Comparative transcriptome analysis reveals carbohydrate and lipid metabolism blocks in Brassica napus L. male sterility induced by the chemical hybridization agent monosulfuron ester sodium
Li et al. BMC Genomics
Comparative transcriptome analysis reveals carbohydrate and lipid metabolism blocks in Brassica napus L. male sterility induced by the chemical hybridization agent monosulfuron ester sodium
Zhanjie Li 0 2
Yufeng Cheng 0 2
Jianmin Cui 0 1
Peipei Zhang 0 2
Huixian Zhao 0 2
Shengwu Hu 0 1
0 State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University , Yangling, Shaanxi 712100 , P. R. China
1 College of Agronomy, Northwest A&F University , Yangling, Shaanxi 712100 , P.R. China
2 College of Life Sciences, Northwest A&F University , Yangling, Shaanxi 712100 , P.R. China
Background: Chemical hybridization agents (CHAs) are often used to induce male sterility for the production of hybrid seeds. We previously discovered that monosulfuron ester sodium (MES), an acetolactate synthase (ALS) inhibitor of the herbicide sulfonylurea family, can induce rapeseed (Brassica napus L.) male sterility at approximately 1% concentration required for its herbicidal activity. To find some clues to the mechanism of MES inducing male sterility, the ultrastructural cytology observations, comparative transcriptome analysis, and physiological analysis on carbohydrate content were carried out in leaves and anthers at different developmental stages between the MES-treated and mock-treated rapeseed plants. Results: Cytological analysis revealed that the plastid ultrastructure was abnormal in pollen mother cells and tapetal cells in male sterility anthers induced by MES treatment, with less material accumulation in it. However, starch granules were observed in chloroplastids of the epidermis cells in male sterility anthers. Comparative transcriptome analysis identified 1501 differentially expressed transcripts (DETs) in leaves and anthers at different developmental stages, most of these DETs being localized in plastid and mitochondrion. Transcripts involved in metabolism, especially in carbohydrate and lipid metabolism, and cellular transport were differentially expressed. Pathway visualization showed that the tightly regulated gene network for metabolism was reprogrammed to respond to MES treatment. The results of cytological observation and transcriptome analysis in the MES-treated rapeseed plants were mirrored by carbohydrate content analysis. MES treatment led to decrease in soluble sugars content in leaves and early stage buds, but increase in soluble sugars content and decrease in starch content in middle stage buds. Conclusions: Our integrative results suggested that carbohydrate and lipid metabolism were influenced by CHA-MES treatment during rapeseed anther development, which might responsible for low concentration MES specifically inducing male sterility. A simple action model of CHA-MES inducing male sterility in B. napus was proposed. These results will help us to understand the mechanism of MES inducing male sterility at low concentration, and might provide some potential targets for developing new male sterility inducing CHAs and for genetic manipulation in rapeseed breeding.
Brassica napus L; Chemical hybridization agent; Male sterility; Monosulfuron ester sodium; Expression profile; Carbohydrate and lipid metabolism
In plants, the hybrid F1 progeny usually exhibits
heterosis (hybrid vigour) relative to the inbred parents [1,2].
Accordingly, the productivity of many crops has been
boosted by introducing hybrid varieties . An effective
pollination control system is a prerequisite for heterosis
utilization. In 1950, it was reported that the plant growth
regulator maleic hydrazide can induce male sterility in
corn plants [4,5]. This initial finding led to the induction
of male sterility by a chemical hybridization agent (CHA),
which became an important tool for crop heterosis. CHAs
are not restricted to particular species and do not require
the laborious practice of transferring sterility and fertility
genes from one species/line to another, unlike the other
two popular pollination control systems in hybrid
breeding, i.e. cytoplasmic male sterility (CMS) and nuclear male
sterility (NMS). In addition, CHAs enable breeders to
develop hybrids with a higher heterosis level in a shorter
time . The technique is now widely used in crops
heterosis, particularly in rapeseed (Brassica napus L.) [6,7]. Till
date, several dozens of commercial hybrids based on
CHA-induced male sterility have been registered
according to the data from the bulletins of Chinese National
Crop Variety Approval Committee. The availability of safe
and selective chemicals capable of inducing male sterility
without causing any significant adverse effect on plant
growth and development has been the necessary
prerequisite in the pursuit of this approach. We previously
found that monosulfuron ester sodium (MES) can induce
complete male sterility in rapeseed at a concentration
below 1% of that required for its herbicide activity and it
has no significant influence on plant vegetative growth .
In the herbicide field, sulfonylurea is well known for
its eco-friendly, extreme low toxicity towards mammals,
and ultralow dosage application . MES is a new
sulfonylurea herbicide that inhibits acetolactate synthase
(ALS, EC184.108.40.206, also known as acetohydroxyacid
synthase, AHAS), an enzyme in the first step of the
branched-chain amino acids (BCAAs; including valine,
leucine, and isoleucine) biosynthesis pathway . Plant
ALSs are encoded by nuclear genes, and their
Nterminal signal peptide sequence is required for
translocating the protein to the chloroplast . In addition,
ALS is the target of four other classes of herbicides in
addition to the sulfonylurea class, including
sulfonylaminocarbonyltriazolinones, and imidazolinones . Several
ALS inhibitor herbicides are exploited as CHAs in crop
breeding . Previous studies suggested several
biochemical and physiological effects as consequence of
the primary action of ALS inhibitors when it was used
at lethal concentration: a quick accumulation of
pyruvate (the main substrate of ALS) [13,14]; increase in
free amino acid pool likely through protein turnover
[15-18]; a rapid accumulation of carbohydrate in leaves
 related to decreased photoassimilate translocation
to sink tissues  due to a decreased sink strength
; and induction of fermentative metabolism [13,22].
Two other studies reported genome-wide gene expression
responses to different ALS-inhibitor herbicides in
Arabidopsis thaliana using the Affymetrix ATH1 microarray
[23,24]. Till date, very few studies were carried out to
investigate the mechanism of ALS inhibitor CHAs inducing
male sterility .
In flowering plants, the development of the male
gametophyte occurs in the anther, and it is a
wellprogrammed and elaborate process [25-27]. In
Arabidopsis, anther development consists of two phases
divided into 14 stages [27,28]. During phase I, from
stage 1 to 8, the four lobes of the anther are formed,
each containing reproductive cells (microspore mother
cells) and nonreproductive cell layers. The lobe is
organised and includes the following from the exterior to
the interior: the epidermis, endothecium, middle layer,
and tapetum [27,28]. The developing pollen is immersed
in locular fluid containing nutrients such as sugars and
lipids from the sporophytic (somatic) tissue tapetum .
The early stages of pollen development are characterized
by active growth and high metabolic activity in the anther.
Thus, anthers have the highest sink strength in developing
flowers, and large amounts of sugars are mobilized to
anthers for supporting their early development . During
phase II, microspores undergo meiosis to form the tetrads
enclosed in a thick shell composed of a callose (-1,3
glucan) wall and a pollen mother cell (PMC) wall composed
of cellulose, hemicelluloses, and pectins . The timely
degradation of the callose and PMC walls is critical for
microspore release from the tetrads . At least three
cell wall enzymes are involved in this process, including
-1,3-glucanase [32,33], endocellulase [34,35], and
polygalacturonase (PG) . During the maturation of
pollen grains, the grains accumulate an energy reserve
in the form of starch for germination and starch thus
serves as a marker of pollen maturity . On the other
hand, at the late unicellular stage or early bicellular
stage, tapetal cells degrade and their remnants are
deposited on the pollen exine . Sporopollenin, the
major component of exine, is a complex polymer
primarily composed of fatty acids and phenolic compounds
. Therefore, the biosynthesis and export processes
of fatty acids are essential for exine formation. The
development of microgametogenesis involves numerous
genes expression and a large part of the metabolism
coordinated by a complex regulation network in both
somatic and gametophytic cells .
To better understand the mechanism how ALS
inhibitor CHAs induced male sterility in rapeseed, we treated
the rapeseed plants at the bolting stage with 0.1 g mL1
MES to induce male sterility during the entire flowering
period without significantly affecting other tissues growth
and development. The objectives of this study were to
investigate 1) whether the ultrastructure of the anthers was
affected in MES treated plants 2) which set of genes
differentially expressed might be associated with the
ultrastructure changes of MES treated anthers 3) how these
cytological and transcriptome changes relate to
modification of physiological processes in rapeseed plants after
MES treatment. This study will provide some clues to
the mechanism of MES inducing male sterility, and
provide some potential targets for developing new CHAs
and for genetic manipulation during rapeseed breeding.
Cytological studies reveal that MES treatment affects the
plastid ultrastructure and metabolite accumulation in the
We previously showed that MES treatment causes two
typical defects in sterile anthers: type I with early broken
down tapetum at the PMC stage and type II with
abnormal nondegraded tapetum at the mature pollen stage
. To better understand these phenomena, we observed
the ultrastructure of fertile and sterile anthers from the
mock-treated and MES-treated plants, respectively, during
their development. The results showed that MES
treatment affected the plastid ultrastructure and metabolite
accumulation in the developing anthers (Figure 1). At the
PMC stage, numerous plastids are dispersed in the
cytoplasm of PMCs and tapetal cells in the mock-treated
plants (Figure 1AC). However, serious plasmolysis in
PMCs and slight plasmolysis in tapetal cells were observed
in the MES-treated male sterile plants (Figure 1D, E, and
black arrow in 1F), and the cytoplasm of meiocytes and
tapetal cells exhibited low electron density, with less
plastids dispersed in them. At the vacuolated-microspore
stage, the tapetum cells began to degrade and a number of
elaioplasts and tapetosomes with abundant lipid
compounds were formed in the tapetum of the mock-treated
fertile anther (Figure 1G, H, white arrow). Besides, another
type of plastids located in a crown, started to accumulate
low electron-dense material and was surrounded by the
rich ER (Figure 1I, white arrow). In contrast, in the
MEStreated sterile plants, two types of abnormal tapetum were
observed, as shown by Cheng et al. (2013) . In type II
abnormal tapetum, a number of elaioplasts and
tapetosomes were formed, as seen in fertile plants; however, the
tapetum cells did not degrade, the crowned plastids
showed an irregular shape and were not well developed
(Figure 1J, K, L). Type I abnormal tapetum was degraded
and released noncompact elaioplasts and low
electrondense tapetosomes (Figure 1M, N). At the mature-pollen
stage, the fertile pollen grains showed profuse globular
particles (Figure 1O, P, Q); however, the sterile pollen
grains were almost empty, type II tapetum still showed an
intact and visible tapetal cell wall (Figure 1R, S), and type I
tapetum showed solidified bulks (Figure 1T).
Furthermore, at the vacuolated-microspore stage, the
chloroplastids in the epidermis and endothecium cells of
the MES-treated plants exhibited defects. In fertile
plants, the epidermis and endothecium cells showed
normal oval-shaped chloroplastids with a distinct
thylakoid structure and little starch granules in the thylakoid
(Figure 1U, V, W); however, in the MES-treated sterile
plants, the chloroplastids of the epidermis cells showed
large starch granules in the thylakoid and the
endothecium cells displayed fusiform-shaped chloroplastids with
a linear thylakoid structure (Figure 1X, Y, Z).
Identification of transcripts differentially expressed between
the MES-treated and mock-treated rapeseed plants
To obtain genome-wide gene expression profiles in
the MES-treated and mock-treated plants of B. napus,
the Agilent Single Channel Brassica Oligo Microarray
(4 44 K) was used. Three independent biological
replicates of four pairs of tissues (organs) from the
MES-treated and mock-treated plants were collected
for gene expression analysis, resulting in a dataset of
24 microarrays. The four tissues (organs) (Figure 2)
included the leaves from main inflorescences (Ls), the
small buds less than 1 mm in length containing
microgametocytes before and during pollen mother stage
(SBs), the anthers from middle buds with length
between 1 mm and 3 mm containing microgametocytes
from meiosis to early uninucleate microspore stage
(An-MBs), and the anthers from large buds more than
3 mm in length containing microgametocytes from
vacuolated stage to mature pollen stage (An-LBs). The
data quality was assessed using two measurements: (1)
correlation coefficients between biological replicates,
which ranged from 0.8495 to 0.9906, with a mean of
0.9447 (Additional file 1), and (2) quantitative real time
RT-PCR (qRT-PCR), which was performed on 62
randomly selected genes. The results of qRT-PCT analysis
showed a high degree of concordance (R2 = 0.8775) with
microarray results (Additional file 2). Taken together,
these demonstrated that the microarray results obtained
in this study were reliable.
To identify differentially expressed transcripts (DETs)
between the MES-treated and mock-treated groups
involved in microgametogenesis, two sets of Students t-test
comparisons were performed (Figure 3). Firstly, vertical
comparisons (comparisons within groups, Figure 3A) were
conducted to identify DETs involved in anther
development, 11,428 and 3,786 DETs being identified within the
mock-treated group and MES-treated group, respectively
(right and left circle in Figure 3C). Secondly, horizontal
comparisons (comparisons between groups, Figure 3B)
Figure 1 (See legend on next page.)
(See figure on previous page.)
Figure 1 Transmission Electron Microscope (TEM) micrographs of the anthers from the mock-treated (fertile) and MES-treated (sterile)
plants. (A) The fertile anthers at pollen mother cell (PMC) stage; (B) Enlarged fertile meiocytes in (A); and (C) Enlarged fertile tapetum in (A)
showing numerous plastids dispersed in cytoplasm (white arrow). (D) The sterile anthers at PMC stage; (E) Enlarged sterile meiocytes in (D)
showing less plastids in condensed cytoplasm separated from the cell wall; (F) Enlarged sterile tapetum in (D) showing little abnormal plastids
(white arrow) and more large vacuoles in cytoplasm, and with a little plasmolysis at meiocyte side (black arrow). (G) The fertile anthers at
vacuolated-microspore stage; (H) The degraded tapetum in (G) showing elaioplasts and tapetsomes with abundant lipids; (I) Plastids in tapetum
located in a crown showing filled with globular low electron-dense metabolites and surrounded by rich endoplasmic reticulum (ER). (J) The sterile
anthers at vacuolated-microspore stage (type I); (K) The undegraded tapetum in (J) showing elaioplasts and tapetsomes with abundant lipids; (L)
Plastids in tapetum located in a crown showing irregular shaped low electron-dense material. (M) The sterile anthers at vacuolated-microspore
stage (type II); (N) The degraded tapetum in (M) showing scattered elaioplasts and tapetsomes with fuzzy structure; (O) The fertile anthers at
mature pollen grain stage; (P) The pollen grain in (O) showing profuse globular particles; (Q) The enlarged globular particles in (P). (R) The sterile
anthers at mature pollen grain stage (type II); (S) The undegraded tapetum in (R) died but cell wall still existed (black arrow); (T) The sterile
anthers at mature pollen grain stage (type I). (U) The epidermis and endothecium cells in fertile plants at vacuolated-microspore stage; (V) The
epidermis cells in (U) showing normal oval-shaped chloroplastids with distinct thylakoid structure and little starch granules in thylakoid; (W) The
endothecium cells in (U) showing oval-shaped chloroplastids with distinct thylakoid structure. (X) The epidermis and endothecium cells in sterile
plants at vacuolated-microspore stage; (Y) The epidermis cells in (X) showing abnormal chloroplastids with large starch granules in thylakoid; (Z)
The endothecium cells in (X) showing fusiform-shaped chloroplastids with linear thylakoid structure. PMC, pollen mother cell; N, nucleus; T,
tapetum; Msp, microspore; Ep, elaioplast; Ts, tapetosome; PG, pollen grain; TCW, tapetum cell wall; E, epidermis; En, endothecium; Ch, chloroplast.
Scale bars = 10 m (A, D, G, J, M, N, O and T), 5 m (C, F, P, R, S, U and X), 2 m (B, E and K), and 1 m (H, I, L, Q, V, W, Y and Z).
were conducted to identify DETs caused by
MEStreatment in the tissues (organs) tested, 1011 up-regulated
and 1218 down-regulated transcripts being identified in
the four-pair tissues (organs) (up and bottom circle in
Figure 3C). The common DETs identified in both the
vertical and horizontal comparisons described above were
considered as anther development related genes affected
by MES treatment (Figure 3C). Therefore, 102 + 108 + 332
and 31 + 84 + 846 DETs (red and green parts in venn
diagram in Figure 3C) corresponding to 1501 unique DETs
(Additional file 3) were selected for further analysis in this
study, in order to reveal the alteration of gene expressions
induced by MES treatment in the tissues (organs) tested.
Distribution of these 1501 DETs indicated that over 64%
(961/1501) were down-regulated and only 36% (542/1501)
were up-regulated in at least one tissue (organ) in the
MES-treated plants (Table 1). In addition, small
fractions of these DETs were in Ls and SBs (77 and 60,
respectively) with 2 ~ 5-fold change (up-regulation or
down-regulation), while the majority were found in
An-MBs and An-LBs (127 and 898, respectively) mainly
with 10-fold or more down-regulation. This suggested that
MES treatment led to expression alterations of a small
number of genes in Ls and SBs of rapeseed plants and a
large number of genes in An-MBs and An-LBs.
Subcellular localization and functional category analysis
of differentially expressed genes
To reveal the functions of these DETs identified above,
we annotated them by BLASTN against Arabidopsis
Information Resource (TAIR, http://www.arabidopsis.org/
Blast/index.jsp), considering the limited information of
gene functional annotations in B. napus and the
sufficient information in Arabidopsis as well as very high
coding-sequence similarity (approximately 85%) between
these two species . Of the 1501 DETs, 1379 (91.87%)
Figure 2 Photographs of the leaves and developmental anthers for microarrays from the mock-treated (A, C, E, G) and MES-treated (B,
D, F, H) plants. A-B, young leaves from the main inflorescences (Ls); C-D, small buds (SBs); E-F, anthers from middle buds (An-MBs); G-H, anthers
form large buds (An-LBs). Scale bar in leaves was 1 cm, scale bars in SBs, An-MBs, and An-LBs were 1 mm.
Figure 3 Strategies for identification of differentially expressed transcripts (DETs) involved in microgametogenesis between the
MES-treated and mock-treated plants by two sets of students t-test comparisons. (A) Comparisons within groups. The pair-wise
comparisons of Students t-test between tissues (organs) were carried out within mock-treatment groups and MES-treatment groups,
respectively, to detected DETs related to anther development under mock-treatment (control, fertile) and MES-treatment (male sterile)
conditions. The criteria for screening DETs were p-value <0.001 and fold change 2. mock, mock-treatment; MES, MES-treatment; (B)
Comparisons between the MES-treated and mock-treated groups. The pair-wise comparisons of Students t-test were performed between
the corresponding tissues (organs) of the mock-treated group and the MES-treated group to identify DETs related to MES-treatment. The
screen criteria were same as above. (C) Venn diagram showing the DETs involved in microgametogenesis between the MES-treated and
mock-treated groups. Comparisons within groups produced two sets of DETs, development-related genes in the MES-treated plants and in
the mock-treated plants (the left and right cycles). Comparisons between groups also produced two sets of DETs, up-regulated genes and
down-regulated genes in MES-treated group (the up and down cycles). These four sets of DETs were all collected, respectively. The common
sections (totally 1501 unique DETs, the red and green parts, (2) indicates 2 DETs existing in the both data sets) were considered to be anther
development-related genes affected by MES-treatment.
*two DETs (A_46_P229574, A_46_P381563) existed both in the up-regulated
and down-regulated probe sets.
Table 1 Distribution of differentially expressed transcripts (DETs) in MES-treated rapeseed plants
were highly similar to 1807 A. thaliana genes (AGI
identifiers) (BLSATN; E-value < 105 for nucleic acids) and
could be thus annotated. The remaining 122 DETs did
not find a close orthologue in the TAIR database and
therefore was not be annotated (Additional file 4).
The localization of these DETs might provide clues to
where they function. To gain insight into the biological
functions of these 1087 annotated unigenes, subcellular
localization and functional category analysis were
conducted according to the information from Munich
Information Center for Protein Sequences (MIPS) (Figure 4).
The 1087 unigenes could be distributed into 14
subcellular localizations, with the largest three categories being
the plastid/chloroplast (31.95%), mitochondrion (23.82%),
and nucleus (13.23%), followed by the eukaryotic plasma
membrane (7.37%), cell wall (6.99%), and cytoplasm
(6.24%) (Figure 4A). Furthermore, the 1087 unigenes were
classified into 21 functional categories, and the top five
categories were proteins with binding functions,
metabolism, unclassified proteins, cellular transport, and protein
fate, representing approximately 70% of the total
differentially expressed unigenes (Figure 4B). In order to
Figure 4 Subcellular localization and functional categories of the 1087 differentially expressed unigenes between the MES-treated and
the mock-treated rapeseed plants. (A) Subcellular localization, (B) Functional categories, (C) Enrichment analysis of the functional categories
list in B. The scale bar indicates -log (P-value), with highly enriched categories in red color, and invalid values in gray, whereas the P-value was
calculated according to a hypothesis test using cumulative hypergeometric distribution. Left panel, enrichment analysis of all the unigenes in
functional categories listed in B, except for those in unclassified category; the right panels, enrichment analysis of the sub-categories from
metabolism and cellular transport (blue rectangles), respectively. The prominent enriched sub-categories were circled in black ellipses.
understand the functions of DETs in different tissues,
we performed the category enrichment analysis in four
tissues separately (Figure 4C). The results of the
functional category (Figure 4B) and enrichment analysis
(Figure 4C) suggested that two categories, namely
metabolism and cellular transport, were particularly
affected by the treatment. They were not only
overrepresented in all four tissues but also in the top
four functional categories for relative abundance. The
other overrepresented classes with only few genes
falling in a certain functional category, including energy,
cell rescue, and interaction with the environment etc.,
wont be given further consideration in this study.
Furthermore, detailed subcategory analysis of metabolism
revealed that carbohydrate metabolism and lipid
metabolism were significantly enriched (top of the right panel in
Figure 4C), while detailed subcategory analysis of cellular
transport exhibited that electron transport was enriched
in all the tissues, along with carbohydrate transport, lipid
transport, and hormone transport that were highly
enriched in SBs (bottom of the right panel in Figure 4C).
Furthermore, to get the overview of the pathways where
the DETs are taking part in, the 1087 unigenes described
above were further analyzed by MapMan software. The
biotic stress and metabolism pathway visualization are
shown in Figure 5. In the biotic stress visualization
(Figure 5A), most of the genes whose expressions were
altered by MES treatment were involved in signaling,
proteolysis, and cell wall. Detail information of these
pathways revealed that 23 of the genes in the signaling
pathway belonged to the protein kinase signaling, and
21 belonged to calcium signaling. However, most of
these genes in signaling were differentially expressed in
An-LBs, except for eight genes altered in other tissues
(Additional file 5). In proteolysis pathway, 32 of 56
protein degradation related genes were coding for the
26S proteasome complex, which mediates
ubiquitindependent protein degradation. Interestingly, eight of
the 56 protein degradation related genes were
upregulated in An-MBs, while most of the others were
down-regulated in An-LBs (Additional file 5). This
indicated that MES treatment might invoke activation of
protein degradation process in the An-MBs of the
MES-treated plants. In addition, a large number of cell
wall related genes were down-regulated, this might be
related to metabolism regulation instead of stress
response, and it will be further analyzed below. In
metabolism pathway visualization (Figure 5B), though the
differentially expressed genes were dispersed in various
primary and secondary metabolism pathways, a large
number of genes were down-regulated in major and
minor CHO, cell wall, and lipid metabolism in all four
tissues. This result was consistent with the functional
category and enrichment analysis mentioned above.
Detail information of these pathways revealed that a
large part of genes were differentially expressed in Ls,
SBs, and An-MBs (Additional file 5). Interestingly, the
few genes involved in amino acid metabolism were
mainly up-regulated in An-LBs (Figure 5B, Additional
file 5). Overall, these findings suggested that MES induced
a tightly regulated gene network for metabolism
reprogramming in the MES-treated plant anthers, especially for
carbohydrate, cell wall, and lipid metabolism pathways.
Expression changes of genes involved in carbohydrate,
cell wall, and lipid metabolism
Combining functional category analysis results with
cytological observation that severe damage occurred at late
stage anthers (Figure 1J-N, R-T), we considered that most
of the differentially expressed genes in An-LBs were the
consequence of male sterility. On the contrary, early
responses in SBs and An-MBs might provide some
important clues to how low concentration of MES is
inducing male sterility. Therefore, we paid more
attention to the differentially expressed genes in Ls, SBs,
and An-MBs. To further corroborate the view that
expressions of carbohydrate, cell wall, and lipid
metabolism-related genes were significantly altered by
MES treatment during anther development process,
detailed alternations of gene expressions were further
analyzed in Ls, SBs, and An-MBs.
In the leaves of the MES-treated plants, the expressions
of several carbohydrate metabolism-related genes, two cell
wall-related genes, and one lipid metabolism-related gene
were altered, of which three genes were localized in plastid
(Table 2, Additional file 6). Four genes involved in starch
biosynthesis and degradation pathway, including ADP
glucose pyrophosphorylase (AGP, AT5G48300),
phosphoglucomutase (PGM, AT5G51820), disproportionating enzyme
(DPE1, 4--gluca-notransferase, AT5G64860), and
alphaglucan phosphorylase 2 (PHS2, AT3G46970), were
downregulated by approximately 2 ~ 3-fold. AGP has been
shown to be one of the key regulatory enzymes
catalyzing the first committed step of starch biosynthesis in
higher plants [42,43]. PGM plays a pivotal role in the
allocation of carbon between polysaccharide formation
and energy production, and it is located in plastid .
DPE1 and PHS2 are enzymes catalyzing the breakdown of
starch into maltose and glucose in the chloroplast at night
[45,46]. In addition, another carbohydrate
metabolismrelated gene (AT3G60750), encoding transketolase in
Calvin cycle, was down-regulated by 3.6-fold (Table 2).
Importantly, a vital gene encoding sweet protein 11
(AT3G48740) was down-regulated by approximately
3fold, which has been recently identified to mediate
sucrose efflux in leaves as a key step for phloem
transport . These results indicated that the genes related
to both starch biosynthesis and degradation processes
as well as sugar transport were affected by MES
treatment in leaves, suggesting that the transitory starch
mobilization regulation network might be disturbed in
the leaves of the MES-treated plants.
In SBs of the MES-treated plants, expression of four
genes related to carbohydrate and cell wall metabolism,
and several lipid related genes was altered, with one
gene being localized in mitochondrion and two in plastid
(Table 2, Additional file 6). Two genes related to cell
wall metabolism, BXL1 (beta-xylosidase, AT5G49360)
and pectin lyase-like superfamily protein (AT5G17200)
were down-regulated by 38.9-fold and 602.82-fold,
respectively (Table 2). The latter gene exhibits
polygalacturonase (PG) activity in cell wall metabolism and is
Figure 5 Transcripts involved in stress (A) and metabolism (B) assigned by MapMan in rapeseed leaves and developmental anthers
treated by MES. Positive fold change values (red) indicate up-regulation, whereas negative fold change values (blue) denote down-regulation.
Color saturates at 4.5-fold change. Each square represents a differentially expressed transcript.
highly expressed at early stages of flower development,
which is essential for anther development . In addition,
expression of several lipid metabolism-related genes was
altered, two of these genes being down-regulated and three
up-regulated (Table 2). KAT5 (AT5G48880), a thiolase, was
down-regulated by 5.9-fold (Table 2). This gene is strongly
expressed during flower development in Arabidopsis
and can partially complements KAT2, which mutant
exhibited partly male sterility . In addition, one
lipid transport gene (AT3G18280) and two lipid
degradation genes (AT1G20130, AT3G02040) were up-regulated
In the An-MBs of the MES-treated plants, a cluster of
cell wall-related genes, one gene involved in Calvin
cycle, and several lipid related genes were differentially
expressed (Table 2, Additional file 6). Most of the cell
wall-related genes were significantly down-regulated,
including cell wall precursor synthesis genes FLA5 and
UGE3, and pectin metabolism genes, such as
polygalacturonase 4, the pectate lyase family protein, plant
invertase/pectin methylesterase inhibitor superfamily, and
VANGUARD 1. Previous studies revealed that pectin
metabolism-related genes played important roles during
late stages of pollen development [49,50]. Besides,
another cell wall related-gene, UGE3 (AT1G63180) was
down-regulated by 19.5-fold (Table 2). It is reported that
UGE2 together with UGE3 affected pollen development
. In addition, several lipid metabolism-related genes
were altered in the An-MBs of the MES-treated plants,
two (AT2G25890, AT2G25890) of them involved in lipid
biosynthesis were down regulated, and three (AT4G35790,
AT3G50660, and AT1G30040) involved in lipid
degradation were up regulated (Table 2).
The alteration of the expression profiles of the genes
described above indicated that there was a dramatically
transcriptome reprogramming on carbohydrate and lipid
metabolism, especially on biosynthesis and degradation
of cell wall and lipid in the anthers of the MES-treated
rapeseed plants during anther development process. And
the carbohydrate mobilization pathway in leaves was
Functional categories enrichment analysis revealed
that cellular transport, particularly material transport
and electron transport function, was enriched in leaves
and developing anthers under MES treatment (Figure 4C).
It was found that detoxification-related genes such as
ABC transporter, heavy metal transport, and MATE efflux
family were up-regulated in the four tissues. Besides,
metabolite transporters for sugars, peptides, amino acids, and
nitrate were up-regulated in SBs and An-MBs (Additional
file 5). However, energy metabolism-related genes about
H+-exporting ATPase such as H(+)-ATPase 3 and H
(+)-ATPase 9 were significantly down-regulated in the
MES-treated plant anthers at late stage (Additional file 5).
These results indicated that substrate transport pathway
was activated, but energy production system might be
repressed in the MES-treated plant anthers.
Analysis of carbohydrate contents confirms that MES
treatment influences carbon metabolism
The ultrastructure and transcriptome analysis suggested
that carbohydrate metabolism might be affected in the
MES-treated plants. To confirm these findings, the
carbohydrate contents, including soluble sugars,
reducing sugars, sucrose, and starch, were analyzed in Ls,
SBs, and MBs of both MES-treated and mock-treated
plants, respectively (Figure 6). Compared with the
mocktreated plants, the contents of soluble sugars and reducing
sugars were significantly reduced in the Ls and SBs of the
MES-treated plants. However, in the MBs of the
MEStreated plants, the contents of soluble sugars and sucrose
were increased but the starch content was decreased,
compared with those in the same tissue of the
mocktreated plants. These data confirmed that the
carbohydrate metabolism in rapeseed leaves and anthers was
significantly influenced by MES treatment, particularly in
the late stage flower buds (MBs).
In this study, cytological observation, comparative
transcriptome, and physiological analysis were conducted to
reveal the mechanism of CHA-MES inducing male
sterility in rapeseed. Cytological results showed that the
ultrastructure of plastids/chloroplastids in the
MEStreated plants was abnormal, and substances in plastids
were deficient in pollen mother cells and tapetal cells
but accumulated in epidermis and endothecium cells
during anther development process. To gain a deeper
insight into the effects of MES treatment on these
processes, a comparative transcriptome analysis was
performed between male sterility and fertility plant leaves and
anthers. Functional analysis of the differentially expressed
genes revealed that the carbohydrate, cell wall, lipid
metabolism, and cellular transport processes were
enriched. Detailed expression of these genes was
analyzed also in leaves, small buds, and anthers from
middle buds. Carbohydrate content analysis further
confirmed the results of cytological observation and
MES treatment disturbes plastid and mitochondrion
functions, probably through acetolactate synthetase (ALS)
MES is an inhibitor of ALS , localized in the plastid/
chloroplast . ALS is universally expressed in plant
tissues, including leaves, seeds, young siliques, and
flower buds, but its highest expression level is in
mature pollen grains in Arabidopsis (Additional file 7A, B).
Co-expression analysis of Arabidopsis ALS (At3g48560)
revealed a network of 10 genes directly or indirectly
related to ALS (Additional file 7C): plastidic pyruvate kinase
beta subunit 1 (At5g52920), acetyl Co-enzyme a
carboxylase biotin carboxylase subunit (At5g35360), acetyl
Coenzyme a carboxylase carboxyltransferase alpha subunit
(At2g38040), ketol-acid reductoisomerase (At3g58610),
pyruvate dehydrogenase E1 alpha (At1g01090),
semialdehyde dehydrogenase family protein (At1g14810),
isopropylmalate dehydrogenase 2 (At1g80560),
adenylosuccinate synthase (At3g57610), transketolase family
protein (At2g34590), and pyruvate kinase family
protein (At3g22960). Most of these genes are expressed in
Figure 6 Comparison of carbohydrate content between mock-treated and MES-treated plants. Ls, young leaves from the main inflorescences;
SBs, small buds with length less than 1 mm; MBs, middle buds with length of 13 mm in. *, **, represents significant difference at 0.05 level and at 0.01
the plastid (six genes) or mitochondrion (three genes)
and are involved in five main pathways, including
biosynthesis of secondary metabolites; purine metabolism;
valine, leucine, and isoleucine biosynthesis, and two
carbohydrate-related pathways (pyruvate metabolism
and glycolysis/gluconeogenesis). In flowering plants,
plastids are the primary organelles that accumulate
carbohydrates and lipid compounds in the tapetum
 and perform essential metabolic functions in the
synthesis of lipid and secondary products . In the
present study, the MES-treated plants showed an
abnormal plastid development from the PMC stage, and
the plastid/chloroplastid structure exhibited obvious
defects in tapetal cells, epidermis, and endothecium
cells at the vacuolated-microspore stage (Figure 1). In
addition, subcellular localization analysis of the
differentially expressed genes showed that most of them
were localized in the plastid and mitochondrion
(Figure 4A). We proposed that MES treatment might
disturb the normal functions of the plastid and
mitochondrion, two functionally communicating organelles
in plant cells, through targeting ALS.
MES treatment influences carbohydrate and lipid
metabolism during anther development
In this study, TEM analysis showed substrate deficient
to form normal plastids containing starch or lipids in
male gametophyte cells, and an abnormal accumulation
of large starch granules in epidermis cells in the
MEStreated anthers at the vacuolated-microspore stage
(Figure 1). These results suggested that there was a
metabolism block during anther development process
in the MES-treated plants. Furthermore, functional
categories and pathway analysis of differentially expressed
genes related to anther development showed that
carbohydrate, cell wall, and lipid metabolism pathways were
significantly affected in the MES-treated plants (Figure 4C,
Figure 5, and Additional file 5). Detailed analysis of gene
expression alternation showed that starch and sucrose
metabolism-related genes (AGP, PGM, DPE1, and PHS2)
were down-regulated in young leaves of the main
inflorescences in the MES-treated plants. In addition, an
important gene (sweet protein 11, AT3G48740)
encoding a protein for sucrose phloem transport was also
down-regulated (Table 2, Additional file 5). In contrast,
several substrate transport-related genes (including
sugar, lipid, peptide, amino acid, and nitrate
transports) were up-regulated in the early development
stage anthers (SBs) of the MES-treated plants. Furthermore,
lipid biosynthesis related genes were down-regulated during
anther development process, companying with lipid
degradation related genes up-regulated (Additional file 5). This
was consistent with our cytological analysis that two
lipidstorage plastids, elaioplast and tapetosome, were impaired
at different degrees under MES treatment (Figure 1G-Q).
Therefore, carbohydrate transport from vegetative to
reproductive tissues was likely to be slightly suppressed at
early anther developmental stages, and carbohydrate and
lipid metabolism was abnormal in the MES-treated plants
Pollen wall consisting of intine and exine needs sugars
and lipids for its formation. Intine is secreted by the
microspore when released from the callose wall,
comprising cellulose, pectin, and various proteins .
Previous investigations indicated that at least three types of
cell wall-related enzymes functioned in pollen
development process, including beta-1,3-glucanase [32,33],
endocellulase [34,35], and polygalacturonase (PG) .
Several genes responsible for genic male sterility in B.
napus mutants were map-based cloned, and were found
to be lipid metabolism related genes [55-57]. It was
reported that the PG family protein was associated with
pollen intine development in B. campestris, and its
mutant displayed male sterility [49,50]. Recently, analysis of
gene expression profiles between genic male sterile
plants and their fertile counterparts in B. napus  and
cotton  by high-throughput digital gene expression
technique revealed that numerous genes involved in
starch and sucrose metabolism were also altered. These
suggested that expression alteration of the genes in
carbohydrate and lipid metabolism could result in male
sterility in plants. In this study, several pectin-related
genes were down-regulated in the developing anther of
the MES-treated plants. Furthermore, lipid-transfer
protein and cyclopropane-fatty-acyl-phospholipid synthase
(CAF, AT3G23510), associated with cell wall and
membrane biogenesis, were down-regulated. So both
carbohydrate and lipid nutrient were deficient in the
developing anthers of MES-treated plants, which might
contribute to rapeseed male sterility.
Furthermore, in this study, H(+)-ATPase 3 and H
(+)-ATPase 9 were down-regulated in Ls and An-MBs
(Table 2) from the MES-treated plants, and several other
ATPase-related genes were down regulated in An-LBs
(Additional file 5). We inferred that down regulation of
these ATPases might be one of the consequent effects
caused by MES treatment, which could also contribute
to rapeseed male sterility, because mitochondrial gene
rearrangements affecting ATP production have been
reported to be the reason for cytoplasmic male sterility
Taken together, carbohydrate and lipid metabolism
was blocked in the MES-treated rapeseed plants, which
may be the effect of MES treatment mainly responsible
for B. napus male sterility.
Similar action mode of CHA-MES as that of ALS inhibitor
herbicides but different organ effects
Till date, several ALS inhibitor herbicides, including
tribenuron-methyl, amidosulphuron, and
monosulfuronester sodium, have been found to induce complete male
sterility in rapeseed when applied at a concentration
below 1% of that required for their herbicidal activities
[6,8,11]. To investigate whether these ALS inhibitor
herbicides work as CHA in the same manner as herbicides,
we compared our results with previous reports on the
action mode of herbicides. When ALS inhibitors are
applied as herbicides, following the inhibition of ALS,
plants respond quickly to renew BCAAs level by
increasing protein turnover, so that the BCAA pool does not
decline to a level that would affect protein synthesis,
leading to an increase in the total free amino acid pool
[15-18,61]. This phenomenon was also observed in the
ALS inhibitor inducing male sterility plants . In
addition, a rapid increase in the level of carbohydrate in
leaves of plants treated with ALS inhibitors was reported
, and this effect was related to a decreased
photoassimilate translocation to sink tissues  due to a
decreased sink strength . In this study, we detected a
decrease in the content of soluble and reducing sugars
and a slight but not significant increase in starch content
in the leaves of the MES-treated plants. While in
flowering organs of the MES-treated plants, the soluble sugars
content was decreased at early stage (SBs) but increased
at late stage (MBs), starch content in both SBs and MBs
was continuously decreased. These results suggested that
carbohydrates translocation between vegetative and
reproductive organs was slightly blocked in the
MEStreated plants, but this seemed not to be the essential
reason for inducing male sterility. Because late increase
of soluble sugars content in MBs and continuous
decrease in starch content from SBs to MBs indicated that
the sugars did transport to reproductive organs from
leaves, but the assimilation of carbon in flowers was
weak, which might be contributed to male sterility.
Qian et al.(2011)  reported that imazethapyr (IM)
affected carbohydrate metabolism in chloroplasts,
including starch and other sugars, and IM treatment resulted in
the accumulation of glucose, maltose, and sucrose in the
cytoplasm or chloroplast and disturbed carbohydrate
utilization. They confirmed that metabolic pathways,
including amino acid metabolism, photosynthesis, starch
and sugar metabolism, and the tricarboxylic acid cycle
were altered in IM-treated rice . Manabe et al.(2007)
 confirmed that CSR1, the catalytic subunit of ALS,
was the sole target of imidazolinone herbicide in A.
thaliana using microarray analysis. Das et al. (2010) 
identified 478 genes significantly and coordinately regulated by
four ALS-inhibiting herbicides, including one
imidazolinone, one triazolopyrimidine, and two sulfonylureas at the
EC50 concentration. Among their 478 genes identified,
only 28 with the same AGI number were differentially
expressed in our data, functionally involved in
photosynthesis, OPP (oxidative pentose phosphate) cycle, TCA,
mitochondrial electron transport/ATP synthesis,
secondary metabolism and stress and so on. However, the
function categories influenced by ALS inhibitors used as
herbicides (Dass study) and CHA (our study) were similar,
though the specific genes affected were different. We
assumed that this was caused by two main reasons. First is
that different plant species and different tissues (organs)
were tested in both studies. In our study, we used
rapeseed flower buds, anthers, and leaves from the main
inflorescences, including vegetative and reproductive tissues,
whereas, in Dass study, they used leaves of 14-day-old
seedlings of A. thaliana as experimental materials. Second
is the very different concentration of ALS inhibitor used
in the two studies. We treated rapeseed plants with
0.24 g ha1 MES (approximately 1% of MES concentration
required to control broadleaf weeds in wheat field) for
inducing male sterility. However, Das et al. (2010) 
treated A. thaliana plants with four ALS-inhibiting
herbicides at their EC50 concentrations, ranging from
0.131 g ha1 for sulfometuron-methyl to 0.586 g ha1
Taken together, the general action mode of ALS
inhibitor herbicides on plants was seemly the same
whether they were used as herbicides or as CHAs at very
low concentration. However, why vegetative and
reproductive organs exhibited different responses (vegetative
normal but reproductive male sterility) to the ALS
inhibitor MES using as CHA at low concentration?
Previous study indicated that ALS expression level is the
highest in mature pollen grains in Arabidopsis (Additional
file 7), so the developing anthers might be more
sensitive to MES treatment than other vegetative tissues
(organs). We speculated that two aspect effects might
contribute to this phenomenon. Firstly, the ALS
inhibition might immediately affect synthesis of some anther
specific BCAAs-enriched enzymes or proteins, or
facilitate the degradation of BCAAs-enriched enzymes or
proteins to renew BACCs levels, considering that the
free amino acids content was higher in ALS inhibitor
inducing male sterility plant anthers than in the control
plant anthers , and several genes involved in protein
degradation were up-regulated in An-MBs in this study
(Additional file 5). Secondly, when ALS was inhibited in
developing anthers, large amount of the substrate
(pyruvate and 2-ketobutyric acid) might be accumulated and
then might disturb other metabolism pathways such as
carbohydrate and lipid metabolism through metabolism
reprogramming, which was essential for anther
development. Since carbon skeletons are necessary to synthesis
several fundamental materials including amino acids, fatty
acids, and secondary metabolites. Thus, low concentration
of MES induces specifically male sterility, but has no
serious effect on vegetative tissues (organs). However, all
these inferences need further experiments to verify.
Putative action of MES-induced male sterility in B. napus
Combining with ultrastructural cytological observation,
systematic comparative transcriptome analysis,and
metabolic analysis, we updated the simple action model of
CHA-MES inducing male sterility in rapeseed, which
was proposed in our previous investigation  (Figure 7).
In this study, we speculated that, following the inhibition
of ALS, two aspect of effects might be caused in
development anthers by MES treatment at low concentration:
One was proteolysis of some essential enzymes related
with carbohydrate and lipid metabolism to renew BCAAs
level; another was accumulation of pyruvate, this
disturbing the normal level of carbon skeleton substrates in
plastids/chloroplasts and leading to pyruvate diverting to the
closely related metabolism pathways such as carbohydrate
and lipid metabolism. Therefore, the metabolism of
carbohydrate and lipids, two types of macromolecules playing
crucial roles during anther development,was blocked
during anther development process in the MES-treated
plants. Furthermore, mitochondria were functionally
disturbed by MES treatment at low concentration based on
our comparative transcriptome analysis. This organelle
was functionally coupled with the plastid/chloroplast and
supplies with metabolism substrates and energy for plant
development. Taken together, carbohydrate and lipid
metabolism blocks in the development anthers of the
MEStreated rapeseed plants might be mainly responsible for B.
napus male sterility induced by MES treatment, along
with energy deficiency and perturbed network regulation.
In leaves, though several genes involved in starch and
sucrose metabolism were detected to be down-regulated in
the MES-treated plants, and the content of soluble sugars
decreased and starch content slightly increased, vegetative
tissues of the MES-treated plants exhibited no obvious
difference to those of the mock-treated plants. However,
how the inhibition of ALS in anthers actually affected
carbohydrate and lipid metabolism in development
anthers remains to be further study.
This study carried out a systematic analysis of
effects caused by CHA-MES treatment at ultrastructure,
Figure 7 A putative action model for MES-treatment inducing male sterility. Some important functions and genes affected by MES
treatment in leaf tissue (left rectangle) and developing anther tissues (right rectangle) are listed (see text for details). Two vertical dashed lines in
the right rectangle separate three anther tissues. after function categories or genes means up-regulation; after function categories or genes
means down-regulation. Two aspects of putative reasons for carbohydrate and lipid metabolism alteration were showed by dashed arrows ?
represents unclear MES transport pathway. MES, Monosulfuron Ester Sodium; ALS, acetolactate synthase; BCAAs: Branch-Chain Amino Acids; AGP,
ADP glucose pyrophosphorylase; PMG, Phosphoglucomutase; DPE1, Disproportionating enzyme; PHS2, alpha-glucan phosphorylase 2; SWEET 11,
Nodulin MtN3 family protein; BXL1, beta-xylosidase; pectin lyases, pectin lyase superfamily protein; VGDH2, VANGUARD 1 homolog 2; PPME1:
Pectin lyase-like superfamily protein; FLA5: FASCICLIN-like arabinogalactan protein 5 precursor; VGDH1: Plant invertase/pectin methylesterase
inhibitor; VGD1: Plant invertase/pectin methylesterase inhibitor; PGA4, Polygalacturonase 4; UGE3, UDP-D-glucose/UDP-D-galactose 4-epimerase 3;
HA9, H(+)-ATPase 9.
transcriptome, and physiological levels, which revealed
that the carbohydrate and lipid metabolism was altered in
rapeseed male sterility plants induced by MES treatment
at low concentration. Accordingly, we proposed a simple
action model for CHA-MES inducing male sterility in B.
napus. These results will provide some clues to the
mechanism of MES inducing male sterility, and give insights
into the complex gene regulation network during anther
development. Besides, these results might provide more
potential targets for developing new male sterility
inducing CHAs and for genetic manipulation in rapeseed
Plant material and experimental setup for MES treatment
The rapeseed cultivar Zhongshuang No.9, developed by
the Oil Crops Research Institute of Chinese Academy of
Agricultural Sciences (Wuhan, China) and selfed for
eight generations before being used in the present
experiment, was planted in the experimental field of
Northwest A&F University, Yangling, Shaanxi, China
(longitude 108E, latitude 3415N) during a natural
growth season on 23rd September 2009. Optimal
agronomic practices were followed.
The experimental plot contained approximately 2,400
plants grown in 120 rows (2-m long each), with a space
of 50 cm between rows and 10 cm between plants within
a row. When the rapeseed plants were at the bolting
stage with the longest floral bud being 2 mm, the plot
was divided into two groups: MES-treated group and
mock-treated group, each containing 60 rows. MES was
kindly provided by Professor Zhengming Li of NanKai
University, Tianjin, China. The plants of MES-treated
group were foliar sprayed with 0.1 g mL1 MES
solution containing 50 ppm DMF and 5 ppm Tween 80 for
approximately 15 mL per plant (approximately 1% of the
concentration that is required for its herbicide action in
wheat field to control broadleaf weeds) for inducing
male sterility during the entire flowering period without
affecting the growth and development of other rapeseed
plants tissues. Meanwhile, the plants of the
mocktreated group were foliar sprayed with the same amount
of solution only containing 50 ppm DMF and 5 ppm
Tween 80 as the control.
The protocol used for cytological studies was described
in the previous report . In brief, when the fertility of
the first opened flower of each MES-treated plant was
visually detectable for male sterility, the main
inflorescences of uniform plants in the MES-treated and
mocktreated groups were collected into plastic bags and
quickly transported to the laboratory on ice.
Acetocarmine staining was performed to examine the correlation
of the microspore developmental stage with the bud
length. Bud samples of the MES-treated and
mocktreated plants at different microspore developmental
stages were treated according to Gonzlez-Melendi et al.
(2008)  for cytological observation. After treatment,
the specimens were sectioned with Ultramicrotome
Leica EM UC7 (Leica Microsystems, Germany).
Ultrathin sections (70 nm) were observed and photographed
with a transmission electron microscope (JEM-1230,
JEOl, Tokyo, Japan) on 600 mesh formvar-coated copper
Plant sample collection for microarray study
Plant sample collection for microarray study was the
same as that for the previous proteomic study . In
brief, based on cytological observation results of
acetocarmine staining, the collected inflorescence samples of
the MES-treated and mock-treated groups were
classified into three subgroups according to their bud length,
namely small buds (SBs) with length below 1 mm
(before and during the pollen mother cell (PMC) stage),
medium buds (MBs) with 13 mm in length (from
meiosis to the early-uninucleate-microspore stage), and
large buds (LBs) with length over 3 mm (from the
vacuolated-microspore to the mature-pollen stages). In
the MB and LB subgroups, anthers were dissected from
the buds, designed as An-MBs and An-LBs, respectively.
Young leaves (Ls) from the main inflorescences of the
MES-treated or mock-treated plants were also collected
as vegetative tissue control. All samples were prepared
on ice, immediately frozen in liquid nitrogen and then
stored at 80C for later use. Mixture samples collected
from every 20 rows of the MES-treated or mock-treated
plants were used as one biological replicate, and three
independent biological replicates were then prepared for
Microarray experiment and data acquisition
The Agilent Single Channel Brassica Oligo Microarray
(4 44 K) was used in this study; the chip contains
43,803 probe sets designed on the basis of ESTs of B.
napus, mRNAs, and predicted gene sequences from
databases such as NCBI, TIGRI, and UniGene. Total RNAs
of 24 samples, four pair tissues from the MES-treated
and mock-treated plants with three biological replicates,
were extracted using TRIzol reagent (Invitrogen Life
Technologies, Carlsbad, CA, US) and purified using
the QIAGEN RNeasy Mini Kit (QIAGEN, GmBH,
Germany). In total, 1.65 g cRNA was used for
hybridization, and washing, staining, and scanning
were performed according to instructions. Three
independent biological replicates were included in each
microarray experiment. The hybridization signals were
normalised by Quantile algorithm  using Gene
Spring Software 11.0 (Agilent technologies, Santa
Clara, CA, US) and log2 transformed.
For the identification of DETs involved in
microgametogenesis between the MES-treated and mock-treated
groups, two sets of Students t-test comparisons were
performed (Figure 3). First, comparisons within groups,
named vertical comparisons, were performed. Pairwise
comparisons of Students t-test between tissues (organs)
were performed within the mock-treated group and
MES-treated group to detect DETs related to anther
development under mock treatment (control) (fertile)
and MES treatment (male sterile) conditions (Figure 3A).
Second, comparisons between groups, named horizontal
comparisons, were performed. These set of comparisons
were performed in the four pairs of corresponding
tissues (organs) between the MES-treated and
mocktreated groups to identify DETs related to MES
treatment (Figure 3B). The results of all these comparisons
were filtered with the constraint of fold change 2 and
p-value 0.001. To focus on genes presumably related to
anther development, which are influenced by MES
treatment, the common DETs in two sets of comparisons
(the red and green parts in Figure 3C) were considered
to be anther development-related genes affected by MES
treatment. Microarray data were deposited to the
database of the National Center for Biotechnology
Information (NCBI) with the accession number GSE53468
Annotation and functional analysis
The identified differently expressed transcripts (DETs) of
B. napus were annotated by BLASTN against TAIR
(http://www.arabidopsis.org/Blast/index.jsp) in the present
study. The unigenes (AGI identifers) with BLASTN
expectation values (E-values) <105  were used to
annotate the target transcripts. Subsequently, the DETs with
AGI identifers were used for further functional analysis.
To categorize differentially expressed genes based on
their subcellular localization and biological functions,
the rapeseed DETs with unique AGIs were submitted to
the Munich Information Center for Protein Sequences
(MIPS) catalogue of A. thaliana genome . In addition,
pathway visualization and analysis was performed
using MapMan . Furthermore, to obtain more cell
wall biogenesis and lipid metabolism related genes, the
differentially expressed genes with AGIs were also
compared to Cell Wall Genomics database (http://
The Arabidopsis Lipid Gene Database (http://lipids.
Data validation by quantitative real-time PCR (qRT-PCR)
To confirm the differential expression pattern of DETs
detected in the microarray experiments, qRT-PCR
analyses were performed. Gene-specific primers were
designed according to the reference unigene sequences
(Additional file 8). Total RNAs were isolated using
TRIzol reagent from the same plant samples as those used
in the above mentioned microarray experiment. For each
sample, cDNA was generated from 1 mg of total RNA
using the MMLV Reverse Transcriptase TIANScript RT
Kit (TIANGEN, China) according to instructions. The B.
napus -actin (accession no. AF111812.1) gene was used
as a reference , and the relative gene expression
levels were calculated using the2Ct method . The
Carbohydrate content analysis
To determine the composition changes in carbohydrate,
such as soluble sugars, sucrose, and starch contents in
both MES-treated and mock-treated groups, we analyzed
three independent biological replicates for each tissue.
The sugar content was measured according to Dorion
et al. (1996) . 1.03.0 g (fresh weight) of each
tissue tested was used for sugar extraction and for starch
content analysis. Total soluble sugars and reducing
sugars were determined using the anthrone method
 and 3,5-dinitrosalicylic acid method , respectively.
The difference between the total soluble sugars content
and the reducing sugars content was the amount of
nonreducing sugars, which was recognized to be sucrose. The
starch content was determined by hydrolyzing it to soluble
sugars and calculated .
Additional file 1: Correlation coefficients between the three
biological replicates of 24 samples.
Additional file 4: Annotation result of the 1501 differentially
expressed transcripts (DETs) according to Arabidopsis Information
Additional file 5: Functional pathways of significantly differentially
regulated genes assigned by MapMan.
Additional file 6: Carbohydrate metabolism, cell wall formation,
lipid metabolism, and cellular transport related genes influenced by
Additional file 8: Primers for quantitative real time PCR (qRT-PCR).
SH and HZ conceived and designed the experiments. ZL, YC, and JC
performed the experiments. ZL and PZ analysed the data. ZL, SH, and HZ
wrote the manuscript. All authors read and approved the final manuscript.
We express deep gratitude to Professor Zhengming Li of Nankai Univeristy,
Tianjin, China for providing the monosulfuron ester sodium, an ALS inhibitor
herbicide. This work was supported by the earmarked fund for China
Agriculture Research System [CARS-13], the National Key Technology R&D
Program [2010BAD01B02] and a grant of Northwest A&F University to SW
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