Abstract
Background
Histone deacetylases (HDACs) are key enzymes catalyzing the removal of
acetyl groups from histones. HDACs act in concert with histone
acetyltransferases (HATs) to regulate histone acetylation status, which
modifies chromatin structure, affecting gene transcription and thus
regulating multiple biological processes such as plant growth and
development. Over a decade, certain HDACs in herbaceous plants have
been deeply studied. However, functions of HDACs in woody plants are
not well understood.
Results
Histone deacetylase specific inhibitor trichostatin A (TSA) was used to
investigate the role of HDACs in organogenesis of roots and root
development in Populus trochocarpa. The adventitious roots were
regenerated and grown on medium supplemented with 0, 1, and 2.5 μM TSA.
TSA treatment delayed root regeneration and inhibited primary root
growth. To examine the genes modified by TSA in the regenerated roots,
tag-based digital gene expression (DGE) analysis was performed using
Illumina HiSeqTM 2000. Approximately 4.5 million total clean tags were
mapped per library. The distinct clean tags for the three libraries
corresponding to 0, 1 and 2.5 μM TSA treatment were 166167, 143103 and
153507, from which 38.45 %, 31.84 % and 38.88 % were mapped
unambiguously to the unigene database, respectively. Most of the tags
were expressed at similar levels, showing a < 5-fold difference after
1 μM and 2.5 μM TSA treatments and the maximum fold-change of the tag
copy number was around 20. The expression levels of many genes in roots
were significantly altered by TSA. A total of 36 genes were
up-regulated and 1368 genes were down-regulated after 1 μM TSA
treatment, while 166 genes were up-regulated and 397 genes were
down-regulated after 2.5 μM TSA treatment. Gene ontology (GO) and
pathway analyses indicated that the differentially expressed genes were
related to many kinds of molecular functions and biological processes.
The genes encoding key enzymes catalyzing gibberellin biosynthesis were
significantly down-regulated in the roots exposed to 2.5 μM TSA and
their expression changes were validated by using real-time PCR.
Conclusions
HDACs were required for de novo organogenesis and normal growth of
populus roots. DGE data provides the gene profiles in roots probably
regulated by histone acetylation during root growth and development,
which will lead to a better understanding of the mechanism controlling
root development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2407-x)
contains supplementary material, which is available to authorized
users.
Keywords: Populus trichocarpa, Histone deacetylase, Trichostatin A,
Digital gene expression
Background
Histone acetylation, as a major and important post-translational
modification of core histones, was started to be investigated early in
1964 [[33]1]. Histone acetylation modifies chromatin structure,
affecting gene transcription and thus regulating multiple cellular
processes. Histone acetylation and deacetylation were regulated by
histone acetyltransferases (HATs) and histone deacetylases (HDACs),
respectively. HATs add acetyl groups to lysines on core histones, while
HDACs remove the acetyls from histones. Histone acetylation catalyzed
by HATs leads to the expanded structure of chromatin, while
hypoacetylation of histones mediated by HDACs is generally associated
with the condensed structure of chromatin and repression/silencing of
genes [[34]2]. HDACs are widely distributed in eukaryotes, including
animals, plants and fungi. To date, HDACs in human and animals have
been more widely and deeply investigated than plants. In 1988, HDAC
enzyme activity was first detected in pea [[35]3]. However, only in
recent years, HDACs in plants have attracted more attention and certain
HDAC genes in Arabidopsis and crops have been deeply studied [[36]4].
The available data from these plants showed that HDACs played a key
role in plant growth, development and stress responses [[37]4]. Based
on sequence homology to yeast HDACs, HDACs in plants were divided into
three major groups, namely reduced potassium dependency 3/histone
deacetylase 1 (RPD3/HDA1), histone deacetylase 2 (HD2) and silent
information regulator 2 (SIR2). The enzyme activity of RPD3/HDA1- and
HD2-type histone deacetylases could be inhibited by HDAC specific
inhibitors trichostatin A (TSA) and butyrate (NaB) [[38]2]. The genome
of Black cottonwood (Populus trichocarpa Torr. & Gray) was sequenced in
2006 [[39]5] and eleven HDAC genes were identified in the Populus
genus. However, functions of these HDACs are remaining to be
characterized.
In plants, root system development such as root hair development,
lateral root formation and primary root growth were epigenetically
regulated by HDACs. Early in 2000, Murphy et al. found that TSA and
helminthosporium carbonum (HC) toxin were able to halt mitosis in
cultured root meristems of Pisum sativum [[40]6]. Recently, HDACs have
been reported to be involved in root hair development. For example, in
Arabidopsis, TSA treatment altered the cellular pattern of root
epidermis and induced hair cell development at non-hair positions
[[41]7]. Moreover, HDA18 was further identified to be a key regulator
of root development. Ethylene insensitive 3 (EIN3) and its closest
homolog ein3-like1 (EIL1) are two transcription factors that integrate
ethylene signaling and jasmonic acid (JA) signaling in root
development. Arabidopsis RPD3-type histone deacetylase HDA6 was able to
repress EIN3/EIL1-dependent transcription and inhibit JA signaling
[[42]8]. When the Arabidopsis seedlings were subjected to TSA, root
hair formation was drastically induced. HDACs also play an important
role in lateral root (LR) development. LR formation is a progress
regulated by auxin and auxin response factors (ARFs), such as ARF7 and
ARF19. In Arabidopsis, arf7/arf19 double mutants had few LRs [[43]9,
[44]10]. IAA14 is an indole-3-acetic acid (IAA) regulatory protein and
functions as a repressor of ARF proteins in Arabidopsis. Its
gain-of-function mutation in domain II, slr-1, improved its stability
and resulted in constitutive inactivation of ARF functions [[45]11]. As
a result, auxin-induced pericycle cell divisions for LR initiation were
blocked. However, the LR formation in slr-1 mutant plants was promoted
by TSA treatment [[46]11]. Thus HDACs appeared to be required for the
LR phenotype in slr-1 mutants and play a negative role in the
activation of ARF7/19 functions in LR initiation. In addition, HDACs
acted as an important regulator in primary root growth. The
distribution of auxin in primary root tips was mediated by auxin
transporters such as pin-formed (PIN) family and ATP-binding cassette
(ABC) superfamily [[47]12]. In the work of Nguyen et al., the primary
root elongation was significantly inhibited by HDAC inhibitors TSA and
NaB [[48]13]. In response to HDAC inhibitors, the accumulation of
Arabidopsis PIN1 protein in root tips was abolished through the 26S
proteasome-mediated degradation [[49]13]. In rice, overexpression of
OsHDAC1 gene in transgenic seedlings enhanced root growth [[50]14] and
a NAM-ATAF-CUC (NAC) transcription factor OsNAC6, which mediates the
alteration of root development, was identified to be the target of
OsHDAC1 [[51]15]. These findings indicated that HDACs act as key
regulators in root system development.
The Black cottonwood (Populus trichocarpa Torr. & Gray) is of
considerable commercial and ecological importance and accepted as a
model system for biological study of trees [[52]16]. In the Populus
genus, an increasing number of genes involved in development and stress
responses have been identified at genome level. For example, genes
encoding heat shock proteins (Hsps) and heat shock factors (Hsfs) in
response to drought stress have recently been identified and
comprehensively characterized [[53]17–[54]19]. Meanwhile, using
high-throughput RNA-Seq method, long intergenic non-coding RNAs
(lncRNAs) [[55]20], drought responsive microRNAs [[56]21] and
alternative splicing of xylem-expressed genes [[57]22] were
genome-widely identified. Thus the high-throughput RNA sequence
analysis is an effective method to identify genes involved in
development and stress responses in the Populus.
For woody plants, de novo organogenesis under tissue culture conditions
is an effective method to reproduce seedlings. However, for many woody
plants, shoot or root regeneration was still difficult, even after
trying many methods such as changing hormones, medium components, or
culture conditions. To date, the role of epigenetic regulation on
organogenesis was less known. Investigation of HDAC functions in root
organogenesis and development and identification of genes regulated by
HDACs at genome-wide level will provide valuable information for
understanding root development mechanism. In this study, histone
deacetylase specific inhibitor TSA was used to investigate the role of
HDACs in populus root regeneration and development. Our results showed
that TSA treatment decreased HDAC activity in roots, delayed root
regeneration and inhibited primary root growth. A digital gene
expression (DGE) analysis was performed to examine the differentially
expressed genes in roots when subjected to different concentrations of
TSA. Our findings suggested that root organogenesis and development
were epigenetically regulated in Populus trichocarpa.
Results
TSA modified root regeneration and root system development
The populus shoots were transferred onto the rooting medium (woody
plant medium, WPM) supplemented with 0, 1 and 2.5 μM TSA to examine the
role of TSA on root regeneration, growth and development. At each
concentration, at least 45 shoots were cultured on the medium for root
regeneration and the regenerated roots showed the same morphological
traits. After shoots being transferred onto the rooting medium without
TSA supplemented for 6 d, roots were regenerated from the bottom of
shoots and reached around 1 cm in length, while in presence of TSA, the
regeneration of roots was delayed. On the medium containing 1 μM TSA,
the length of the regenerated roots was about 0.5 cm, while no root was
regenerated on the medium containing 2.5 μM TSA (Fig. [58]1). The
growth of the regenerated roots was inhibited by TSA after shoots being
transferred onto the rooting medium for 2 weeks (Fig. [59]2a). HDAC
activities in the regenerated roots were 1.9-fold and 2.6-fold
decreased after 1 μM and 2.5 μM TSA treatment, respectively
(Fig. [60]2b). The length (Fig. [61]2c) and number (Fig. [62]2d) of the
regenerated roots were significantly reduced by TSA in a dose-dependent
manner. In addition, the regenerated roots growing on the medium
containing 2.5 μM TSA were much thicker than control roots. To know the
reason why the roots were so thick, analysis of semithin sections was
performed. The morphological analysis showed that the number of cells
in cortex was increased, while the size of the cells appeared not to be
significantly altered (Fig. [63]3). These findings suggested that HDACs
were required for root organogenesis, growth and development in
populus.
Fig. 1.
Fig. 1
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Inhibition of root regeneration by TSA. Roots were regenerated from the
bottom of shoots which were cultured on medium supplemented with 0 μM
(a), 1 μM (b) and 2.5 μM (c) TSA in Populus trichocarpa
Fig. 2.
Fig. 2
[65]Open in a new tab
Inhibition of root growth by TSA. The growth of regenerated roots was
inhibited when subjected to indicated concentrations of TSA for 2 weeks
(a). TSA inhibited HDAC activities in the regenerated roots (b). The
length of regenerated roots (c) and root number (d) were decreased by
TSA in a dose-dependent manner
Fig. 3.
Fig. 3
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Morphology of roots after growth on medium supplemented with 0, 1 and
2.5 μM TSA, respectively. a-c, cross sections of root tips; d-f, cross
sections in the middle region of the roots. a and d, roots after growth
on medium without TSA supplemented for two weeks; b and e, roots after
growth on medium containing 1 μM TSA; c and f, roots after growth on
medium containing 2.5 μM TSA
Digital gene expression (DGE) libraries and tag mapping
In order to know the possible mechanism by which root growth and
development were regulated by TSA, a DGEs analysis was performed. The
DGE libraries from the roots grown on the WPM medium supplemented with
0, 1, and 2.5 μM TSA were named libraries T0, T1 and T2.5, respectively
(Table [67]1). A total of 4816584, 4906668 and 4805265 raw tags were
sequenced in T0, T1 and T2.5 libraries, respectively. After filtering
out the adaptors, low quality tags containing unknown nucleotides “N”
and tags with copy number < 2, the remaining “clean” tags for the three
libraries were 4453843 (92.4 %), 4620879 (94.2 %) and 4504396 (93.7 %),
respectively. The distinct tags for the three libraries corresponding
to 0, 1 and 2.5 μM TSA treatment were 372060, 322043 and 338137, from
which the distinct clean tags were 166167 (44.7 %), 143103 (44.4 %) and
153507 (45.3 %), respectively. The distribution of the total and
distinct clean tag copy numbers showed highly similar tendencies in the
three libraries (Fig. [68]4). The percentage of distinct clean tags
declined with the increase of tag copy number (Fig. [69]4). For each
library, approximately 71 % of the transcripts were expressed at low
levels (<10 copies), less than 24 % of the distinct clean tags had copy
numbers between 11 and 100, while only ~5 % were expressed at high
levels (>100 copies). Therefore, most of the genes in roots were
expressed at low levels and only a small number of the genes were
expressed at high levels.
Table 1.
Statistics of categorization and abundance of DGE tags
Summary T0 T1 T2.5
Raw Data Total 4816584 4906668 4805265
Distinct Tag 372060 322043 338137
Clean Tag Total number 4453843 4620879 4504396
Distinct Tag number 166167 143103 153507
All Tag Mapping to Gene Total number 1211139 889173 1180631
Total % of clean tag 27.19 % 19.24 % 26.21 %
Distinct Tag number 64057 45678 59848
Distinct Tag % of clean tag 38.55 % 31.92 % 38.99 %
Unambiguous Tag Mapping to Gene Total number 1207720 885861 1176439
Total % of clean tag 27.12 % 19.17 % 26.12 %
Distinct Tag number 63895 45557 59683
Distinct Tag % of clean tag 38.45 % 31.84 % 38.88 %
All Tag-mapped Genes number 20423 17990 19966
% of ref genes 45.35 % 39.95 % 44.34 %
Unambiguous Tag-mapped Genes number 20376 17947 19907
% of ref genes 45.25 % 39.85 % 44.21 %
Mapping to Genome Total number 2909567 3341569 3038306
Total % of clean tag 65.33 % 72.31 % 67.45 %
Distinct Tag number 83956 78922 78929
Distinct Tag % of clean tag 50.53 % 55.15 % 51.42 %
Unknown Tag Total number 333137 390137 285459
Total % of clean tag 7.48 % 8.44 % 6.34 %
Distinct Tag number 18154 18503 14730
Distinct Tag % of clean tag 10.93 % 12.93 % 9.60 %
[70]Open in a new tab
Fig. 4.
Fig. 4
[71]Open in a new tab
Distribution of clean tags in three libraries. Black color represents
the distribution of total clean tags and blue color represents the
distribution of distinct clean tags in T0, T1 and T2.5 libraries,
corresponding to 0 μM (T0), 1 μM (T1), and 2.5 μM TSA treatment (T2.5),
respectively
To reveal the molecular events behind DGE profiles, clean tags of the
three DGE libraries were mapped to the Populus trichocarpa genome,
allowing only a 1-bp mismatch (Table [72]1). For T0, T1 and T2.5 DGE
libraries, 50.53 %, 55.15 % and 51.42 % of the distinct clean tags were
mapped to the populus genome database, 38.45 %, 31.84 % and 38.88 % of
the distinct clean tags were mapped unambiguously to the unigene
database, and 10.93 %, 12.93 % and 9.6 % of the distinct clean tags did
not map to the unigene virtual tag database, respectively.
Differentially expressed genes
To identify the differentially expressed genes (DEGs) in T0, T1 and
T2.5 libraries, a rigorous algorithm was developed. The tag frequencies
that appeared in libraries were used for estimating gene expression
levels. The distribution of fold-changes of tag copy number showed that
most of the tags were expressed at similar levels, showing a < 5-fold
difference after 1 μM and 2.5 μM TSA treatments and the maximum
fold-change of the tag copy number was around 20 (Fig. [73]5). After
1 μM TSA treatment, the expression levels of 99.67 % tags were < 5-fold
changed, while only 0.08 % tags were up-regulated by at least five
folds and 0.25 % tags were down-regulated by at least five folds
(Fig. [74]5a). After 2.5 μM TSA treatment, the expression levels of 99.
76 % tags were < 5-fold changed, while only 0.1 % tags were
up-regulated by at least five folds and 0.14 % tags were down-regulated
by at least five folds (Fig. [75]5b). In our study, a threshold of
false discovery rate (FDR) ≤ 0.001 and 2- fold change in expression
level were used to judge the significance of gene expression difference
(Fig. [76]6a and b). The red dots represented the abundance of
transcripts higher than two folds and green dots represented
transcripts lower than two folds in T1 and T2.5 libraries in comparison
with T0 library. The blue dots represented the abundance of transcripts
less than two folds. A total of 1404 genes and 563 genes were detected
to be differentially expressed in T1 and T2.5 libraries in comparison
with T0 data set, respectively (Fig. [77]6c). Of the differentially
expressed genes, 313 genes were present in both T1 and T2.5 libraries
(Fig. [78]6d). After TSA treatment, most of the genes in roots were
down-regulated. Among all differentially expressed genes, 36 were
up-regulated and 1368 were down-regulated after 1 μM TSA treatment,
while 166 were up-regulated and 397 were down-regulated after 2.5 μM
TSA treatment (Fig. [79]6c). Most of the differentially expressed genes
had functional annotations, while some of the genes were not well
characterized and of unknown functions.
Fig. 5.
Fig. 5
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Differentially expressed tags in two libraries. The tags were expressed
at similar levels in T1 (a) and T2.5 (b) libraries. The “x” axis
represents fold-change of differentially expressed distinct tags in two
libraries. The “y” axis represents the number of distinct tags. The
region in red color indicates distinct tags with a <5-fold fold-change
in T1 and T2.5 libraries. The regions in green and blue indicate the
distinct tags up- and down-regulated for more than 5-folds in T1 and
T2.5 libraries, respectively
Fig. 6.
Fig. 6
[81]Open in a new tab
Differential expression analysis of unigenes by DGE. The expression
level for each unigene in roots after growth on medium supplemented
with TSA is shown in the volcano plots (a) and (b). The “x” axis
represents the log10 of transcripts per million of the control (0 μM
TSA treatment) and the “y” axis represents the log10 of transcripts per
million of samples treated by 1 μM TSA (a) and 2.5 μM TSA (b). The red
dots indicate the abundance of transcripts higher than two folds and
green dots indicate transcripts lower than two folds in T1 and T2.5
libraries in comparison with T0 library. FDR ≤ 0.001 and the absolute
value of log2Ratio ≥ 1 were used as the threshold to judge the
significance of gene expression difference. The number of up-regulated
and down-regulated unigenes in roots after TSA treatments (c) and the
common differentially expressed genes after 1 μM TSA and 2.5 μM TSA
treatments (d) were summarized
Gene ontology functional analysis of DEGs
To better understand the biological functions of the differentially
expressed genes (DEGs), gene ontology (GO) enrichment analysis was
performed. GO includes three ontologies, cellular component, molecular
function and biological process, describing properties of genes and
their products in any organism. Each of the ontologies is composed of
GO-terms. GO enrichment analysis applies a hypergeometric test to map
all DEGs to terms in the GO database, searching for significantly
enriched GO terms which are defined by a threshold of corrected-P
value ≤ 0.05. A total of 1182 DEGs in T1 library and 448 DEGs in T2.5
in comparison with T0 library were classified into the three main
ontologies, respectively. For T1 library in comparison with T0, 616
genes were mapped to the significantly enriched GO terms (P ≤ 0.05) and
could be categorized into 22 functional groups (Fig. [82]7). For T2.5
library in comparison with T0, 208 genes were mapped to the
significantly enriched GO terms (P ≤ 0.05) and could be categorized
into 9 functional groups (Fig. [83]7). In the presence of 1 μM TSA,
most of the differentially expressed genes were associated with
cellular component or involved in biological processes. However, under
2.5 μM TSA treatment, most of the differentially expressed genes were
specially categorized into the ontology of molecular function,
including GO terms “active transporter activity”, “iron ion binding”,
“antioxidant activity”, “oxidoreductase activity”, “oxidoreductase
activity acting on paired donors” and “copper-transporting ATPase
activity”. For T1 and T2.5 libraries, the common GO terms include
“active transporter activity”, “response to stimulus” and “response to
stress”.
Fig. 7.
Fig. 7
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Histogram showing Gene Ontology functional enrichment analysis of DEGs.
Transcripts were classified into three different categories, cellular
component, molecular function and biological process
Pathway analysis for DEGs
To further characterize the function of the differentially expressed
genes in T1 and T2.5 libraries, we mapped the genes to terms in kyoto
encyclopedia of genes and genomes (KEGG) database to identify
significantly enriched metabolic or signal transduction pathways
(Fig. [85]8). Among the mapped pathways, four pathways were
significantly enriched (Q value ≤ 0.05) in roots grown on the medium
containing 1 μM TSA. Most of the genes in the four enriched pathways,
including nitrogen metabolism (25 members) (Additional file [86]1),
ribosome (44 members) (Additional file [87]2), fructose and mannose
metabolism (12 out of 14 members) (Additional file [88]3), and
glutathione metabolism (18 out of 19 members) (Additional file [89]4),
were down-regulated. Nitrogen metabolism is important for plant growth
and development. Proteins are synthesized by ribosomes. Thus the
down-regulation of the genes involved in nitrogen metabolism and
ribosomal protein synthesis may impair normal growth and development of
roots. For the roots grown on the medium supplemented with 2.5 μM TSA,
ten pathways were significantly enriched (Q value ≤ 0.05) and the top
two abundant pathways were metabolic (101 members) and biosynthesis of
secondary metabolites (63 members). Most of the genes in the ten
pathways were down-regulated. The genes in the nitrogen metabolism
pathway (10 members), photosynthesis-antenna proteins (4 members),
anthocyanin biosynthesis (3 members), diterpenoid biosynthesis (6 out
of 7 members), phenylpropanoid biosynthesis pathway (17 out of 26), and
flavones and flavonol biosynthesis (5 out of 7 members) were
down-regulated. Some genes in phenylalanine metabolism (9 out of 17
members) and glutathione metabolism (6 out of 9) were up-regulated. The
genes in the metabolic pathway, diterpenoid/GA, and nitrogen metabolic
may play a significant role in plant growth and development. The
flavones and flavonol biosynthesis, anthocyanin biosynthesis, and
phenypropanoid biosynthesis were important for plant stress tolerance
[[90]23, [91]24].
Fig. 8.
Fig. 8
[92]Open in a new tab
Histogram illustrating pathway enrichment analyses
Effect of TSA on the GA biosynthesis pathway
Gibberellins (GAs), as a large family of tetracyclic diterpenoid,
regulate plant growth and development such as seed germination, stem
elongation, flowering, fruit development, and circadian and light
regulation [[93]25]. The growth and development of root system were
regulated by GA as well. In our study, diterpenoid/GA biosynthesis
pathway was significantly enriched after roots were grown on the medium
supplemented with 2.5 μM TSA for two weeks. A total of four genes
participating in GA biosynthesis, including KO, KAO, GA20ox and GA3ox,
were detected in T2.5 library and all of them were down-regulated
(Fig. [94]9). KO (Potri.002G129700) encoded enzyme ent-kaurene oxidase
and KAO (Potri.014G179100) encoded ent-kaurenoic acid hydroxylase.
Genes Potri.001G176000 and Potri.001G175800 were homologues of
Arabidopsis GA20ox and GA3ox which encoded GA 20-oxidase and GA
3-oxidase. The expression levels of these genes were confirmed by
real-time PCR (Fig. [95]10). The real-time PCR results were consistent
with the data obtained by DGE analysis. These findings revealed that
inhibition of HDAC enzyme activity by TSA resulted in the
down-regulation of genes in GA biosynthesis pathway.
Fig. 9.
Fig. 9
[96]Open in a new tab
Gibberellin biosynthesis pathway in populus roots in response to 2.5 μM
TSA treatment. The expression of four genes encoding enzymes catalyzing
GA biosynthesis (marked with green) was down-regulated on exposure to
2.5 μM TSA for 2 weeks
Fig. 10.
Fig. 10
[97]Open in a new tab
Real-time PCR validations of GA biosynthesis genes. The expression
levels of genes participating in GA biosynthesis, Potri.002G129700
(KO), Potri.014G179100 (KAO), Potri.001G176000 and Potri.001G175800
(GA20ox and GA3ox), were analyzed by real-time PCR. The PCR results
were consistent with the data in DGE analysis
Confirmation of differentially expressed genes by quantitative real-time PCR
To verify the DGE data, nine genes were selected for quantitative
real-time PCR analysis (Fig. [98]11). The representative genes selected
for the analysis included two genes related to root development,
Potri.006G138500 (auxin response factor 7, ARF7)) and Potri.003G133900
(tiny root hair 1, TRH1)), and two populus HDAC genes, Potri.009G170700
(HDA902) and Potri.001G460000 (HDA904). The expression changes of the
nine genes in DGE analysis were consistent with the results obtained by
real-time PCR. It is interesting that populus HDA902 and HDA904 genes
were able to be regulated by TSA.
Fig. 11.
Fig. 11
[99]Open in a new tab
Real-time PCR validations of tag-mapped genes. Relative level, 2^−△△CT;
TPM, transcript per million mapped reads
Discussion
TSA modified the de novo organogenesis of roots
Tissue culture is an effective and fast method to obtain seedlings for
many woody plants. In woody plants such as populus and birch, detached
shoots are generally cultured on medium containing appropriate plant
hormones and nutrient to regenerate roots. In order to improve the
regeneration rate of roots, the hormones such as auxin and cytokinin,
components of medium, or culture conditions such as temperature,
humanity and lightening were usually adjusted. Nevertheless, for many
woody plants, regeneration of adventitious roots from detached tissues
or organs is still difficult. To date, it has not been fully understood
that root regeneration may be epigenetically regulated. In our work, we
examined the role of histone deacetylases in de novo root organogenesis
using HDAC specific inhibitor TSA. TSA treatment inhibited populus root
organogenesis in a dose-dependent manner (Fig. [100]1), suggesting that
HDACs were required for populus root regeneration from detached shoots
under tissue culture conditions. This finding might shed light on the
organogenesis of those woody plants which are difficult to obtain
regenerated seedlings by tissue culture method.
Differentially expressed genes (DEGs)
Histone deacetylases (HDACs) catalyze histone deacetylation and are
usually associated with the repression of gene transcription. TSA, as a
HDAC specific inhibitor, is expected to have a role to increase histone
acetylation levels, thus leading to up-regulation of genes. However,
interestingly, most of the differentially expressed genes in roots were
down-regulated on exposure to TSA, especially when roots were exposed
to 1 μM TSA. It appeared that histone acetylation may have a positive
or negative role in gene activation. In yeast, histone deacetylation
not only repress genes but can be required for gene activation
[[101]26]. Using spotted oligo-gene microarray, Tian et al.
investigated the expression of genes in AtHDA19 T-DNA insertion mutant
(athd1-t1) [[102]27]. Over 7 % of the transcriptome was modified. In
leaves and flowers of the athd1-t1 mutant, relatively equal numbers of
genes were up- or downregulated. These findings indicate that histone
acetylation may activate or repress the transcription of genes, which
is consistent with our result. In our study, the populus roots were
regenerated on WPM medium containing different concentrations of TSA
(0, 1 and 2.5 μM). Organogenesis and development of the roots were
inhibited by TSA in a dose-dependent manner, which were consistent with
the finding in Arabidopsis [[103]28]. In order to know whether the
genes were modified by TSA in a dose-dependent manner, the expression
levels of DEGs in the libraries were compared. After comparison, only
three genes exhibited dose-dependent manner when roots were subjected
to different concentrations of TSA, suggesting that the expression of
genes in response to TSA was not in a dose-dependent manner. In T1
library, most of the DEGs (1091) were not found in T2.5 library, while
almost half of the DEGs in T2.5 library were not included in T1
library, indicating that different sets of genes in roots were modified
at each concentration (1 and 2.5 μM TSA). The morphological difference
of the roots under 1 and 2.5 μM TSA treatments might be due to
different genes were modified during root development.
Stress-responsive genes
In our experiment, the growth of populus roots was inhibited after
long-term growth on medium supplemented with TSA (Fig. [104]2). It is
well known that stresses such as salt, cold, drought and heavy metals
were able to induce the accumulation of reactive oxygen species (ROS)
and inhibit root growth. We examined the ROS accumulation in roots on
exposure to different concentrations of TSA for 2 weeks. No significant
increases of ROS were observed in roots in response to TSA treatment
(Fig. [105]12). Additionally, we examined the expression levels of
genes encoding ROS scavenging enzymes such as superoxide dismutase
(SOD), catalase (CAT), peroxidase (POD) and glutathione S-transferase
(GTS) in the three DGE libraries and their expression levels were
proved by using real-time PCR. A total of 10 genes encoding the four
ROS scavenging enzymes were available by searching national center for
biotechnology information (NCBI) and they could be detected in the T1
and T2.5 DGE libraries (Additional file [106]5). Of the ten genes, no
one was significantly up-regulated. However, four genes encoding CATs
(Potri.002G009800 and Potri.005G100400), PO2 and GST U45, respectively
were significantly down-regulated (Fig. [107]13). Whereas, the
expression of the four genes appeared not to be dose-dependent since
their expression levels were not significantly different under 1 and
2.5 μM TSA treatments. Base on the analysis of ROS accumulation and the
expression of ROS scavenging genes in the roots, we proposed that the
inhibition of root growth on exposure to TSA was not due to ROS
accumulation.
Fig. 12.
Fig. 12
[108]Open in a new tab
ROS accumulation in roots in response to TSA treatments. The detached
shoots were transferred onto medium supplemented with indicated
concentrations of TSA. After 2 weeks the regenerated roots were
collected for DAB staining to check the accumulation level of H[2]O[2]
Fig. 13.
Fig. 13
[109]Open in a new tab
Real-time PCR validations of ROS scavenging genes. Relative level,
2^−△△CT; TPM, transcript per million mapped reads
In our study, the common GO terms for T1 and T2.5 libraries included
“active transporter activity”, “response to stimulus” and “response to
stress”, implying a relationship between HDACs and stimuli/stress
responses in populus roots. Current available data from herbaceous
plants such as Arabidopsis, rice and maize have shown that HDACs are
involved in stress responses. In plants, the expression of HDAC genes
was regulated by abscisic acid (ABA), jasmonic acid (JA), salicylic
acid (SA), ethylene, biotic (salt, drought and cold) or abiotic
stresses [[110]29–[111]34]. Meanwhile, the alteration of HDAC levels
because of overexpression, mutation or RNAi-mediated repression could
affect the expression levels of some stress-responsive genes [[112]4].
In our experiment, HDAC activity was inhibited by TSA and some genes
encoding ROS scavenging enzymes such as CAT, POD and GST were proved to
be down-regulated, suggesting that HDACs were required for ROS
scavenging. Based on the evidence from other plants and our current
findings, we hypothesized that the inhibition of HDAC activity by TSA
might alleviate the control of HDACs on target genes and, as a result,
the transcription of some genes involved in stress/stimulus responses
were altered. Our data suggested the possibility that stimulus- and
stress-responsive genes were directly or indirectly regulated by HDACs
in populus roots.
Root development genes
Root development is a complex process for many plants. Studies of root
system in the model plant Arabidopsis advanced the understanding of
root development. To date, functions of genes involved in root
development, gene regulatory network, mechanisms regulating root
development, and root development under stress conditions have been
well characterized in Arabidopsis [[113]35, [114]36]. In Arabidopsis,
many genes involved in root development had been identified and they
participated in different processes of root development, including
patterning and maintenance of the stem cell niche, meristem size
control, xylem patterning, root hair pattering, lateral root initiation
and patterning, lateral root emergence and auxin pathway [[115]35,
[116]36]. We checked the expression of root development genes in the
DGE libraries. A total of 12 genes in T1 and T2.5 libraries were
available to be annotated to corresponding genes in Arabidopsis
(Additional file [117]6). During root embryogenesis, root apical
meristem (RAM) is established and provides new cells for root formation
and growth. At the tip of the RAM, a single layer of initial cells
(stem cells) surrounding the quiescent center (QC), a group of less
mitotically active cells, form the stem cell niche [[118]35, [119]36].
Stem cells produce the vascular, endodermal, cortex, epidermal, lateral
root cap cells, and columella root cap. QC has a role to maintain the
identity of surrounding stem cells by the expression of wuschel-related
homeobox 5 (WOX5), which is controlled by clavata3/embryo surrounding
region (CLE) peptide CLE40 and the receptor-like kinase Arabidopsis
crinkly 4 (ACR4) [[120]35, [121]36]. The QC identity is specified by
plethora (PLT) pathways and short root (SHR)/scarecrow (SCR),
transcription factors belonged to the GRAS [gibberellin insensitive
(GAI), repressor of ga1–3 (RGA), SCR] family. In Arabidopsis, ACR4
acted as a key factor in promoting formative cell divisions in the
pericycle [[122]37] and SHR mutation (shr) highly reduced root growth
[[123]38]. Vascular system of the plants is consisted of two types of
tissues, xylem and phloem, to transport water, nutrients and
photosynthates to and from the shoot. Arabidopsis ATHB-8, a member of a
small homeodomain-leucine zipper family, is expressed in the vascular
tissue and regulates cell proliferation and differentiation.
Over-expression of ATHB-8 in transgenic Arabidopsis reduced the number
of lateral roots and higher order roots [[124]39]. Meanwhile, the
diameter of the transgenic root was much larger than that of wild-type,
suggesting the role of ATHB-8 in secondary growth of root. In populus,
in our study, the expression levels of ACR4 and SHR in roots were
down-regulated and ATHB-8 was up-regulated by TSA (Additional file
[125]6). Based on the expression change and corresponding morphological
alteration in root development in Arabidopsis, the expression
alterations of the genes were consistent with the developmental
inhibition and morphological change of root system.
In the differentiation zone of root, one of the key features is the
development of root hairs. Root hairs are important for water and
nutrient uptake and soil anchoring. Epidermal cells produced in the RAM
may become hair cells or nonhair cells based on their relative
positions to cells in the underlying cortical layer of the roots. An
epidermal cell lies between underlying cortical cells (outside an
anticlinal cortical cell wall) will develop as a root hair cell, while
an epidermal cell adjacent to a single cortical cell (outside a
periclinal cortical cell wall) will develop as a nonhair cell [[126]35,
[127]36]. In Arabidopsis, the cellular pattern of root was determined
by six patterning genes, caprice (CPC), enhancer of try and cpc (ETC.),
glabra 2 (GL2), GL3, enhancer of glabra 3 (EGL3), and transparent testa
glabra (TTG). In nonhair cell, a complex of transcription factors GL3,
TTG1, EGL3 and WER directly activate transcription of the hair cell
fate repressor GL2 and CPC. CPC moves into neighboring cells and
inactivates the complex by replacing WER, resulting in the inactivation
of GL2 and hair cell specification. Xu et al. (2005) reported that TSA
treatment significantly altered the expression of genes CPC, GL2 and
WEREWOLF (WER) in Arabidopsis [[128]7]. In our work, the patterning
genes TTG1 [[129]40–[130]42] and GL2 [[131]40, [132]43, [133]44] were
significantly down-regulated after 1 μM and 2.5 μM TSA treatment,
respectively. These results suggested the involvement of HDACs in the
regulation of root hair patterning in populus.
Root system includes primary roots and LRs. LRs were initiated from
pericycle cells adjacent to the xylem poles in differentiation zone.
Pericycle cells initiate a series of asymmetric transverse and
periclinal divisions and, as a result, a dome-shaped lateral root
primordium (LRP) is formed, which leads to the emergence of lateral
root [[134]45]. LR development is regulated by auxin, involved complex
regulation of auxin biosynthesis, auxin transportation and cellular
appropriate response to auxin. The transport of auxin is important for
LR development. Arabidopsis AUX1 is a putative auxin influx carrier.
Mutation of AUX1 resulted in a reduction of IAA in root and the mutant
(aux1) had a reduced number of LRP and fewer lateral roots than wild
type [[135]45]. In LR formation and development, many genes are
regulated by auxin. The expression of auxin-responsive genes is
regulated by two families of important proteins, auxin-response factors
(ARFs) and auxin/indole-3-aceticacids (Aux/IAAs). ARFs are
transcriptional activator of auxin-responsive genes and positively
regulate LR formation, while Aux/IAAs can inhibit the activity of
specific ARFs. In presence of auxin, auxin binds to its receptor
transport inhibitor response1 (TIR1), which promotes the degradation of
Aux/IAA proteins by ubiquitin-ligase complex. The degradation of
Aux/IAA proteins derepresses the activity of ARFs, such as ARF7 and
ARF19, and allow auxin-responsive genes to be expressed, which leads to
the initiation of LR formation [[136]35]. In Arabidopsis, ARF7 and
ARF19 double mutation (arf7arf19) strongly inhibited the lateral root
formation at the very early stage of LR initiation [[137]46]. In
addition, monopteros (MP)/ARF5 is another important regulator in LR
development. The ARF5 mutant (arf5-1) failed to form root meristem
[[138]9]. In transgenic Arabidopsis over-expressing ARF5, closely
positioned lateral root initiation sites and aberrantly spaced lateral
root primordia were occasionally observed [[139]47]. These finding
indicated that ARF5 is involved in LR formation. In our study, the LR
formation and root growth were observed to be inhibited by TSA
treatment, especially under 2.5 μM TSA treatment (Fig. [140]2a). After
examination of DEG profiles, several genes related to LR development
such as ARF5 [[141]11], ARF7 [[142]46] and auxin resistant 1 (AUX1)
[[143]45, [144]48] were found to be differentially down-regulated, even
though the fold-changes of the genes were not statistically significant
(Additional file [145]6). Based on functions of the corresponding genes
in Arabidopsis, the down-regulation of these genes in populus might
contribute, at least in part, to the inhibition of lateral root
formation. Additionally, in Arabidopsis, S-Phase Kinase-Associated
Protein 2B (SKP2B), encoding an F-box protein, has recently been
reported to play a negatively regulatory role in cell cycle and LR
formation [[146]49]. In this study, the promoter of SKP2B was regulated
by H3 acetylation in an auxin- and IAA14-dependent manner and skp2b
mutant has longer roots and more LRs than control plants [[147]49]. In
our experiment, the expression of skp2b-like gene (Potri.005G185700)
was not detectable in the roots of plants without TSA treatment, while
its expression levels in the roots treated by different concentrations
of TSA were much higher, especially under 2.5 μM TSA treatment
(Additional file [148]6). The induction of SKP2B might be related to
the short root and less LR observed in populus roots after TSA
treatments (Fig. [149]2).
GA signaling pathway
GAs, as phytohormones, played an essential role in both primary root
elongation and LR development [[150]50–[151]52]. Available evidence
showed that root growth of many plants was altered due to the change of
GA levels such as application of GAs to root system, supplementation of
GA-biosynthesis inhibitors, alteration of genes in GA pathway, or
mutations of genes involved in GA biosynthesis. GAs may play a negative
or positive role in promoting root growth. The work of Gou and
colleagues (2010) showed that shortage of GAs promoted root growth and
LR development [[152]52]. GA-deficient (35S: PcGA2ox1) and
GA-insensitive (35S: rgl1) transgenic populus exhibited increased LR
proliferation and elongation, and these effects were reversed by
exogenous GA treatment [[153]52]. Although GAs appeared to have a
negative role in root growth and development in Gou and colleagues’
work, GAs played a positive role in promoting root system development
in many plants. DELLA proteins function as growth repressors to repress
GA signaling. In Arabidopsis, application of GAs to root system
promoted root growth by targeting the degradation of DELLA proteins,
repressors GA1–3 (RGA) and gibberellin insensitive (GAI), in elongation
zone tissues such as epidermal, cortical, endodermal and stele tissues
[[154]53]. GAI mutation, gai, rendered the GAI protein resistance to
GA-dependent disruption and the root growth of gai mutants was
significantly reduced. Uniconazole P (Un-P) is a GA biosynthesis
inhibitor. Un-P at the concentrations of 10 and 100 nM were able to
significantly inhibit the growth of roots in Lemna minor [[155]50]. In
pea, the genes na, lh-2, and ls-1 encode enzymes catalyzing GA
biosynthesis [[156]54]. Mutation of the genes, na, lh-2, and ls-1,
reduced GA levels in roots and the length of roots was 50 %, < 15 %
and < 15 % decreased, respectively [[157]51]. These findings indicated
that GAs played a positive role in promoting root growth. In
Arabidopsis, GA biosynthesis was majorly catalyzed by enzymes such as
copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), KO, KAO,
GA 20-oxidase, GA 3-oxidase and GA 2-oxidase [[158]52]. In our work, GA
biosynthesis pathway in the populus roots subjected to 2.5 μM TSA was
significantly altered (Fig. [159]9). Four genes involved in GA
biosynthesis, including KO, KAO, GA20ox and GA3ox, were detected to be
down-regulated in the roots (Fig. [160]10). We speculate that the
repression of genes involved in GA biosynthesis might be associated
with the inhibition of root growth by TSA. Our findings suggested the
regulatory role of HDACs in GA biosynthesis.
Conclusions
Regeneration of roots from shoots and root growth were inhibited by TSA
in populus. A digital gene expression (DGE) approach was used to
identify differentially expressed genes in the populus roots exposed to
different concentrations of TSA. In comparison with the control sample,
a total of 1404 and 563 genes were detected to be differentially
expressed in the roots subjected to 1 μM and 2.5 μM TSA, respectively.
Most of the differentially expressed genes were down-regulated on
exposure to TSA. GO and pathway analyses showed that the DEGs were
related to many kinds of molecular functions and biological processes.
The DGE data provides a large set of candidate genes probably regulated
by HDACs in root development.
Methods
Plant growth and TSA treatment
Seedlings of Populus trichocarpa were cultured under the 25 ± 2 °C,
70–80 % relative humidity, and 16 h light/8 h dark condition. Stems of
the populus seedlings were cut into segments each with an axillary bud
and cultured on WPM medium containing 0.1 mg/L IBA. Three weeks later,
the shoots developed from the axillary buds were cut off and
transferred onto the WPM medium supplemented with 0, 1 and 2.5 μM TSA
(Sigma) for root regeneration. The populus explants including stem
segments, shoots, and regenerated seedlings were cultured under the
aforementioned condition. After 2 weeks of rooting, number of roots and
root length of regenerated seedlings were determined. Data were
statistically analyzed using one-way analysis of variance (one-way
ANOVA) followed by Tukey’s multiple comparisons test with significance
level set as 5 %. The experiment was repeated three times. The roots
grown on medium containing different concentrations of TSA were frozen
in liquid nitrogen and kept at −80 °C until RNA isolation.
HDAC activity assay
Roots grown on the WPM medium supplemented with different
concentrations of TSA (0, 1, and 2.5 μM) were collected. HDAC activity
in root was measured using the HDAC Colorimetric Assay/Drug Discovery
Kit following the manufacturer’s instructions (Enzo Life Sciences). The
protein samples were incubated with substrate comprising an acetylated
lysine at 37 °C for 30 min. The reaction was stopped by adding
developer and incubating the plate at 37 °C for 15 min. The HDAC
activity was then measured by microtiter-plate reader at 405 nm. HeLa
nuclear extracts were used as the positive control and a blank sample
(without enzyme) was used as the negative control. The data were
statistically analyzed using one-way ANOVA followed by Tukey’s multiple
comparisons test (P level ≤ 5 %).
Semithin sections
Root sections taken 1 cm from root cap and in the middle region of the
roots were fixed immediately in formalin-acetic acid-alcohol (FAA,
[[161]55]) for 24 h. After fixation, the samples were dehydrated in
ethanol followed by 10 h in 100 % isopropanol and 10 h in 100 %
1-butanol. The dehydrated tissues were then placed in glycol
methacrylate (GMA) for infiltration. After this infiltration, the
specimens were transferred to GMA and left to polymerize overnight at
60 °C. Sections were stained with toluidine blue and photographs were
taken using a microscope (BX43F, Olympus, JP).
RNA preparation, Solexa/illumine sequencing and data processing
Total RNA was isolated from roots using Trizol reagent (Invitrogen).
RNA concentration was determined using a Qubit Fluorometer and RNA
integrity was assessed with the Bioanalyzer 2100 (Agilent
Technologies). The A260/A280 ratio and A260/A230 ratio of all RNA
samples were around 2.1. For Solexa sequencing, the DGE libraries were
prepared using Illumina Gene Expression Sample Prep Kit. The
single-strand molecules were fixed onto a Solexa sequencing chip
(flowcell) and then sequenced by Illumina HiSeq^TM 2000 system. In
details, mRNA was purified from 6 μg of total RNAs by Oligo (dT)
magnetic beads adsorption method. The first and second-strand cDNAs
were synthesized using Oligo (dT) primers and digested with restriction
enzyme NlaIII, which recognizes the CATG sites. The 3’ cDNA fragments
were purified and the Illumina adaptor 1 was ligated to the 5’ end of
the fragments through CATG sticky site. The junction of Illumina
adaptor 1 and CATG site is the recognition site of MmeI, which is a
type of endonuclease with separated recognition sites and digestion
sites. It cuts at 17 bp downstream of the CATG site, producing tags
with adaptor 1. After removing 3’ fragments with magnetic beads
precipitation, Illumina adaptor 2 was ligated to the 3’ ends of the
tags, thus generating a tag library with different adaptors at both
ends of the tags. After 15 cycles of linear PCR amplification, 105 bp
fragments were purified by 6 % TBE PAGE gel electrophoresis. After
denaturation, the single-chain molecules were fixed onto the Illumina
Sequencing Chip (flowcell). Each molecule turned into a single-molecule
cluster sequencing template through in situ amplification. Then four
types of nucleotides labeled by four different colors were added in,
and sequencing was performed with the method of sequencing by synthesis
(SBS).
Data analysis
Raw image data obtained from sequencing was transformed by base calling
into sequence data, also called raw data or raw reads. Of the raw data,
empty tags (no tag sequence between the adaptors), adaptors, low
quality tags (tags containing unknown nucleotides “N”), abnormal tags
(too long or too short tags), and single copy tags were removed to
obtain clean tags (21 bp). To identity the gene expression patterns in
populus roots, all clean tags were annotated by mapping to the
sequenced genome of populus trichocarpa which covered all possible
CATG + 17-nt tag sequences, allowing only 1 bp mismatch. The clean tags
mapped to multiple reference sequences were filtered and the remaining
clean tags were designated as unambiguous tags. For gene expression
analysis, the number of unambiguous clean tags for each gene was
calculated and then normalized to transcripts per million clean tags
(TPM) [[162]56, [163]57].
Analysis and screening of DEGs
Based on the method described by Audic and Claverie [[164]58], a
rigorous algorithm was used to identify differentially expressed genes
(DEGs) between two samples. The P-value corresponds to differential
gene expression test. The false discovery rate (FDR) was used to
determine the threshold of P-Value in multiple tests. FDR ≤ 0.001 and
the absolute value of | log2Ratio | ≥ 1 were used as the threshold to
judge the significance of gene expression difference.
GO and pathway enrichment analysis
To classify the functions of DEGs, gene ontology (GO) analysis was
performed by mapping the DEGs to terms in GO database
([165]http://www.geneontology.org/). For further understanding the
functions of the DEGs, pathway enrichment analysis was conducted by
searching the KEGG database ([166]http://www.genome.jp/kegg/)
[[167]59]. Significantly enriched metabolic pathways or signal
transduction pathways in DEGs were identified in comparison to the
whole genome background. The calculation formula used for this analysis
was as follows:
[MATH: P=1−∑i=0
m−1MiN−Mn<
/mi>−iNn
:MATH]
Here N is the number of all genes with a KEGG annotation, n is the
number of DEGs in N, M is the number of all genes annotated to specific
pathways, and m is the number of DEGs in M. For GO and pathway
enrichment analyses, a P-value of 0.05 was selected as the threshold
for considering a gene set as significantly enriched.
Quantitative real-time PCR analysis
The quantitative real-time PCR was set up using SYBR Premix Ex Taq II
Kit (TaKaRa) in a volume of 20 μl. The reactions were performed in
triplicate for each run and three biological replicates were included.
The conditions for the PCR reactions were as follows: 95 °C for 3 m,
followed by 44 amplification cycles at 95 °C for 30 s, 55 °C for 30 s,
and 72 °C for 30 s. The specific primers used for the selected genes
were listed in Additional file [168]7. For each gene, the pair of
primers was designed on different exons using online program Primer 3
([169]http://bioinfo.ut.ee/primer3/). The Ct values obtained for all
the genes were normalized to that of the internal control 18S RNA. For
the gene expression analysis, the transcript amount of these genes was
determined using 2^-ΔΔCt calculations. The transcript level of each
gene without TSA treatment (0 μM) was indicated as 1. Transcript levels
(n-fold) of the examined genes under TSA treatment conditions were
obtained by comparison with their transcript levels in the control
sample (0 μM).
Diaminobenzidine (DAB) stain for hydrogen peroxide
The populus roots were immersed in DAB solution (1 mg/mL, pH 3.8) for
overnight. Then the samples were de-stained by soaking in 95 % ethanol
and boiling for 10 min.
Availability of supporting data
The sequence data associated with this study has been deposited to the
NCBI Sequence Read Archive (SRA) under the BioProject ID PRJNA304268.
Additional supporting data are included as additional files.
Acknowledgement