BACKGROUND
the life cycle of flowering plants alternates between a prominent diploid sporophyte generation and a short-lived haploid gametophyte generation.
the haploid gametophytes are derived from the haploid spores that are produced by diploid megasporocytes  and microsporocyte parent cells
<cit> .
during female gametophyte development, the megasporocyte undergoes meiosis to produce a tetrad of four haploid spores.
three of the spores degenerate, and one proceeds through three sequential rounds of mitotic division, forming the female gametophyte , which consists of seven cells with four cell types
<cit> .
during male gametophyte development, microsporocytes undergo meiosis to form a tetrad of four haploid microspores.
each microspore undergoes two mitotic divisions to form the male gametophyte  consisting of a vegetative cell and two sperm cells
<cit> .
following pollination, the pollen grain lands on the pistil and extends a pollen tube that allows the delivery of the two sperm cells into the female gametophyte, and then gives rise to the diploid zygote to begin the sporophytic generation
<cit> .
female and male gametophyte development differ considerably, but at the same time share the same fundamental hallmark of being haploid organs: it is therefore logical that they might require the same basal machinery and share a number of common regulators
<cit> .
meiosis is a specialized cellular division that is conserved among most eukaryotes.
this process is indispensable for formation of viable offspring.
it consists of two rounds of chromosome segregation after a single round of dna replication, giving rise to four haploid daughter cells.
during meiosis i homologous chromosomes pair, undergo recombination and then segregate, whereas sister chromatids separate during meiosis ii
<cit> .
recombination is initiated by the formation of spo11-induced dna double strand-breaks , and dsbs in meiosis are repaired by homologous recombination
<cit> .
disruption of meiotic homologous recombination could result in chromosome anomalies, which could lead to mis-segregation and aneuploidy
<cit> .
the faithful transmission of chromosomes during meiosis is essential for the survival and reproduction of flowering plants.
a critical aspect of chromosome dynamics is structural maintenance of chromosome  proteins, which are responsible for sister chromatid cohesion, chromosome condensation and homologous recombination  during meiosis
<cit> .
the evolutionarily conserved smc gene family encodes members of the three complexes: the cohesin, the condensin and the smc5/ <dig> complex.
in arabidopsis, the cohesin complex consists of the smc <dig>  smc <dig>  scc <dig>  and four α-kleisin subunits: syn <dig>  syn <dig>  syn <dig> and syn4
<cit> .
evidence from mutants  defective in meiosis have shown that cohesin is essential for the control of chromosome structure and many subsequent meiotic events
<cit> .
the arabidopsis condensin complex consists of the smc <dig>  smc <dig>  and β-kleisin subunit cap-d <dig>  data from mutants  with gametophytic defects have shown that condensin is required for chromosome condensation and segregation during mitosis, meiosis and embryo development
<cit> .
in plants, knowledge about the role of smc5/ <dig> complex is still limited.
the arabidopsis smc5/ <dig> complex presumably consists of smc <dig>  one of two alternative smc <dig> proteins and four nse proteins
<cit> .
it has been shown in arabidopsis that smc <dig> and smc <dig> enhances sister chromatid alignment after dna damage and thereby facilitates correct dsb repair via hr between sister chromatids
<cit> .
although the arabidopsis nse <dig> and smc <dig> are essential for seed development
<cit> , the role smc5/ <dig> complex in gametophyte development is still unknown.
the arabidopsis sumo e <dig> ligase atmms21/hpy <dig>  a homologue of nse2/mms <dig>  has been identified recently as participating in root development.
loss of atmms21/hpy <dig> function results in premature mitotic-to-endocycle transition, defective cytokinin signaling, and impaired cell cycle, leading to severe dwarfism with compromised meristems
<cit> .
recent data demonstrate that atmms21/hpy <dig> functions as a subunit of the smc5/ <dig> complex through its interaction with smc <dig>  atmms <dig> acts in dsb amelioration and stem cell niche maintenance during root development
<cit> .
hence, atmms <dig> is involved in cell division, differentiation, expansion and survival during plant development.
the highly coordinated processes of cell division, differentiation, and expansion that take place during gametophyte development require precise fine-tuning of gene regulatory networks
<cit> .
however, whether and how atmms <dig> participates in regulating the gametophyte development and reproductive processes remains unclear.
in the present study, we provide cell-biological and molecular evidence that atmms <dig> is required for fertility in arabidopsis.
mutations in atmms <dig> cause semi-sterility with aberrant gamete, indicating that the gene is essential for gametogenesis.
furthermore, mms21- <dig> mutant cells exhibit chromosome fragmentation and mis-segregation during meiosis.
transcription level for several meiotic genes are also altered in mms21- <dig> buds.
these observations suggest that atmms <dig> plays an important role in meiosis and gametophyte development.
RESULTS
mms21- <dig> mutant shows severely reduced fertility
previous studies showed that mutation of atmms21/hpy <dig> resulted in severe developmental defects
<cit> .
to determine whether atmms <dig> regulates the reproductive development, we first analyzed the fertility of mms21- <dig> and wild-type plants.
in their reproductive phase, mms21- <dig> plants were bushy with short siliques .
mean silique length was reduced to  <dig>  ±  <dig>  mm in mms21- <dig>  compared with  <dig>  ±  <dig>  mm in the wild-type .
ten days after pollination, dissected siliques from mms21- <dig> plants showed severely reduced seed-set and unfertilized ovules .
later in mature siliques, the mean seed-set was only  <dig>  ±  <dig>  per silique, accounting for  <dig> % of the normal seed-set in the wild-type .
some of the mutant seeds were abnormal in appearance with a dark and shrunken seed coat .
the percentage of aborted seeds in mms21- <dig> is approximatly  <dig> %, while only  <dig> % in wide-type .
furthermore, we analyzed fertility in the transgenic plants expressing 35s::atmms21-gfp in mms21- <dig>  and found that the expression of atmms21-gfp could rescue the semisterile phenotype of mms21- <dig> , indicating that the impaired fertility of the mms21- <dig> is caused by the absence of atmms <dig>  therefore, these results suggested that atmms <dig> is essential to fertility in arabidopsis.
decreased fertility in mms21- <dig> is caused by both abnormal male and female fertility
to answer the question of whether male or female fertility was affected by the mutation, we performed reciprocal cross-pollinations between mms21- <dig> and wild-type plants.
in reciprocal cross-pollinations, wild-type pollen showed active pollen tube growth to the base of the wild-type pistil in 12 h, and the average size of mature siliques and number of seeds per silique from this cross were equivalent to those of self-pollinated wild-type plants .
by contrast, mms21- <dig> pollen did not show normal fertilization in either mms21- <dig> or wild-type pistils.
unfertilized ovules were random distributed in the mature siliques and a high percentage of shrunken seeds , and short siliques and small numbers of seeds per silique, , indicating that the function of the pollen was compromised in the mms21- <dig> mutants.
cross-pollination of wild-type pollen onto mms21- <dig> pistils resulted in better fertilization and silique sizes .
however, the siliques size and seed number per silique were still decreased in this cross, compared with the wild-type self-cross .
cross-pollination of mms21- <dig> pollen onto wild-type pistil showed a lower percentage in pollen tube growth to the base of the pistil .
taken together, our reciprocal cross-pollination studies suggested that atmms <dig> has a function in both male and female fertility.
mms21- <dig> mutant shows reduced pollen number, viability, germination and abnormal pollen tube growth
to further characterize the semisterile phenotype of mms21- <dig> plant, we first examined the effects of the mms21- <dig> mutation on male fertility.
unlike wild-type , there were few pollen grains produced on the surfaces of anthers and stigma in mms21- <dig> flowers .
861 ±  <dig> pollen grains per wild-type flower but only about 136 ±  <dig> pollen grains were observed in mms21-1flowers.
to assess pollen grain viability, anthers and mature pollen grains from both the wild-type and mutant flowers were stained with alexander’s solution
<cit> .
wild-type mature anthers were in uniform size, and the pollen grains stained red, which indicates viability .
in contrast, mms21- <dig> mature anthers were variable in size and shape .
pollen grains in mms21- <dig> plants were generally bigger with about  <dig> % nonviable pollen grains, as indicated by blue staining, whereas the wild-type produced < <dig> % abnormal pollen .
in vitro pollen germination and pollen tube growth assays were also performed.
in mms21- <dig>  pollen tube initiation and growth occurred in only  <dig> % of the pollen grains while  <dig> % of wild-type pollen germinated .
particularly, mms21- <dig> pollen tubes showed a variable phenotypes and abnormal morphologies.
for example, long pollen tubes exhibiting branched tips  and short swollen pollen tubes were observed .
these results indicated that atmms <dig> mutation affects pollen number, viability, germination and tube growth.
mutation of atmms <dig> causes defects in gametogenesis
to determine which step of pollen development is affected by the mms21- <dig> mutation, paraffin-cross sections of anthers stained with toluidine blue from various developmental stages were analyzed .
in arabidopsis, anther development can be divided into  <dig> well-ordered stages by morphological characteristics
<cit> .
in mms21- <dig>  the early  <dig> stages of pollen development appeared normal compared with wild-type .
alteration were first observed at tetrad stage, wild-type meioses produced four uniform spores, while the mutant produced a mixture of dyads, triads, and tetrads .
at stage  <dig>  microspores are typically released from the tetrads.
microspores were angular in shape in wild-type plants, whereas microspores of mms21- <dig> plants appeared of various sizes .
during stage  <dig>  wild-type microspores underwent asymmetric mitotic divisions and generated a significant pollen wall .
in contrast, in mms21- <dig>  most of the microspores were degenerated .
eventually, pollen grains of the wild-type were released when anther dehiscenced, whereas most of the mutant microspores were degenerated, leaving an empty locule .
to more precisely define the developmental defect in mms21- <dig> pollen, developing mms21- <dig> pollen were stained with dapi and observed at different developmental stages.
the mms21- <dig> meiotic products were a mixture of dyads, triads and tetrads, while the wt tetrads had four equal-sized spores enclosed in a callose wall .
at the uninucleate stage, some abnormal microspores in mms21- <dig> exhibited no fluorescence or contained two nuclei within one exine wall .
in the bicellular pollen stage, some of the mms21- <dig> pollen became shrunken and lacked dapi staining compared with wild-type .
the size difference became more pronounced at the tricellular stage, as normal pollen continued to approach maturity, while mutants were losing nuclear content and becoming distorted .
we also examined expression patten of atmms <dig> by in situ hybridization.
transverse sections of anthers showed strong hybridization signals in pollen mother cells ; no signals were detected when a sense probe was used .
loss of atmms <dig> function also causes defects in female gametogenesis.
normally the diploid megaspore mother cell undergoes meiosis and gives rise to four haploid nuclei.
subsequently, three megaspores undergo cell death, with the remaining, functional megaspore proceeding into megagametogenesis.
examination of cleared ovules in mms21- <dig> mutant plants showed that some megaspore mother cells appeared to abort either before or during meiosis , giving rise to embryo sacs containing one to five nuclei .
these data indicated that atmms <dig> is important for gametogenesis, both during male and female gametophyte development.
disruption of atmms <dig> leads to defects in chromosome behavior during meiosis
the defects in gametophyte development described above could arise from defective meiosis.
therefore we examined chromosome spreads from various stages of meiosis of wild-type and mms21- <dig> anthers.
in wild-type meiosis, chromosomes were clearly single and unpaired in leptotene  and underwent synapsis between homologous chromosomes at the zygotene stage  until its completion in pachytene .
at diakinesis stage, homologous chromosomes desynapsed and then underwent further condensation to form five bivalents .
the five bivalents aligned at the division plane at metaphase i .
at anaphase i homologous chromosomes separated from each other and moved to opposite poles .
subsequently tetrads formed at anaphase ii .
in mm21-1mutant plants, the early development stages of meiotic nuclei, in other words from leptotene to pachytene, appeared to proceed normally .
alterations were observed at diakinesis, in diakinesis, chromosomes were further condensed, but in mms21- <dig> it appeared to be less condensed than wild type .
interestingly, we did not observe distinct metaphase i in the mms21– <dig>  furthermore, chromosome fragments and bridges between bivalents were observed in anaphase i  in anaphase ii, segregation of the sister chromotids were also disturbed leading to irregular meiotic products with variable dna contents .
as some lagging chromosome dispersed throughout the cytoplasm, typical tetrads were rarely found .
these observations showed that loss function of atmms <dig> cause abnormal meiotic chromosome behavior.
mms21- <dig> mutants exhibits altered transcript levels for meiotic genes
meiocytes exhibit abnormal chromosome behavior, we investigated whether loss of atmms <dig> affected meiotic gene expression, which includes asy <dig> <cit> , zyp <dig> <cit> , dmc <dig> <cit> , tam <cit> , msh <dig> <cit> , phs <dig> <cit> , rad <dig> <cit> , rad51c <cit> , rbr <dig> <cit> , spo11- <dig> <cit> , and spo11- <dig> <cit>  by the qrt-pcr.
although transcript levels for dmc <dig>  msh <dig>  spo11- <dig> and tam <dig> were similar in the buds of wild-type , many meiotic gene expression were changed in the flower buds of mms21- <dig>  for example, transcripts for spo11- <dig> and rad <dig> were found to be increased approximately  <dig> and  <dig>  fold, respectively.
rbr is essential for inter-homologue recombination and synapsis
<cit> , and transcripts for rbr were up-regulated in mms21- <dig> mutant.
zyp <dig> encodes a synaptonemal complex protein and asy <dig> encods an axis-associated protein in arabidopsis <cit> ; zyp1a and asy <dig> transcript levels were down-regulated compared with wild-type plants.
in addition, transcripts of cohesin, condensin, smc5/ <dig> complex and smc-like genes in mms21- <dig> plants were elevated, with the exception of syn <dig> and swi1-like .
particularly, the transcript abundance level of swi <dig> was dramatically increased.
mms21- <dig> mutant exhibits reduced sumoylation levels in pollen grains
because atmms <dig> encodes a sumo e <dig> ligase and mutation of atmms <dig> causes defective gametocyte, it is reasonable to assume that accumulation of sumo-protein conjugates in generative cell were altered in mms21- <dig> mutant.
an immunoblot of total pollen grain proteins by anti-atsumo <dig> demonstrated a difference in the sumo conjugates in wild-type and mms21- <dig>  as some sumoylation conjugates were missing in mms21- <dig> pollen grains proteins .
these results suggested that atmms <dig> is involved in the sumoylation of arabidopsis pollen grains proteins, and may regulate the gametophyte developmental process through the sumoylation pathway.
discussion
sumoylation and the components of the sumo conjugation machinery are essential for viability, as shown by the embryo lethality of the mutations in sae <dig> or sce or both in sumo <dig> and sumo <dig> <cit> .
however, the role of sumoylation for gametophyte development is poorly understood because of the zygotic lethality.
here, we used a viable mutant without functional sumo e <dig> ligase atmms <dig> for studying the function of sumoylation in reproductive development.
first, we examined t-dna mutants of atmms <dig> <cit> , focusing on silique size and seed set, which are direct indicators of successful fertility.
the mms21- <dig> mutant exhibits a drastic reduction in fertility, embodied by a shorter silique length with a smaller seed set than those in the wild-type .
examination of the male fertility indicated that mutant anthers produce fewer functional pollen grains .
cytological observation of various developmental stages in mms21- <dig> pollen revealed that the products of meiosis in the mutant were mostly dyads of spores instead of tetrads .
similarly, mutant meiosis produced abnormal embryo sacs during female gametogenesis .
these results are consistent with our reciprocal cross study suggesting that atmms <dig> has crucial roles in both male and female gametogenesis.
gametogenesis is an essential process for plant reproduction and the roles of the ubiquitin system in different processes during gametogenesis have been studied extensively
<cit> .
however, although ubiquitin-like proteins sumo have emerged as a key regulator of plant development and stress response
<cit> , there are currently little data supporting a specific role of the sumo system in the control of reproduction.
our data presented a regulatory framework for the action of atmms21-dependent sumoylation in reproductive development.
the loss of function mutant mms21- <dig> is still viable probably due to the action of another sumo e <dig> ligase siz1
<cit> .
the siz1- <dig> and mms21/hpy <dig> double mutant results in embryonic lethality, supporting the notion that atmms <dig> and siz <dig> have overlapping functions
<cit> .
mature female gametophytes were rapidly disrupted in the absence of the siz <dig> protein, while pollen developed well, indicating that siz <dig> plays important roles in sustaining the stability of the mature female gametophyte
<cit> .
however, unlike the siz <dig>  atmms <dig> is involved in the development and the female and male gametophyte.
interestingly, atmms <dig> was recently shown to be expressed highly in reproductive organs such as anther using a gus reporter construct
<cit> .
the expression data supported the role of atmms <dig> during gametophyte development inferred from the mms21- <dig> mutant lines.
the severe defects in mms21- <dig> gametophyte indicated that atmms <dig> is vital for fundamental processes , thereby ensuring normal reproductive development in arabidopsis.
the precise transmission of chromosomes from mother to daughter cells is a highly controlled process that requires members of the smc  protein family.
although cohesin  and condensin  complexes have been reported to be involved in many aspects of meiosis, the role of the smc5/ <dig> complex in meiosis remains elusive.
here, we demonstrated that a smc5/ <dig> subunit, atmms <dig>  is essential during arabidopsis gametogenesis.
the lack of atmms <dig> resulted in severely disrupted meiosis with bridges between chromosomes and chromosome fragmentations during meiosis i, leading to the unequal distribution of meiotic products and polyad formation during meiosis ii .
atmms <dig> is an important regulator of cell cycle progression
<cit> .
it is possible that the dyad cells may result from delayed progression of meiosis.
during meiosis, a germ cell will purposely create double-strand breaks which is induced by the protein spo <dig> to promote chiasmata formation, and the programmed dsbs which are repaired by hr
<cit> .
if the dsbs remain unrepaired while the cell cycle continues, it will possibly lead to fragmentation and mis-segregation
<cit> .
recent study demonstrated that atmms <dig>  a smc5/ <dig> complex sub-unit, is involved in dsb repair
<cit> .
chromosome fragmentation and mis-segregation observed in meiosis of mms21- <dig> may due to defective dsb repair.
the chromosome fragmentation may result from failure of dsb repair or non-condensed entangled chromosomes pulled to break by the spindle.
this notion is consistent with previous finding that the yeast smc5/ <dig> complex is required to ensure proper chromosome behavior during meiosis
<cit> .
in addition, several genes associated with dsb accumulation or formation, such as rad <dig> rad51c and spo11- <dig> were increased in mms21- <dig> flower buds , suggesting that mms21- <dig> generative cells may contain unrepaired dsbs.
therefore, it will be interesting to examine whether atmms <dig> is recruited to mitotic dsbs and function in meiotic recombination during meiosis.
although the meiotic functions of atmms <dig> need to be investigated, our data demonstrated that atmms <dig> is required for proper chromosome behavior and successful meiotic divisions.
meiotic roles have been discovered for cohesins and condensins in plants
<cit> .
here we show that a smc5/ <dig> associated protein atmms <dig> is important for meiosis.
furthermore, several genes encode various components of the different smc complexes in mms21- <dig> plants display increased transcription level .
smc complexes and their associated proteins are essential for sister chromatid cohesion, chromosome condensation, dna repair and recombination.
it is tempting to speculate that atmms <dig> gene may affect meiotic process indirectly by altering the expression of smc complexes and their associated genes.
when exposured to dna damaging agents all subunits of cohesin become sumoylated, such as the sumoylation of scc <dig> is carried out by the sumo e <dig> ligase mms <dig> in yeast
<cit> .
therefore, further analysis of the protein expression of smc complexes in the mms21- <dig> meiocytes and the relation between atmms <dig> and cohesin/condensin complexes will help to clarify the precise function of atmms <dig> during meiosis.
at the protein level, our results demonstrated that sumoylation level in mms21- <dig> pollen grains proteins is different from the wild-type , indicating that atmms21-mediated sumoylation may participate in generative cell formation.
identification and functional analyses of sumoylated proteins related to atmms <dig> are necessary for the understanding how atmms21-dependent sumoylation participates in meiosis and gametophyte development.
CONCLUSIONS
in conclusion, our studies found that arabidopsis mms <dig> is important for gametogenesis, both during male and female gametophyte development.
the loss of the arabidopsis mms <dig> causes reduced pollen number, viability, germination and abnormal meiotic chromosome behavior.
several transcripts for meiotic genes related to chromosome maintenance and behavior are altered in the mms21- <dig> plant.
futhermore, sumo-protein conjugates in the mms21- <dig> pollen grains are different from those in wild-type.
thus, these results indicated that atmms <dig> mediated sumoylation may stabilize the expression and accumulation of meiotic proteins in the gametophyte development.

 the life cycle of flowering plants alternates between a prominent diploid sporophyte generation and a short-lived haploid gametophyte generation. the haploid gametophytes are derived from the haploid spores that are produced by diploid megasporocytes and microsporocyte parent cells. during female gametophyte development, the megasporocyte undergoes meiosis to produce a tetrad of four haploid spores. three of the spores degenerate, and one proceeds through three sequential rounds of mitotic division, forming the female gametophyte, which consists of seven cells with four cell types. during male gametophyte development, microsporocytes undergo meiosis to form a tetrad of four haploid microspores. each microspore undergoes two mitotic divisions to form the male gametophyte consisting of a vegetative cell and two sperm cells. following pollination, the pollen grain lands on the pistil and extends a pollen tube that allows the delivery of the two sperm cells into the female gametophyte, and then gives rise to the diploid zygote to begin the sporophytic generation. female and male gametophyte development differ considerably, but at the same time share the same fundamental hallmark of being haploid organs: it is therefore logical that they might require the same basal machinery and share a number of common regulators. meiosis is a specialized cellular division that is conserved among most eukaryotes. this process is indispensable for formation of viable offspring. it consists of two rounds of chromosome segregation after a single round of dna replication, giving rise to four haploid daughter cells. during meiosis i homologous chromosomes pair, undergo recombination and then segregate, whereas sister chromatids separate during meiosis ii. recombination is initiated by the formation of spo11-induced dna double strand-breaks, and dsbs in meiosis are repaired by homologous recombination. disruption of meiotic homologous recombination could result in chromosome anomalies, which could lead to mis-segregation and aneuploidy. the faithful transmission of chromosomes during meiosis is essential for the survival and reproduction of flowering plants. a critical aspect of chromosome dynamics is structural maintenance of chromosome proteins, which are responsible for sister chromatid cohesion, chromosome condensation and homologous recombination during meiosis. the evolutionarily conserved smc gene family encodes members of the three complexes: the cohesin, the condensin and the smc5/ complex. in arabidopsis, the cohesin complex consists of the smc smc scc and four α-kleisin subunits: syn syn syn and syn4. evidence from mutants defective in meiosis have shown that cohesin is essential for the control of chromosome structure and many subsequent meiotic events. the arabidopsis condensin complex consists of the smc smc and β-kleisin subunit cap-d data from mutants with gametophytic defects have shown that condensin is required for chromosome condensation and segregation during mitosis, meiosis and embryo development. in plants, knowledge about the role of smc5/ complex is still limited. the arabidopsis smc5/ complex presumably consists of smc one of two alternative smc proteins and four nse proteins. it has been shown in arabidopsis that smc and smc enhances sister chromatid alignment after dna damage and thereby facilitates correct dsb repair via hr between sister chromatids. although the arabidopsis nse and smc are essential for seed development, the role smc5/ complex in gametophyte development is still unknown. the arabidopsis sumo e ligase atmms21/hpy a homologue of nse2/mms has been identified recently as participating in root development. loss of atmms21/hpy function results in premature mitotic-to-endocycle transition, defective cytokinin signaling, and impaired cell cycle, leading to severe dwarfism with compromised meristems. recent data demonstrate that atmms21/hpy functions as a subunit of the smc5/ complex through its interaction with smc atmms acts in dsb amelioration and stem cell niche maintenance during root development. hence, atmms is involved in cell division, differentiation, expansion and survival during plant development. the highly coordinated processes of cell division, differentiation, and expansion that take place during gametophyte development require precise fine-tuning of gene regulatory networks. however, whether and how atmms participates in regulating the gametophyte development and reproductive processes remains unclear. in the present study, we provide cell-biological and molecular evidence that atmms is required for fertility in arabidopsis. mutations in atmms cause semi-sterility with aberrant gamete, indicating that the gene is essential for gametogenesis. furthermore, mms21- mutant cells exhibit chromosome fragmentation and mis-segregation during meiosis. transcription level for several meiotic genes are also altered in mms21- buds. these observations suggest that atmms plays an important role in meiosis and gametophyte development.  mms21- mutant shows severely reduced fertility previous studies showed that mutation of atmms21/hpy resulted in severe developmental defects. to determine whether atmms regulates the reproductive development, we first analyzed the fertility of mms21- and wild-type plants. in their reproductive phase, mms21- plants were bushy with short siliques. mean silique length was reduced to  ±   mm in mms21- compared with  ±   mm in the wild-type. ten days after pollination, dissected siliques from mms21- plants showed severely reduced seed-set and unfertilized ovules. later in mature siliques, the mean seed-set was only  ±  per silique, accounting for % of the normal seed-set in the wild-type. some of the mutant seeds were abnormal in appearance with a dark and shrunken seed coat. the percentage of aborted seeds in mms21- is approximatly %, while only % in wide-type. furthermore, we analyzed fertility in the transgenic plants expressing 35s::atmms21-gfp in mms21- and found that the expression of atmms21-gfp could rescue the semisterile phenotype of mms21-, indicating that the impaired fertility of the mms21- is caused by the absence of atmms therefore, these results suggested that atmms is essential to fertility in arabidopsis. decreased fertility in mms21- is caused by both abnormal male and female fertility to answer the question of whether male or female fertility was affected by the mutation, we performed reciprocal cross-pollinations between mms21- and wild-type plants. in reciprocal cross-pollinations, wild-type pollen showed active pollen tube growth to the base of the wild-type pistil in 12 h, and the average size of mature siliques and number of seeds per silique from this cross were equivalent to those of self-pollinated wild-type plants. by contrast, mms21- pollen did not show normal fertilization in either mms21- or wild-type pistils. unfertilized ovules were random distributed in the mature siliques and a high percentage of shrunken seeds, and short siliques and small numbers of seeds per silique,, indicating that the function of the pollen was compromised in the mms21- mutants. cross-pollination of wild-type pollen onto mms21- pistils resulted in better fertilization and silique sizes. however, the siliques size and seed number per silique were still decreased in this cross, compared with the wild-type self-cross. cross-pollination of mms21- pollen onto wild-type pistil showed a lower percentage in pollen tube growth to the base of the pistil. taken together, our reciprocal cross-pollination studies suggested that atmms has a function in both male and female fertility. mms21- mutant shows reduced pollen number, viability, germination and abnormal pollen tube growth to further characterize the semisterile phenotype of mms21- plant, we first examined the effects of the mms21- mutation on male fertility. unlike wild-type, there were few pollen grains produced on the surfaces of anthers and stigma in mms21- flowers. 861 ±  pollen grains per wild-type flower but only about 136 ±  pollen grains were observed in mms21-1flowers. to assess pollen grain viability, anthers and mature pollen grains from both the wild-type and mutant flowers were stained with alexander’s solution. wild-type mature anthers were in uniform size, and the pollen grains stained red, which indicates viability. in contrast, mms21- mature anthers were variable in size and shape. pollen grains in mms21- plants were generally bigger with about % nonviable pollen grains, as indicated by blue staining, whereas the wild-type produced < % abnormal pollen. in vitro pollen germination and pollen tube growth assays were also performed. in mms21- pollen tube initiation and growth occurred in only % of the pollen grains while % of wild-type pollen germinated. particularly, mms21- pollen tubes showed a variable phenotypes and abnormal morphologies. for example, long pollen tubes exhibiting branched tips and short swollen pollen tubes were observed. these results indicated that atmms mutation affects pollen number, viability, germination and tube growth. mutation of atmms causes defects in gametogenesis to determine which step of pollen development is affected by the mms21- mutation, paraffin-cross sections of anthers stained with toluidine blue from various developmental stages were analyzed. in arabidopsis, anther development can be divided into well-ordered stages by morphological characteristics. in mms21- the early stages of pollen development appeared normal compared with wild-type. alteration were first observed at tetrad stage, wild-type meioses produced four uniform spores, while the mutant produced a mixture of dyads, triads, and tetrads. at stage microspores are typically released from the tetrads. microspores were angular in shape in wild-type plants, whereas microspores of mms21- plants appeared of various sizes. during stage wild-type microspores underwent asymmetric mitotic divisions and generated a significant pollen wall. in contrast, in mms21- most of the microspores were degenerated. eventually, pollen grains of the wild-type were released when anther dehiscenced, whereas most of the mutant microspores were degenerated, leaving an empty locule. to more precisely define the developmental defect in mms21- pollen, developing mms21- pollen were stained with dapi and observed at different developmental stages. the mms21- meiotic products were a mixture of dyads, triads and tetrads, while the wt tetrads had four equal-sized spores enclosed in a callose wall. at the uninucleate stage, some abnormal microspores in mms21- exhibited no fluorescence or contained two nuclei within one exine wall. in the bicellular pollen stage, some of the mms21- pollen became shrunken and lacked dapi staining compared with wild-type. the size difference became more pronounced at the tricellular stage, as normal pollen continued to approach maturity, while mutants were losing nuclear content and becoming distorted. we also examined expression patten of atmms by in situ hybridization. transverse sections of anthers showed strong hybridization signals in pollen mother cells; no signals were detected when a sense probe was used. loss of atmms function also causes defects in female gametogenesis. normally the diploid megaspore mother cell undergoes meiosis and gives rise to four haploid nuclei. subsequently, three megaspores undergo cell death, with the remaining, functional megaspore proceeding into megagametogenesis. examination of cleared ovules in mms21- mutant plants showed that some megaspore mother cells appeared to abort either before or during meiosis, giving rise to embryo sacs containing one to five nuclei. these data indicated that atmms is important for gametogenesis, both during male and female gametophyte development. disruption of atmms leads to defects in chromosome behavior during meiosis the defects in gametophyte development described above could arise from defective meiosis. therefore we examined chromosome spreads from various stages of meiosis of wild-type and mms21- anthers. in wild-type meiosis, chromosomes were clearly single and unpaired in leptotene and underwent synapsis between homologous chromosomes at the zygotene stage until its completion in pachytene. at diakinesis stage, homologous chromosomes desynapsed and then underwent further condensation to form five bivalents. the five bivalents aligned at the division plane at metaphase i. at anaphase i homologous chromosomes separated from each other and moved to opposite poles. subsequently tetrads formed at anaphase ii. in mm21-1mutant plants, the early development stages of meiotic nuclei, in other words from leptotene to pachytene, appeared to proceed normally. alterations were observed at diakinesis, in diakinesis, chromosomes were further condensed, but in mms21- it appeared to be less condensed than wild type. interestingly, we did not observe distinct metaphase i in the mms21– furthermore, chromosome fragments and bridges between bivalents were observed in anaphase i in anaphase ii, segregation of the sister chromotids were also disturbed leading to irregular meiotic products with variable dna contents. as some lagging chromosome dispersed throughout the cytoplasm, typical tetrads were rarely found. these observations showed that loss function of atmms cause abnormal meiotic chromosome behavior. mms21- mutants exhibits altered transcript levels for meiotic genes meiocytes exhibit abnormal chromosome behavior, we investigated whether loss of atmms affected meiotic gene expression, which includes asy, zyp, dmc, tam, msh, phs, rad, rad51c, rbr, spo11-, and spo11- by the qrt-pcr. although transcript levels for dmc msh spo11- and tam were similar in the buds of wild-type, many meiotic gene expression were changed in the flower buds of mms21- for example, transcripts for spo11- and rad were found to be increased approximately and fold, respectively. rbr is essential for inter-homologue recombination and synapsis, and transcripts for rbr were up-regulated in mms21- mutant. zyp encodes a synaptonemal complex protein and asy encods an axis-associated protein in arabidopsis; zyp1a and asy transcript levels were down-regulated compared with wild-type plants. in addition, transcripts of cohesin, condensin, smc5/ complex and smc-like genes in mms21- plants were elevated, with the exception of syn and swi1-like. particularly, the transcript abundance level of swi was dramatically increased. mms21- mutant exhibits reduced sumoylation levels in pollen grains because atmms encodes a sumo e ligase and mutation of atmms causes defective gametocyte, it is reasonable to assume that accumulation of sumo-protein conjugates in generative cell were altered in mms21- mutant. an immunoblot of total pollen grain proteins by anti-atsumo demonstrated a difference in the sumo conjugates in wild-type and mms21- as some sumoylation conjugates were missing in mms21- pollen grains proteins. these results suggested that atmms is involved in the sumoylation of arabidopsis pollen grains proteins, and may regulate the gametophyte developmental process through the sumoylation pathway. discussion sumoylation and the components of the sumo conjugation machinery are essential for viability, as shown by the embryo lethality of the mutations in sae or sce or both in sumo and sumo. however, the role of sumoylation for gametophyte development is poorly understood because of the zygotic lethality. here, we used a viable mutant without functional sumo e ligase atmms for studying the function of sumoylation in reproductive development. first, we examined t-dna mutants of atmms, focusing on silique size and seed set, which are direct indicators of successful fertility. the mms21- mutant exhibits a drastic reduction in fertility, embodied by a shorter silique length with a smaller seed set than those in the wild-type. examination of the male fertility indicated that mutant anthers produce fewer functional pollen grains. cytological observation of various developmental stages in mms21- pollen revealed that the products of meiosis in the mutant were mostly dyads of spores instead of tetrads. similarly, mutant meiosis produced abnormal embryo sacs during female gametogenesis. these results are consistent with our reciprocal cross study suggesting that atmms has crucial roles in both male and female gametogenesis. gametogenesis is an essential process for plant reproduction and the roles of the ubiquitin system in different processes during gametogenesis have been studied extensively. however, although ubiquitin-like proteins sumo have emerged as a key regulator of plant development and stress response, there are currently little data supporting a specific role of the sumo system in the control of reproduction. our data presented a regulatory framework for the action of atmms21-dependent sumoylation in reproductive development. the loss of function mutant mms21- is still viable probably due to the action of another sumo e ligase siz1. the siz1- and mms21/hpy double mutant results in embryonic lethality, supporting the notion that atmms and siz have overlapping functions. mature female gametophytes were rapidly disrupted in the absence of the siz protein, while pollen developed well, indicating that siz plays important roles in sustaining the stability of the mature female gametophyte. however, unlike the siz atmms is involved in the development and the female and male gametophyte. interestingly, atmms was recently shown to be expressed highly in reproductive organs such as anther using a gus reporter construct. the expression data supported the role of atmms during gametophyte development inferred from the mms21- mutant lines. the severe defects in mms21- gametophyte indicated that atmms is vital for fundamental processes, thereby ensuring normal reproductive development in arabidopsis. the precise transmission of chromosomes from mother to daughter cells is a highly controlled process that requires members of the smc protein family. although cohesin and condensin complexes have been reported to be involved in many aspects of meiosis, the role of the smc5/ complex in meiosis remains elusive. here, we demonstrated that a smc5/ subunit, atmms is essential during arabidopsis gametogenesis. the lack of atmms resulted in severely disrupted meiosis with bridges between chromosomes and chromosome fragmentations during meiosis i, leading to the unequal distribution of meiotic products and polyad formation during meiosis ii. atmms is an important regulator of cell cycle progression. it is possible that the dyad cells may result from delayed progression of meiosis. during meiosis, a germ cell will purposely create double-strand breaks which is induced by the protein spo to promote chiasmata formation, and the programmed dsbs which are repaired by hr. if the dsbs remain unrepaired while the cell cycle continues, it will possibly lead to fragmentation and mis-segregation. recent study demonstrated that atmms a smc5/ complex sub-unit, is involved in dsb repair. chromosome fragmentation and mis-segregation observed in meiosis of mms21- may due to defective dsb repair. the chromosome fragmentation may result from failure of dsb repair or non-condensed entangled chromosomes pulled to break by the spindle. this notion is consistent with previous finding that the yeast smc5/ complex is required to ensure proper chromosome behavior during meiosis. in addition, several genes associated with dsb accumulation or formation, such as rad rad51c and spo11- were increased in mms21- flower buds, suggesting that mms21- generative cells may contain unrepaired dsbs. therefore, it will be interesting to examine whether atmms is recruited to mitotic dsbs and function in meiotic recombination during meiosis. although the meiotic functions of atmms need to be investigated, our data demonstrated that atmms is required for proper chromosome behavior and successful meiotic divisions. meiotic roles have been discovered for cohesins and condensins in plants. here we show that a smc5/ associated protein atmms is important for meiosis. furthermore, several genes encode various components of the different smc complexes in mms21- plants display increased transcription level. smc complexes and their associated proteins are essential for sister chromatid cohesion, chromosome condensation, dna repair and recombination. it is tempting to speculate that atmms gene may affect meiotic process indirectly by altering the expression of smc complexes and their associated genes. when exposured to dna damaging agents all subunits of cohesin become sumoylated, such as the sumoylation of scc is carried out by the sumo e ligase mms in yeast. therefore, further analysis of the protein expression of smc complexes in the mms21- meiocytes and the relation between atmms and cohesin/condensin complexes will help to clarify the precise function of atmms during meiosis. at the protein level, our results demonstrated that sumoylation level in mms21- pollen grains proteins is different from the wild-type, indicating that atmms21-mediated sumoylation may participate in generative cell formation. identification and functional analyses of sumoylated proteins related to atmms are necessary for the understanding how atmms21-dependent sumoylation participates in meiosis and gametophyte development.  in conclusion, our studies found that arabidopsis mms is important for gametogenesis, both during male and female gametophyte development. the loss of the arabidopsis mms causes reduced pollen number, viability, germination and abnormal meiotic chromosome behavior. several transcripts for meiotic genes related to chromosome maintenance and behavior are altered in the mms21- plant. futhermore, sumo-protein conjugates in the mms21- pollen grains are different from those in wild-type. thus, these results indicated that atmms mediated sumoylation may stabilize the expression and accumulation of meiotic proteins in the gametophyte development. 
the mms21- mutant shows reduced pollen number, and viability, and germination and abnormal pollen tube growth. atmms deficient plants are semisterile with shorter mature siliques and abortive seeds. thus, these results suggest that atmms mediated sumoylation may stabilize the expression and accumulation of meiotic proteins and affect gametophyte development. moreover, accumulation of sumo-protein conjugates in the mms21- pollen grains is distinct from that in wild-type. several transcripts for meiotic genes related to chromosome maintenance and behavior are altered. mms is a sumo e ligase that is conserved in eukaryotes, and has previously been shown to be required for dna repair and maintenance of chromosome integrity. here, we show a role for atmms during gametophyte development.
 mms is a sumo e ligase that is conserved in eukaryotes, and has previously been shown to be required for dna repair and maintenance of chromosome integrity. loss of the arabidopsis mms causes defective meristems and dwarf phenotypes.  here, we show a role for atmms during gametophyte development. atmms deficient plants are semisterile with shorter mature siliques and abortive seeds. the mms21- mutant shows reduced pollen number, and viability, and germination and abnormal pollen tube growth. embryo sac development is also compromised in the mutant. during meiosis, chromosome mis-segregation and fragmentation is observed, and the products of meiosis are frequently dyads or irregular tetrads. several transcripts for meiotic genes related to chromosome maintenance and behavior are altered. moreover, accumulation of sumo-protein conjugates in the mms21- pollen grains is distinct from that in wild-type.  thus, these results suggest that atmms mediated sumoylation may stabilize the expression and accumulation of meiotic proteins and affect gametophyte development. atmms21sumoylationgametophyte developmentmeiosisarabidopsis thaliana 