BACKGROUND
in plants, the hybrid f <dig> progeny usually exhibits heterosis  relative to the inbred parents  <cit> .
accordingly, the productivity of many crops has been boosted by introducing hybrid varieties  <cit> .
an effective pollination control system is a prerequisite for heterosis utilization.
in  <dig>  it was reported that the plant growth regulator maleic hydrazide can induce male sterility in corn plants  <cit> .
this initial finding led to the induction of male sterility by a chemical hybridization agent , 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  and nuclear male sterility .
in addition, chas enable breeders to develop hybrids with a higher heterosis level in a shorter time  <cit> .
the technique is now widely used in crops heterosis, particularly in rapeseed   <cit> .
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  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  <cit> .
in the herbicide field, sulfonylurea is well known for its eco-friendly, extreme low toxicity towards mammals, and ultralow dosage application  <cit> .
mes is a new sulfonylurea herbicide that inhibits acetolactate synthase , an enzyme in the first step of the branched-chain amino acids  biosynthesis pathway  <cit> .
plant alss are encoded by nuclear genes, and their n-terminal signal peptide sequence is required for translocating the protein to the chloroplast  <cit> .
in addition, als is the target of four other classes of herbicides in addition to the sulfonylurea class, including triazolopyrimidines, pyrimidinylthiobenzoates, sulfonylamino-carbonyltriazolinones, and imidazolinones  <cit> .
several als inhibitor herbicides are exploited as chas in crop breeding  <cit> .
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   <cit> ; increase in free amino acid pool likely through protein turnover  <cit> ; a rapid accumulation of carbohydrate in leaves  <cit>  related to decreased photoassimilate translocation to sink tissues  <cit>  due to a decreased sink strength  <cit> ; and induction of fermentative metabolism  <cit> .
two other studies reported genome-wide gene expression responses to different als-inhibitor herbicides in arabidopsis thaliana using the affymetrix ath <dig> microarray  <cit> .
till date, very few studies were carried out to investigate the mechanism of als inhibitor chas inducing male sterility  <cit> .
in flowering plants, the development of the male gametophyte occurs in the anther, and it is a well-programmed and elaborate process  <cit> .
in arabidopsis, anther development consists of two phases divided into  <dig> stages  <cit> .
during phase i, from stage  <dig> to  <dig>  the four lobes of the anther are formed, each containing reproductive 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  <cit> .
the developing pollen is immersed in locular fluid containing nutrients such as sugars and lipids from the sporophytic  tissue tapetum  <cit> .
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  <cit> .
during phase ii, microspores undergo meiosis to form the tetrads enclosed in a thick shell composed of a callose  wall and a pollen mother cell  wall composed of cellulose, hemicelluloses, and pectins  <cit> .
the timely degradation of the callose and pmc walls is critical for microspore release from the tetrads  <cit> .
at least three cell wall enzymes are involved in this process, including β- <dig> -glucanase  <cit> , endocellulase  <cit> , and polygalacturonase   <cit> .
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  <cit> .
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  <cit> .
sporopollenin, the major component of exine, is a complex polymer primarily composed of fatty acids and phenolic compounds  <cit> .
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  <cit> .
to better understand the mechanism how als inhibitor chas induced male sterility in rapeseed, we treated the rapeseed plants at the bolting stage with  <dig>  μg ml− <dig> 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.
RESULTS
cytological studies reveal that mes treatment affects the plastid ultrastructure and metabolite accumulation in the developing anthers
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  <cit> .
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 .
at the pmc stage, numerous plastids are dispersed in the cytoplasm of pmcs and tapetal cells in the mock-treated plants .
however, serious plasmolysis in pmcs and slight plasmolysis in tapetal cells were observed in the mes-treated male sterile plants , 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 .
besides, another type of plastids located in a crown, started to accumulate low electron-dense material and was surrounded by the rich er .
in contrast, in the mes-treated sterile plants, two types of abnormal tapetum were observed, as shown by cheng et al.
<cit> .
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 .
type i abnormal tapetum was degraded and released noncompact elaioplasts and low electron-dense tapetosomes .
at the mature-pollen stage, the fertile pollen grains showed profuse globular particles ; however, the sterile pollen grains were almost empty, type ii tapetum still showed an intact and visible tapetal cell wall , and type i tapetum showed solidified bulks .figure  <dig>
transmission electron microscope  micrographs of the anthers from the mock-treated  and mes-treated  plants.
the fertile anthers at pollen mother cell  stage;  enlarged fertile meiocytes in ; and  enlarged fertile tapetum in  showing numerous plastids dispersed in cytoplasm .
the sterile anthers at pmc stage;  enlarged sterile meiocytes in  showing less plastids in condensed cytoplasm separated from the cell wall;  enlarged sterile tapetum in  showing little abnormal plastids  and more large vacuoles in cytoplasm, and with a little plasmolysis at meiocyte side .
the fertile anthers at vacuolated-microspore stage;  the degraded tapetum in  showing elaioplasts and tapetsomes with abundant lipids;  plastids in tapetum located in a crown showing filled with globular low electron-dense metabolites and surrounded by rich endoplasmic reticulum .
the sterile anthers at vacuolated-microspore stage ;  the undegraded tapetum in  showing elaioplasts and tapetsomes with abundant lipids;  plastids in tapetum located in a crown showing irregular shaped low electron-dense material.
the sterile anthers at vacuolated-microspore stage ;  the degraded tapetum in  showing scattered elaioplasts and tapetsomes with fuzzy structure;  the fertile anthers at mature pollen grain stage;  the pollen grain in  showing profuse globular particles;  the enlarged globular particles in .
the sterile anthers at mature pollen grain stage ;  the undegraded tapetum in  died but cell wall still existed ;  the sterile anthers at mature pollen grain stage .
the epidermis and endothecium cells in fertile plants at vacuolated-microspore stage;  the epidermis cells in  showing normal oval-shaped chloroplastids with distinct thylakoid structure and little starch granules in thylakoid;  the endothecium cells in  showing oval-shaped chloroplastids with distinct thylakoid structure.
the epidermis and endothecium cells in sterile plants at vacuolated-microspore stage;  the epidermis cells in  showing abnormal chloroplastids with large starch granules in thylakoid;  the endothecium cells in  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 μm , 5 μm , 2 μm , and 1 μm .
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 ; 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 .
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  was used.
three independent biological replicates of four pairs of tissues  from the mes-treated and mock-treated plants were collected for gene expression analysis, resulting in a dataset of  <dig> microarrays.
the four tissues   included the leaves from main inflorescences , the small buds less than 1 mm in length containing microgametocytes before and during pollen mother stage , the anthers from middle buds with length between 1 mm and 3 mm containing microgametocytes from meiosis to early uninucleate microspore stage , and the anthers from large buds more than 3 mm in length containing microgametocytes from vacuolated stage to mature pollen stage .
the data quality was assessed using two measurements:  correlation coefficients between biological replicates, which ranged from  <dig>  to  <dig> , with a mean of  <dig>  , and  quantitative real time rt-pcr , which was performed on  <dig> randomly selected genes.
the results of qrt-pct analysis showed a high degree of concordance  with microarray results .
taken together, these demonstrated that the microarray results obtained in this study were reliable.figure  <dig>
photographs of the leaves and developmental anthers for microarrays from the mock-treated  and mes-treated  plants.
a-b, young leaves from the main inflorescences ; c-d, small buds ; e-f, anthers from middle buds ; g-h, anthers form large buds .
scale bar in leaves was  <dig> cm, scale bars in sbs, an-mbs, and an-lbs were  <dig> mm.
to identify differentially expressed transcripts  between the mes-treated and mock-treated groups involved in microgametogenesis, two sets of student’s t-test comparisons were performed .
firstly, vertical comparisons  were conducted to identify dets involved in anther development,  <dig>  and  <dig>  dets being identified within the mock-treated group and mes-treated group, respectively .
secondly, horizontal comparisons  were conducted to identify dets caused by mes-treatment in the tissues  tested,  <dig> up-regulated and  <dig> down-regulated transcripts being identified in the four-pair tissues  .
the common dets identified in both the vertical and horizontal comparisons described above were considered as anther development related genes affected by mes treatment .
therefore, 102 + 108 +  <dig> and 31 + 84 +  <dig> dets  corresponding to  <dig> unique dets  were selected for further analysis in this study, in order to reveal the alteration of gene expressions induced by mes treatment in the tissues  tested.
distribution of these  <dig> dets indicated that over 64%  were down-regulated and only 36%  were up-regulated in at least one tissue  in the mes-treated plants .
in addition, small fractions of these dets were in ls and sbs  with 2 ~ 5-fold change , while the majority were found in an-mbs and an-lbs  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.figure  <dig>
strategies for identification of differentially expressed transcripts  involved in microgametogenesis between the mes-treated and mock-treated plants by two sets of student’s t-test comparisons.
comparisons within groups.
the pair-wise comparisons of student’s t-test between tissues  were carried out within mock-treatment groups and mes-treatment groups, respectively, to detected dets related to anther development under mock-treatment  and mes-treatment  conditions.
the criteria for screening dets were p-value < <dig>  and fold change ≥  <dig>  mock, mock-treatment; mes, mes-treatment;  comparisons between the mes-treated and mock-treated groups.
the pair-wise comparisons of student’s t-test were performed between the corresponding tissues  of the mock-treated group and the mes-treated group to identify dets related to mes-treatment.
the screen criteria were same as above.
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 .
comparisons between groups also produced two sets of dets, up-regulated genes and down-regulated genes in mes-treated group .
these four sets of dets were all collected, respectively.
the common sections  indicates  <dig> dets existing in the both data sets) were considered to be anther development-related genes affected by mes-treatment.
distribution of differentially expressed transcripts  in mes-treated rapeseed plants
fold change
ls
sbs
an-mbs
an-lbs
total unique
up-regulated
2 ~ 5
5 ~ 10
≥10
total-up
down-regulated
2 ~ 5
5 ~ 10
≥10
total-down
total
*two dets  existed both in the up-regulated and down-regulated probe sets.
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 , 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  between these two species  <cit> .
of the  <dig> dets,  <dig>  were highly similar to  <dig> a. thaliana genes   and could be thus annotated.
the remaining  <dig> dets did not find a close orthologue in the tair database and therefore was not be annotated .
the localization of these dets might provide clues to where they function.
to gain insight into the biological functions of these  <dig> annotated unigenes, subcellular localization and functional category analysis were conducted according to the information from munich information center for protein sequences  .
the  <dig> unigenes could be distributed into  <dig> subcellular localizations, with the largest three categories being the plastid/chloroplast , mitochondrion , and nucleus , followed by the eukaryotic plasma membrane , cell wall , and cytoplasm  .
furthermore, the  <dig> unigenes were classified into  <dig> 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 .
in order to understand the functions of dets in different tissues, we performed the category enrichment analysis in four tissues separately .
the results of the functional category  and enrichment analysis  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., won’t be given further consideration in this study.
furthermore, detailed subcategory analysis of metabolism revealed that carbohydrate metabolism and lipid metabolism were significantly enriched , 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 .figure  <dig>
subcellular localization and functional categories of the  <dig> differentially expressed unigenes between the mes-treated and the mock-treated rapeseed plants.
subcellular localization,  functional categories,  enrichment analysis of the functional categories list in b. the scale bar indicates -log , 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 , respectively.
the prominent enriched sub-categories were circled in black ellipses.
furthermore, to get the overview of the pathways where the dets are taking part in, the  <dig> unigenes described above were further analyzed by mapman software.
the biotic stress and metabolism pathway visualization are shown in figure  <dig>  in the biotic stress visualization , 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  <dig> of the genes in the signaling pathway belonged to the protein kinase signaling, and  <dig> 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 .
in proteolysis pathway,  <dig> of  <dig> protein degradation related genes were coding for the 26s proteasome complex, which mediates ubiquitin-dependent protein degradation.
interestingly, eight of the  <dig> protein degradation related genes were up-regulated in an-mbs, while most of the others were down-regulated in an-lbs .
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 , 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 .
interestingly, the few genes involved in amino acid metabolism were mainly up-regulated in an-lbs .
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.figure  <dig>
transcripts involved in stress  and metabolism  assigned by mapman in rapeseed leaves and developmental anthers treated by mes.
positive fold change values  indicate up-regulation, whereas negative fold change values  denote down-regulation.
color saturates at  <dig> -fold change.
each square represents a differentially expressed transcript.
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 , 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 .
four genes involved in starch biosynthesis and degradation pathway, including adp glucose pyrophosphorylase , phosphoglucomutase , disproportionating enzyme , and alpha-glucan phosphorylase  <dig> , were down-regulated 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  <cit> .
pgm plays a pivotal role in the allocation of carbon between polysaccharide formation and energy production, and it is located in plastid  <cit> .
dpe <dig> and phs <dig> are enzymes catalyzing the breakdown of starch into maltose and glucose in the chloroplast at night  <cit> .
in addition, another carbohydrate metabolism-related gene , encoding transketolase in calvin cycle, was down-regulated by  <dig> -fold .
importantly, a vital gene encoding sweet protein  <dig>  was down-regulated by approximately 3-fold, which has been recently identified to mediate sucrose efflux in leaves as a key step for phloem transport  <cit> .
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.table  <dig>
representative differentially expressed genes involved in carbohydrate and lipid metabolism in ls, sbs, and an-mbs
probename
agi
symble
short description
location*
fold change
ls
sbs
an-mbs
c-compound and carbohydrate metabolism
cell wall
lipid, fatty acid and isoprenoid metabolism
*the localization information was from mips  website.
fold change was calculated between mes-treated plants and mock-treated plants; ls, sbs, and an-mbs represents leaves, small buds, and anthers of middle buds, respectively; “-” represents that the gene was not significantly and differentially expressed in the specific tissue between the mes-treated and mock-treated plants.
all the carbohydrate, cell wall, and lipid metabolism, and cellular transport related genes identified in this study were deposited in additional file  <dig>
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 .
two genes related to cell wall metabolism, bxl <dig>  and pectin lyase-like superfamily protein  were down-regulated by  <dig> -fold and  <dig> -fold, respectively .
the latter gene exhibits polygalacturonase  activity in cell wall metabolism and is highly expressed at early stages of flower development, which is essential for anther development  <cit> .
in addition, expression of several lipid metabolism-related genes was altered, two of these genes being down-regulated and three up-regulated .
kat <dig> , a thiolase, was down-regulated by  <dig> -fold .
this gene is strongly expressed during flower development in arabidopsis and can partially complements kat <dig>  which mutant exhibited partly male sterility  <cit> .
in addition, one lipid transport gene  and two lipid degradation genes  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 .
most of the cell wall-related genes were significantly down-regulated, including cell wall precursor synthesis genes fla <dig> and uge <dig>  and pectin metabolism genes, such as polygalacturonase  <dig>  the pectate lyase family protein, plant invertase/pectin methylesterase inhibitor superfamily, and vanguard  <dig>  previous studies revealed that pectin metabolism-related genes played important roles during late stages of pollen development  <cit> .
besides, another cell wall related-gene, uge <dig>  was down-regulated by  <dig> -fold .
it is reported that uge <dig> together with uge <dig> affected pollen development  <cit> .
in addition, several lipid metabolism-related genes were altered in the an-mbs of the mes-treated plants, two  of them involved in lipid biosynthesis were down regulated, and three  involved in lipid degradation were up regulated .
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 slightly repressed.
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 .
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 .
however, energy metabolism-related genes about h+-exporting atpase such as h-atpase  <dig> and h-atpase  <dig> were significantly down-regulated in the mes-treated plant anthers at late stage .
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 .
compared with the mock-treated 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 mes-treated 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 mock-treated 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 .figure  <dig>
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 1–3 mm in.
*, **, represents significant difference at  <dig>  level and at  <dig>  level, respectively.
discussion
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 mes-treated 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 transcriptome analysis.
mes treatment disturbes plastid and mitochondrion functions, probably through acetolactate synthetase
mes is an inhibitor of als  <cit> , localized in the plastid/chloroplast  <cit> .
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 .
co-expression analysis of arabidopsis als  revealed a network of  <dig> genes directly or indirectly related to als : plastidic pyruvate kinase beta subunit  <dig> , acetyl co-enzyme a carboxylase biotin carboxylase subunit , acetyl co-enzyme a carboxylase carboxyltransferase alpha subunit , ketol-acid reductoisomerase , pyruvate dehydrogenase e <dig> alpha , semialdehyde dehydrogenase family protein , isopropylmalate dehydrogenase  <dig> , adenylosuccinate synthase , transketolase family protein , and pyruvate kinase family protein .
most of these genes are expressed in the plastid  or mitochondrion  and are involved in five main pathways, including biosynthesis of secondary metabolites; purine metabolism; valine, leucine, and isoleucine biosynthesis, and two carbohydrate-related pathways .
in flowering plants, plastids are the primary organelles that accumulate carbohydrates and lipid compounds in the tapetum  <cit>  and perform essential metabolic functions in the synthesis of lipid and secondary products  <cit> .
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 .
in addition, subcellular localization analysis of the differentially expressed genes showed that most of them were localized in the plastid and mitochondrion .
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 mes-treated anthers at the vacuolated-microspore stage .
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 .
detailed analysis of gene expression alternation showed that starch and sucrose metabolism-related genes  were down-regulated in young leaves of the main inflorescences in the mes-treated plants.
in addition, an important gene  encoding a protein for sucrose phloem transport was also down-regulated .
in contrast, several substrate transport-related genes  were up-regulated in the early development stage anthers  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 .
this was consistent with our cytological analysis that two lipid-storage plastids, elaioplast and tapetosome, were impaired at different degrees under mes treatment .
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 anthers.
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  <cit> .
previous investigations indicated that at least three types of cell wall-related enzymes functioned in pollen development process, including beta- <dig> -glucanase  <cit> , endocellulase  <cit> , and polygalacturonase   <cit> .
several genes responsible for genic male sterility in b. napus mutants were map-based cloned, and were found to be lipid metabolism related genes  <cit> .
it was reported that the pg family protein was associated with pollen intine development in b. campestris, and its mutant displayed male sterility  <cit> .
recently, analysis of gene expression profiles between genic male sterile plants and their fertile counterparts in b. napus  <cit>  and cotton  <cit>  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 , 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  <dig> and h-atpase  <dig> were down-regulated in ls and an-mbs  from the mes-treated plants, and several other atpase-related genes were down regulated in an-lbs .
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   <cit> .
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 monosulfuron-ester 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  <cit> .
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  <cit> .
this phenomenon was also observed in the als inhibitor inducing male sterility plants  <cit> .
in addition, a rapid increase in the level of carbohydrate in leaves of plants treated with als inhibitors was reported  <cit> , and this effect was related to a decreased photoassimilate translocation to sink tissues  <cit>  due to a decreased sink strength  <cit> .
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  but increased at late stage , 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 mes-treated 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.
<cit>  reported that imazethapyr  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  <cit> .
manabe et al.
<cit>  confirmed that csr <dig>  the catalytic subunit of als, was the sole target of imidazolinone herbicide in a. thaliana using microarray analysis.
das et al.
<cit>  identified  <dig> genes significantly and coordinately regulated by four als-inhibiting herbicides, including one imidazolinone, one triazolopyrimidine, and two sulfonylureas at the ec <dig> concentration.
among their  <dig> genes identified, only  <dig> with the same agi number were differentially expressed in our data, functionally involved in photosynthesis, opp  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  and cha  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  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 das’s 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  <dig>  g ha− <dig> mes  for inducing male sterility.
however, das et al.
<cit>  treated a. thaliana plants with four als-inhibiting herbicides at their ec <dig> concentrations, ranging from  <dig>  g ha− <dig> for sulfometuron-methyl to  <dig>  g ha− <dig> for primisulfuron-methyl.
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  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 , so the developing anthers might be more sensitive to mes treatment than other vegetative tissues .
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  <cit> , and several genes involved in protein degradation were up-regulated in an-mbs in this study .
secondly, when als was inhibited in developing anthers, large amount of the substrate  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 .
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  <cit>  .
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 mes-treated 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.figure  <dig>
a putative action model for mes-treatment inducing male sterility.
some important functions and genes affected by mes treatment in leaf tissue  and developing anther tissues  are listed .
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; dpe <dig>  disproportionating enzyme; phs <dig>  alpha-glucan phosphorylase 2; sweet  <dig>  nodulin mtn <dig> family protein; bxl <dig>  beta-xylosidase; pectin lyases, pectin lyase superfamily protein; vgdh <dig>  vanguard  <dig> homolog 2; ppme1: pectin lyase-like superfamily protein; fla5: fasciclin-like arabinogalactan protein  <dig> precursor; vgdh1: plant invertase/pectin methylesterase inhibitor; vgd1: plant invertase/pectin methylesterase inhibitor; pga <dig>  polygalacturonase 4; uge <dig>  udp-d-glucose/udp-d-galactose 4-epimerase 3; ha <dig>  h-atpase  <dig>
CONCLUSIONS
this study carried out a systematic analysis of effects caused by cha-mes treatment at ultrastructure, 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 breeding.

 in plants, the hybrid f progeny usually exhibits heterosis relative to the inbred parents. 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 it was reported that the plant growth regulator maleic hydrazide can induce male sterility in corn plants. this initial finding led to the induction of male sterility by a chemical hybridization agent, 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 and nuclear male sterility. 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. 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 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, an enzyme in the first step of the branched-chain amino acids biosynthesis pathway. plant alss are encoded by nuclear genes, and their n-terminal 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 triazolopyrimidines, pyrimidinylthiobenzoates, sulfonylamino-carbonyltriazolinones, 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; increase in free amino acid pool likely through protein turnover; 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. two other studies reported genome-wide gene expression responses to different als-inhibitor herbicides in arabidopsis thaliana using the affymetrix ath microarray. 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 well-programmed and elaborate process. in arabidopsis, anther development consists of two phases divided into stages. during phase i, from stage to the four lobes of the anther are formed, each containing reproductive 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. the developing pollen is immersed in locular fluid containing nutrients such as sugars and lipids from the sporophytic 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 wall and a pollen mother cell 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 β- -glucanase, endocellulase, and polygalacturonase. 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  μg ml− 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 developing anthers 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. at the pmc stage, numerous plastids are dispersed in the cytoplasm of pmcs and tapetal cells in the mock-treated plants. however, serious plasmolysis in pmcs and slight plasmolysis in tapetal cells were observed in the mes-treated male sterile plants, 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. besides, another type of plastids located in a crown, started to accumulate low electron-dense material and was surrounded by the rich er. in contrast, in the mes-treated sterile plants, two types of abnormal tapetum were observed, as shown by cheng et al.. 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. type i abnormal tapetum was degraded and released noncompact elaioplasts and low electron-dense tapetosomes. at the mature-pollen stage, the fertile pollen grains showed profuse globular particles; however, the sterile pollen grains were almost empty, type ii tapetum still showed an intact and visible tapetal cell wall, and type i tapetum showed solidified bulks.figure transmission electron microscope micrographs of the anthers from the mock-treated and mes-treated plants. the fertile anthers at pollen mother cell stage; enlarged fertile meiocytes in; and enlarged fertile tapetum in showing numerous plastids dispersed in cytoplasm. the sterile anthers at pmc stage; enlarged sterile meiocytes in showing less plastids in condensed cytoplasm separated from the cell wall; enlarged sterile tapetum in showing little abnormal plastids and more large vacuoles in cytoplasm, and with a little plasmolysis at meiocyte side. the fertile anthers at vacuolated-microspore stage; the degraded tapetum in showing elaioplasts and tapetsomes with abundant lipids; plastids in tapetum located in a crown showing filled with globular low electron-dense metabolites and surrounded by rich endoplasmic reticulum. the sterile anthers at vacuolated-microspore stage; the undegraded tapetum in showing elaioplasts and tapetsomes with abundant lipids; plastids in tapetum located in a crown showing irregular shaped low electron-dense material. the sterile anthers at vacuolated-microspore stage; the degraded tapetum in showing scattered elaioplasts and tapetsomes with fuzzy structure; the fertile anthers at mature pollen grain stage; the pollen grain in showing profuse globular particles; the enlarged globular particles in. the sterile anthers at mature pollen grain stage; the undegraded tapetum in died but cell wall still existed; the sterile anthers at mature pollen grain stage. the epidermis and endothecium cells in fertile plants at vacuolated-microspore stage; the epidermis cells in showing normal oval-shaped chloroplastids with distinct thylakoid structure and little starch granules in thylakoid; the endothecium cells in showing oval-shaped chloroplastids with distinct thylakoid structure. the epidermis and endothecium cells in sterile plants at vacuolated-microspore stage; the epidermis cells in showing abnormal chloroplastids with large starch granules in thylakoid; the endothecium cells in 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 μm, 5 μm, 2 μm, and 1 μm. 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; 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. 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 was used. three independent biological replicates of four pairs of tissues from the mes-treated and mock-treated plants were collected for gene expression analysis, resulting in a dataset of microarrays. the four tissues included the leaves from main inflorescences, the small buds less than 1 mm in length containing microgametocytes before and during pollen mother stage, the anthers from middle buds with length between 1 mm and 3 mm containing microgametocytes from meiosis to early uninucleate microspore stage, and the anthers from large buds more than 3 mm in length containing microgametocytes from vacuolated stage to mature pollen stage. the data quality was assessed using two measurements: correlation coefficients between biological replicates, which ranged from to, with a mean of, and quantitative real time rt-pcr, which was performed on randomly selected genes. the results of qrt-pct analysis showed a high degree of concordance with microarray results. taken together, these demonstrated that the microarray results obtained in this study were reliable.figure photographs of the leaves and developmental anthers for microarrays from the mock-treated and mes-treated plants. a-b, young leaves from the main inflorescences; c-d, small buds; e-f, anthers from middle buds; g-h, anthers form large buds. scale bar in leaves was cm, scale bars in sbs, an-mbs, and an-lbs were mm. to identify differentially expressed transcripts between the mes-treated and mock-treated groups involved in microgametogenesis, two sets of student’s t-test comparisons were performed. firstly, vertical comparisons were conducted to identify dets involved in anther development, and dets being identified within the mock-treated group and mes-treated group, respectively. secondly, horizontal comparisons were conducted to identify dets caused by mes-treatment in the tissues tested, up-regulated and down-regulated transcripts being identified in the four-pair tissues. the common dets identified in both the vertical and horizontal comparisons described above were considered as anther development related genes affected by mes treatment. therefore, 102 + 108 +  and 31 + 84 +  dets corresponding to unique dets were selected for further analysis in this study, in order to reveal the alteration of gene expressions induced by mes treatment in the tissues tested. distribution of these dets indicated that over 64% were down-regulated and only 36% were up-regulated in at least one tissue in the mes-treated plants. in addition, small fractions of these dets were in ls and sbs with 2 ~ 5-fold change, while the majority were found in an-mbs and an-lbs 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.figure strategies for identification of differentially expressed transcripts involved in microgametogenesis between the mes-treated and mock-treated plants by two sets of student’s t-test comparisons. comparisons within groups. the pair-wise comparisons of student’s t-test between tissues were carried out within mock-treatment groups and mes-treatment groups, respectively, to detected dets related to anther development under mock-treatment and mes-treatment conditions. the criteria for screening dets were p-value < and fold change ≥  mock, mock-treatment; mes, mes-treatment; comparisons between the mes-treated and mock-treated groups. the pair-wise comparisons of student’s t-test were performed between the corresponding tissues of the mock-treated group and the mes-treated group to identify dets related to mes-treatment. the screen criteria were same as above. 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. comparisons between groups also produced two sets of dets, up-regulated genes and down-regulated genes in mes-treated group. these four sets of dets were all collected, respectively. the common sections indicates dets existing in the both data sets) were considered to be anther development-related genes affected by mes-treatment. distribution of differentially expressed transcripts in mes-treated rapeseed plants fold change ls sbs an-mbs an-lbs total unique up-regulated  2 ~ 5  5 ~ 10  ≥10  total-up down-regulated  2 ~ 5  5 ~ 10  ≥10  total-down  total *two dets existed both in the up-regulated and down-regulated probe sets. 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, 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 between these two species. of the dets, were highly similar to a. thaliana genes and could be thus annotated. the remaining dets did not find a close orthologue in the tair database and therefore was not be annotated. the localization of these dets might provide clues to where they function. to gain insight into the biological functions of these annotated unigenes, subcellular localization and functional category analysis were conducted according to the information from munich information center for protein sequences. the unigenes could be distributed into subcellular localizations, with the largest three categories being the plastid/chloroplast, mitochondrion, and nucleus, followed by the eukaryotic plasma membrane, cell wall, and cytoplasm. furthermore, the unigenes were classified into 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. in order to understand the functions of dets in different tissues, we performed the category enrichment analysis in four tissues separately. the results of the functional category and enrichment analysis 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., won’t be given further consideration in this study. furthermore, detailed subcategory analysis of metabolism revealed that carbohydrate metabolism and lipid metabolism were significantly enriched, 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.figure subcellular localization and functional categories of the differentially expressed unigenes between the mes-treated and the mock-treated rapeseed plants. subcellular localization, functional categories, enrichment analysis of the functional categories list in b. the scale bar indicates -log, 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, respectively. the prominent enriched sub-categories were circled in black ellipses. furthermore, to get the overview of the pathways where the dets are taking part in, the unigenes described above were further analyzed by mapman software. the biotic stress and metabolism pathway visualization are shown in figure  in the biotic stress visualization, 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 of the genes in the signaling pathway belonged to the protein kinase signaling, and 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. in proteolysis pathway, of protein degradation related genes were coding for the 26s proteasome complex, which mediates ubiquitin-dependent protein degradation. interestingly, eight of the protein degradation related genes were up-regulated in an-mbs, while most of the others were down-regulated in an-lbs. 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, 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. interestingly, the few genes involved in amino acid metabolism were mainly up-regulated in an-lbs. 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.figure transcripts involved in stress and metabolism assigned by mapman in rapeseed leaves and developmental anthers treated by mes. positive fold change values indicate up-regulation, whereas negative fold change values denote down-regulation. color saturates at -fold change. each square represents a differentially expressed transcript. 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, 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. four genes involved in starch biosynthesis and degradation pathway, including adp glucose pyrophosphorylase, phosphoglucomutase, disproportionating enzyme, and alpha-glucan phosphorylase, were down-regulated 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. pgm plays a pivotal role in the allocation of carbon between polysaccharide formation and energy production, and it is located in plastid. dpe and phs are enzymes catalyzing the breakdown of starch into maltose and glucose in the chloroplast at night. in addition, another carbohydrate metabolism-related gene, encoding transketolase in calvin cycle, was down-regulated by -fold. importantly, a vital gene encoding sweet protein was down-regulated by approximately 3-fold, 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.table representative differentially expressed genes involved in carbohydrate and lipid metabolism in ls, sbs, and an-mbs probename agi symble short description location* fold change ls sbs an-mbs c-compound and carbohydrate metabolism cell wall lipid, fatty acid and isoprenoid metabolism *the localization information was from mips website. fold change was calculated between mes-treated plants and mock-treated plants; ls, sbs, and an-mbs represents leaves, small buds, and anthers of middle buds, respectively; “-” represents that the gene was not significantly and differentially expressed in the specific tissue between the mes-treated and mock-treated plants. all the carbohydrate, cell wall, and lipid metabolism, and cellular transport related genes identified in this study were deposited in additional file 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. two genes related to cell wall metabolism, bxl and pectin lyase-like superfamily protein were down-regulated by -fold and -fold, respectively. the latter gene exhibits polygalacturonase activity in cell wall metabolism and is 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. kat, a thiolase, was down-regulated by -fold. this gene is strongly expressed during flower development in arabidopsis and can partially complements kat which mutant exhibited partly male sterility. in addition, one lipid transport gene and two lipid degradation genes 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. most of the cell wall-related genes were significantly down-regulated, including cell wall precursor synthesis genes fla and uge and pectin metabolism genes, such as polygalacturonase the pectate lyase family protein, plant invertase/pectin methylesterase inhibitor superfamily, and vanguard previous studies revealed that pectin metabolism-related genes played important roles during late stages of pollen development. besides, another cell wall related-gene, uge was down-regulated by -fold. it is reported that uge together with uge affected pollen development. in addition, several lipid metabolism-related genes were altered in the an-mbs of the mes-treated plants, two of them involved in lipid biosynthesis were down regulated, and three involved in lipid degradation were up regulated. 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 slightly repressed. 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. 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. however, energy metabolism-related genes about h+-exporting atpase such as h-atpase and h-atpase were significantly down-regulated in the mes-treated plant anthers at late stage. 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. compared with the mock-treated 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 mes-treated 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 mock-treated 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.figure 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 1–3 mm in. *, **, represents significant difference at level and at level, respectively. discussion 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 mes-treated 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 transcriptome analysis. mes treatment disturbes plastid and mitochondrion functions, probably through acetolactate synthetase 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. co-expression analysis of arabidopsis als revealed a network of genes directly or indirectly related to als: plastidic pyruvate kinase beta subunit, acetyl co-enzyme a carboxylase biotin carboxylase subunit, acetyl co-enzyme a carboxylase carboxyltransferase alpha subunit, ketol-acid reductoisomerase, pyruvate dehydrogenase e alpha, semialdehyde dehydrogenase family protein, isopropylmalate dehydrogenase, adenylosuccinate synthase, transketolase family protein, and pyruvate kinase family protein. most of these genes are expressed in the plastid or mitochondrion and are involved in five main pathways, including biosynthesis of secondary metabolites; purine metabolism; valine, leucine, and isoleucine biosynthesis, and two carbohydrate-related pathways. 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. in addition, subcellular localization analysis of the differentially expressed genes showed that most of them were localized in the plastid and mitochondrion. 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 mes-treated anthers at the vacuolated-microspore stage. 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. detailed analysis of gene expression alternation showed that starch and sucrose metabolism-related genes were down-regulated in young leaves of the main inflorescences in the mes-treated plants. in addition, an important gene encoding a protein for sucrose phloem transport was also down-regulated. in contrast, several substrate transport-related genes were up-regulated in the early development stage anthers 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. this was consistent with our cytological analysis that two lipid-storage plastids, elaioplast and tapetosome, were impaired at different degrees under mes treatment. 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 anthers. 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- -glucanase, endocellulase, and polygalacturonase. several genes responsible for genic male sterility in b. napus mutants were map-based cloned, and were found to be lipid metabolism related genes. it was reported that the pg family protein was associated with pollen intine development in b. campestris, and its mutant displayed male sterility. 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, 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 and h-atpase were down-regulated in ls and an-mbs from the mes-treated plants, and several other atpase-related genes were down regulated in an-lbs. 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 monosulfuron-ester 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. 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. 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 but increased at late stage, 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 mes-treated 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. reported that imazethapyr 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. confirmed that csr the catalytic subunit of als, was the sole target of imidazolinone herbicide in a. thaliana using microarray analysis. das et al. identified genes significantly and coordinately regulated by four als-inhibiting herbicides, including one imidazolinone, one triazolopyrimidine, and two sulfonylureas at the ec concentration. among their genes identified, only with the same agi number were differentially expressed in our data, functionally involved in photosynthesis, opp 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 and cha 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 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 das’s 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  g ha− mes for inducing male sterility. however, das et al. treated a. thaliana plants with four als-inhibiting herbicides at their ec concentrations, ranging from  g ha− for sulfometuron-methyl to  g ha− for primisulfuron-methyl. 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 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, so the developing anthers might be more sensitive to mes treatment than other vegetative tissues. 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. secondly, when als was inhibited in developing anthers, large amount of the substrate 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. 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. 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 mes-treated 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.figure a putative action model for mes-treatment inducing male sterility. some important functions and genes affected by mes treatment in leaf tissue and developing anther tissues are listed. 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; dpe disproportionating enzyme; phs alpha-glucan phosphorylase 2; sweet nodulin mtn family protein; bxl beta-xylosidase; pectin lyases, pectin lyase superfamily protein; vgdh vanguard homolog 2; ppme1: pectin lyase-like superfamily protein; fla5: fasciclin-like arabinogalactan protein precursor; vgdh1: plant invertase/pectin methylesterase inhibitor; vgd1: plant invertase/pectin methylesterase inhibitor; pga polygalacturonase 4; uge udp-d-glucose/udp-d-galactose 4-epimerase 3; ha h-atpase  this study carried out a systematic analysis of effects caused by cha-mes treatment at ultrastructure, 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 breeding. 
chemical hybridization agents are often used to induce male sterility for the production of hybrid seeds. we previously discovered that monosulfuron ester sodium, an acetolactate synthase inhibitor of the herbicide sulfonylurea family, can induce rapeseed 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. 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. 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 agentmale sterilitymonosulfuron ester sodiumexpression profilecarbohydrate and lipid metabolismissue-copyright-statement© the author 2015 transcripts involved in metabolism, especially in carbohydrate and lipid metabolism, and cellular transport were differentially expressed. 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. comparative transcriptome analysis identified differentially expressed transcripts in leaves and anthers at different developmental stages, most of these dets being localized in plastid and mitochondrion. pathway visualization showed that the tightly regulated gene network for metabolism was reprogrammed to respond to mes treatment.
 chemical hybridization agents are often used to induce male sterility for the production of hybrid seeds. we previously discovered that monosulfuron ester sodium, an acetolactate synthase inhibitor of the herbicide sulfonylurea family, can induce rapeseed 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.  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 differentially expressed transcripts 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords brassica napus l.chemical hybridization agentmale sterilitymonosulfuron ester sodiumexpression profilecarbohydrate and lipid metabolismissue-copyright-statement© the author 2015 