements
This work is supported by the Major Science Foundation of
Ministry of Education of China (no. 313008), the National Basic
Research Program of China (973 Program 2009CB118500), the
Ministry of Agriculture of China (2011ZX08002-003 and
2009ZX08009-095B) and the China Postdoctoral Science Foun-
dation (2012M520457). The authors declare that there is no
conflict of interest.
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Table S5 The level of gene expression for each gene in Chinese

Supporting Information fir
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