BACKGROUND
phosphomannomutase , catalyzing the interconversion between mannose-6-phosphate and mannose-1-phosphate, is an essential and conserved enzyme in eukaryotic organisms  <cit> .
mannose-1-phosphate is necessary for synthesizing the vital cellular metabolite gdp-mannose, which plays a crucial role in the formation of polysaccharide chains required for the glycosylation of protein and lipid molecules  <cit> .
in higher plants, gdp-mannose also acts as an important precursor for the biosynthesis of the key antioxidant ascorbic acid   <cit> , and the mannose containing polysaccharides are essential for the development of functional cell walls  <cit> .
from available information, the copy number of functional pmm in diploid eukaryotic species varies from one  to two .
pmm proteins from different eukaryotic species are highly similar in primary structure, with more than 50% amino acid sequence identities found among the pmms from s. cerevisiae, arabidopsis and human  <cit> .
the crystal structure and catalytic mechanism of mammalian pmms are well understood  <cit> , and there is also a wealth of information on human pmm <dig>  whose point mutations lead to a genetically inherited disease cdg-ia  <cit> .
two recent molecular genetic studies in arabidopsis show that defect in pmm function reduces asa biosynthesis and protein glycosylation, causing enhanced susceptibility to oxidation stress and plant death at high growth temperature   <cit> .
the requirement of a functional pmm protein for arabidopsis to grow at 28°c is consistent with the pioneering finding of temperature sensitive pmm mutations that arrest s. cerevisiae growth at 37°c  <cit> , suggesting that pmm may be generally important for the temperature adaptability of eukaryotes.
compared to the above progress, little is known about pmm gene and function in crop plants, although broad temperature adaptability is vitally important for crops to achieve high yield potential under diverse environmental conditions.
to improve our understanding on pmm in crop plants, we have embarked on a systematic molecular and biochemical analysis of pmm genes in bread wheat , a polyploid crop species cultivated in both temperate and tropical regions and with harvest possible under a wide range of temperature conditions  <cit> .
bread wheat belongs to the tribe triticeae, which is composed of approximately  <dig> species, and contains both diploid  and polyploid  crop plants  <cit> .
the diploid genomes in triticeae species are homoeologous, and may be combined to form polyploids through natural and artificial hybridizations  <cit> .
bread wheat was evolved through natural hybridization between tetraploid wheat  and aegilops tauschii  about  <dig> years ago  <cit> .
t. turgidum was formed about  <dig>  million years ago  through spontaneous hybridization between an unidentified triticeae species containing the b genome and the wild einkorn wheat t. urartu   <cit> .
the a, b and d genomes diverged from each other around  <dig>  to  <dig>  mya, whereas the divergence of wheat and barley occurred around  <dig> mya  <cit> .
although bread wheat and related triticeae species generally contain large and complex genomes, the advent of structural and functional genomics research in model cereals  has made it possible to conduct relatively detailed molecular genetic and biochemical studies of important genes both within triticeae and among rice, brachypodium and triticeae species.
the genomic resources of rice and b. distachyon  have both been proved to be very useful in the investigations of important triticeae genes, such as ph <dig> and lr34/yr <dig> in common wheat and ppd-h <dig> and cly <dig> in barley  <cit> .
from the information presented above, the main objectives of this work were to 1) determine pmm genes and loci in bread wheat and related triticeae species, and 2) investigate the evolution of triticeae pmm genes and the main active pmm isozymes in bread wheat tissues, with the aid of the genomic knowledge of rice and b. distachyon.
RESULTS
isolation and characterization of pmm genes
two approaches  were used to identify and isolate the pmm genes in b. distachyon, bread wheat and related triticeae species .
in total,  <dig> distinct pmm coding sequences were isolated .
a single pmm gene was found for both rice  and b. distachyon .
the cdna of ospmm was reported previously  <cit> , whereas that of bdpmm was isolated in this work using rt-pcr .
brachypodium distachyon
rice
a the genomic dna  and cdna sequences include start  and stop  codons.
b plant materials from which pmm sequences were isolated.
c orf disrupted by premature stop codon.
d orf interrupted by frameshift mutation.
e the chromosomal locus containing ospmm genomic sequence is os04g <dig>
f the genbank accession number for ospmm cdna is dq <dig>
six different pmm genes were isolated from each of the two bread wheat varieties, xiaoyan  <dig> and chinese spring .
t. urartu, ae.
tauschii and barley were each found to contain two distinct pmm genes, while four different pmm genes were cloned from t. turgidum.
based on the data on exon and intron pattern, nucleotide and deduced protein identities, chromosomal location, and phylogenetic clustering analysis of genomic coding sequences , the a, b and d genomes were each found to contribute two members to the six pmm genes in bread wheat, which were thus named as tapmm-a <dig>  b <dig>  d <dig>  a <dig>  b <dig> and d <dig>  respectively .
the pmm- <dig> genes in bread wheat and its diploid and tetraploid progenitor species and barley were homoeologous.
likewisely, homoeologous relationship existed among the pmm- <dig> genes from these species.
the intron and exon patterns of the pmm genes from rice, b. distachyon, and wheat and relatives were highly similar.
except for ospmm and bdpmm, both of which had  <dig> exons owing to the split of the first exon into two parts, the remaining genes all had  <dig> exons and  <dig> introns .
apart from pmm-a <dig> homoeologs and hvpmm- <dig>  the remaining triticeae pmm genes cloned in this work all contained intact orf.
the coding region of hvpmm- <dig> was disrupted by premature stop codon based on analyzing the pcr products amplified from two barley varieties  .
consistent with our earlier study  <cit> , the proteins deduced from the pmm genes of common wheat and progenitor species, barley and b. distachyon were more than 50% identical to the pmms of human and s. cerevisiae .
the identities among the grass pmms were generally above 90%, with bdpmm showing higher identity to its homologs from bread wheat and related triticeae species than to ospmm.
the identities among the deduced pmm- <dig> proteins of bread wheat and its diploid and tetraploid ancestors were generally 98% or higher, with complete identity found among the deduced tapmm-a <dig>  tupmm-a <dig>  ttpmm-a <dig> and tapmm-b <dig> proteins.
the identities among the deduced pmm-b <dig> and d <dig> proteins of bread wheat and its diploid and tetraploid ancestors were generally 97%.
by contrast, the identities between the deduced pmm- <dig> and  <dig> proteins were comparatively lower, being 95% between b <dig> and b <dig> proteins, and 96% between d <dig> and d <dig> proteins.
the deduced pmm-b <dig>  proteins of tetraploid and bread wheats were 99% identical.
interestingly, tapmm-b <dig> and ttpmm-b <dig> displayed unusual amino acid substitutions.
the first one, involving the substitution of a broadly conserved glycine residue by alanine, was shared between tapmm-b <dig> and ttpmm-b <dig> .
the second one was unique to tapmm-b <dig>  involving the replacement of a highly conserved arginine residue by cysteine.
the four structural and active site motifs , identified by analyzing the crystal structure of hspmm- <dig>  <cit> , were generally conserved among the compared plant pmms .
determination of pmm copy number and chromosomal locations
to investigate the copy number of pmm genes, we made use of intron length polymorphisms and designed a pair of intron flanking  primers , which enabled the amplification of pcr fragments containing introns iii to v .
by carefully analyzing the products amplified with fluorescently labeled if primers, six distinct fragments were found for bread wheat , and their size equaled to that calculated from the six cloned tapmm genomic sequences in all cases .
using the same strategy, two pmm fragments were found for t. urartu , ae.
tauschii  and barley , respectively, and for all three species, the size of the amplified fragments corresponded exactly to the one deduced from the cloned pmm members .
four fragments were amplified from t. turgidum , whose size again showed specific correspondence to that derived from each of the four cloned ttpmm members .
finally, a single fragment was amplified from b. distachyon  and its size agreed with the one calculated from the cloned bdpmm genomic sequence .
the copy number of pmm genes in an additional set of materials  was also investigated by fragment analysis using the if primers .
the results showed that the diploid triticeae species carrying the amam, cc, mm, nn, rr, ss, sbsb, slsl, ssss, sshssh, stst or uu genomes generally had two different pmm fragments, whereas the tetraploid triticeae species containing the aagg, ddcc, ddmm, ddnn or uumm genomes generally possessed four pmm specific fragments .
the fluorescently labeled if primers were further employed for investigating the chromosomal locations of wheat and barley pmm members by pcr amplifications.
the fragments derived from pmm-a <dig>  b <dig> and d <dig> of bread wheat were not amplified from the nt lines lacking 2a, 2b and 2d chromosomes, respectively , indicating that tapmm- <dig> homoeologs were located on group  <dig> chromosomes.
this finding was confirmed by the fragment analysis data obtained with ldn substitution lines .
the fragments derived from pmm-a <dig> and d <dig> members of bread wheat were not amplified from the nt lines lacking 4a and 4d chromosomes, respectively .
moreover, the fragment from ttpmm-b <dig> was absent in the ldn substitution line lacking the 4b chromosome .
together, these results indicated that tapmm- <dig> homoeologs were located on group  <dig> chromosomes.
using a similar strategy, the two barley pmm genes were mapped to the 2h and 4h chromosomes, respectively , which are homoeologous to wheat groups  <dig> and  <dig> chromosomes, respectively  <cit> .
the finding of six pmm genes located on groups  <dig> and  <dig> chromosomes in bread wheat by this work is consistent with the data generated in the wheat expressed sequence tag  mapping project http://wheat.pw.usda.gov/nsf/progress_ mapping.html.
the est be <dig>  a  <dig> bp cdna exhibiting higher than 95% nucleotide sequence identity to the 5' half of the mrnas of the six bread wheat pmm genes, has been used as a probe to hybridize with the genomic dna samples of cs and associated nt lines digested with the restriction enzyme ecori.
the hybridization yields  <dig> positive bands, which are mapped to groups  <dig> or  <dig> chromosomes.
our bioinformatic analysis suggests the presence of three to five ecori sites in the genomic orf of bread wheat pmm genes, with the last ecori site located in the 8th exon .
because the first seven exons give rise to the 5' half of pmm mrnas upon post transcriptional processing, the dna blot hybridization with be <dig> probe is expected to show about  <dig> to  <dig> hybridizing bands from six tapmm gene members.
the hybridization with another pmm est probe  yields approximately seven positive bands, which are mapped mainly to groups  <dig> or  <dig> chromosomes.
consequently, it is suggested that the number of homoeologous pmm genes in bread wheat is likely to be around six.
however, we can not exclude the existence of additional and divergent pmm copies or fragments in bread wheat genome, because they may not be amplified by our pcr strategy , or be distinguishable from one or more of the six completely sequenced copies during fragment analysis .
duplication of pmm in diploid triticeae species
the foregoing experiments indicated that there existed two pmm genes  in diploid triticeae species, and a single pmm gene in either b. distachyon or rice.
these findings raised an important question on how and when the two pmm gene members in diploid triticeae species were evolved.
to investigate this question, phylogenetic analysis was conducted using the genomic dna sequences of pmm genes.
two distinct clades, one composed of triticeae pmm- <dig> homoeologs and the other of the pmm- <dig> homoeologs from common wheat and progenitors, were consistently found in the phylogenetic trees constructed using different tree building methods .
hvpmm- <dig>  bdpmm and ospmm fell outside of the two clades, with hvpmm- <dig> showing a closer association with both clades than bdpmm and ospmm .
these observations, plus the established synteny between barley chromosome  <dig>  and rice chromosome  <dig>   <cit> , indicated that the triticeae pmm- <dig> genes were most likely the orthologs of bdpmm and ospmm, and that the triticeae pmm- <dig> member may be derived from pmm- <dig> through duplication.
trees with topology highly similar to that displayed in figure  <dig> were also obtained when the deduced amino acid sequences of the pmm genes were used for phylogenetic analysis, but they could not reveal the phylogenetic relationships of pmm-a <dig> homoeologs to other pmm genes because tapmm-a <dig>  ttpmm-a <dig> and tupmm-a <dig> could not yield intact proteins upon conceptual translation .
to find the approximate timing at which the duplication of triticeae pmm occurred, we first calculated the number of substitutions per synonymous site for the grass pmm genes characterized in this work.
the values of synonymous substitutions calculated with pmm- <dig> genes were generally lower compared to those obtained with pmm- <dig> members .
this prompted us to calculate the molecular clocks for grass pmm- <dig> and  <dig> genes, respectively.
based on the averaged synonymous substitutions between ospmm and wheat pmm- <dig> or  <dig> genes , and assuming that rice and wheat diverged about  <dig> mya  <cit> , the average nucleotide substitution rate for pmm- <dig> genes was estimated to be  <dig>  × 10- <dig> per site per year, whereas that for pmm- <dig> genes was  <dig>  × 10- <dig> per site per year.
bdpmm and wheat pmm- <dig> genes diverged about  <dig>  ±  <dig>  mya .
the divergence time between wheat pmm- <dig> and  <dig> genes was estimated to be  <dig>  ±  <dig>  or  <dig>  ±  <dig>  mya, depending on whether the clock rates of pmm- <dig> or  <dig> genes were used for the calculation .
a the ds values and their standard deviations were calculated using pmm cdna coding sequences.
tapmm-a <dig> was not included in the calculation owing to the occurrence of mutation in its coding region.
a specific molecular clock used for the estimation:  <dig>  × 10- <dig> per site per year.
b specific molecular clock:  <dig>  × 10- <dig> per site per year.
analysis of evolutionary rate and potential positive selection
the cdna sequence of  <dig> pmm genes  were aligned , and the resultant alignment was used to construct a nj tree .
the non-synonymous/synonymous substitution rate ratios  were calculated for analyzing evolution rate and potential positive selection on amino acid sites by appropriate models .
the analysis with one-ratio model  produced an averaged ω value of  <dig> , indicating that purifying selection dominated the evolution of the examined pmm gene members .
the analysis with two-ratio model revealed that the mean ω value of pmm- <dig> genes was larger than that of pmm- <dig> members, though this difference was not statistically significant by the likelihood-ratio test  comparing one-ratio with two-ratio models .
the analysis with six site specific models, m1a, m2a, m <dig> , m <dig> , m <dig> and m <dig>  suggested that more than 90% of codon sites were under strong purifying selection .
two candidate sites  were found to be under positive selection based on naïve empirical bayes  posterior probability, one of which  was significant .
however, the positive selection on site  <dig> was not significant according to bayes empirical bayes  posterior probability, and neither was it supported by lrt .
a ω values are dn/ds ratios.
p <dig>  p <dig> and p <dig> indicate the proportion of sites in categories  <dig>   <dig> or  <dig>
b the codon sites  were numbered based on tapmm-d <dig> cdna .
asterisk denotes statistical significance level at or above  <dig> .
neb, naïve empirical bayes; beb, bayes empirical bayes; na, not applicable.
transcriptional patterns of six tapmm genes
semiquantitative rt-pcr experiments, using member specific primers, were conducted to compare the transcriptional patterns of six pmm genes in xiaoyan  <dig>  in general, the transcripts of the six pmm members were detectable in both vegetative  and reproductive  organs, with relatively higher transcript levels detected in the seedling and flag leaves .
the transcript levels of tapmm- <dig> members  were generally and relatively higher than those of tapmm- <dig> members  .
the transcriptional profiles of the three tapmm- <dig> genes were basically similar, except that the transcript level of d <dig> in the immature spikes was much lower relative to those of a <dig> or b <dig> .
among the three tapmm- <dig> members, b <dig> and d <dig> showed a highly similar transcriptional profile .
the transcript levels of a <dig> in the roots, culms and immature spikes were substantially lower than those of b <dig> and d <dig> in the same set of organs .
biochemical activity of tapmm proteins
first, yeast complementation assays were carried out to test if the proteins expressed from tapmm-a <dig>  b <dig>  d <dig>  b <dig> or d <dig> may complement the temperature sensitivity of the sec53- <dig> mutant of s. cerevisiae.
tapmm-a <dig> was not included because it did not possess an intact orf .
from figure  <dig>  it is clear that the expression of tapmm-a <dig>  b <dig>  d <dig> or d <dig> restored the growth of sec53- <dig> cells at  <dig> or 37°c, and to a similar degree.
by contrast, the expression of tapmm-b <dig> complemented pmm deficiency of sec53- <dig> only weakly at 30°c, with no complementation observed at 37°c .
subsequently, tapmm-a <dig>  b <dig>  b <dig>  d <dig> and d <dig> were individually expressed in the bacterial cells, and the resultant recombinant proteins were purified using nickel affinity chromatography .
the recombinant proteins of a <dig>  b <dig>  d <dig> and d <dig> were examined for catalytic activity using mannose-1-phosphate as the substrate.
the b <dig> recombinant protein was not included in this analysis because its deduced amino acid sequence was identical to that of a <dig> .
in three independent experiments, b <dig> did not show detectable catalytic activity.
by contrast, the recombinant a <dig>  d <dig> and d <dig> proteins were all catalytically active.
the average km  values calculated for recombinant a <dig>  d <dig> and d <dig> proteins were  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
temperature-activity profiles of the recombinant pmms of rice, b. distachyon, barley and bread wheat
ospmm, bdpmm and hvpmm- <dig> were expressed in the bacterial cells, and the resultant recombinant proteins were purified as described above.
the main temperature-activity characteristics of the three pmms and tapmm-a <dig>  d <dig> and d <dig> were compared using  <dig> mm mannose-1-phosphate as the substrate and at three test temperatures .
from figure  <dig>  it is evident that the activity level of ospmm was significantly higher at  <dig> and 37°c than at 24°c.
moreover, increasing the test temperature from  <dig> and 37°c did not decrease ospmm activity significantly .
bdpmm was most active at 30°c, but its activity level was reduced by nearly 95% as the temperature increased from  <dig> to 37°c .
hvpmm- <dig> was most active at 24°c, and its activity level also decreased sharply  as the temperature shifted from  <dig> to 37°c .
the three bread wheat pmms exhibited different temperature-activity characteristics.
tapmm-a <dig> was less active than bdpmm and hvpmm- <dig> at  <dig> and 30°c, but its activity level was substantially higher than those of bdpmm and hvpmm- <dig> at 37°c, owing to a 51% increase of its activity as the temperature changed from  <dig> to 37°c .
tapmm-d <dig>  although less active than bdpmm and hvpmm- <dig> at  <dig> and 30°c, displayed substantially higher activity levels than bdpmm and hvpmm- <dig> at 37°c, because the change of test temperature from  <dig> to 37°c did not reduce tapmm-d <dig> activity as strongly as that occurred to bdpmm and hvpmm- <dig> .
the activity level of tapmm-d <dig> was lower than those of bdpmm and hvpmm- <dig> but higher than those of tapmm-a <dig> and d <dig> at 24°c .
however, tapmm-d <dig> was highly active at 30°c, with an activity level statistically comparable to that of ospmm .
the shift of assay temperature from  <dig> to 37°c reduced the activity of tapmm-d <dig> by about 40%, but the scale of this reduction was much lower compared to that observed for bdpmm, hvpmm- <dig> or tapmm-d <dig>
discussion
pmm genes and chromosomal loci in triticeae species
through the molecular cloning experiments in this work, the following suggestions may be made regarding pmm genes and chromosome loci in triticeae species.
first, there exist two distinct pmm gene members  in the progenitor diploid genomes  of bread wheat.
there are four and six pmm genes  in tetraploid and hexaploid wheats, respectively.
second, in bread wheat and its diploid and tetraploid progenitors, the loci containing pmm- <dig> or  <dig> genes are located on homoeologous groups  <dig> and  <dig> chromosomes, respectively.
third, two pmm genes  exist in diploid barley.
the loci harboring hvpmm- <dig> or  <dig> are located on the 2h and 4h chromosomes, respectively.
finally, judging from the fragment analysis data of pmm genes described in additional file  <dig>  it is probable that the basic organization of pmm gene family, as defined for the a, b, d and h genomes , is conserved in the diploid triticeae species possessing amam, cc, mm, nn, rr, ss, sbsb, slsl, ssss, sshssh, stst or uu genomes.
however, further work is needed to verify if in the  <dig> triticeae genomes the two pmm genes may also be located on homoeologous group  <dig> and  <dig> chromosomes.
the existence of two pmm genes in diploid triticeae species contrasts with the finding of a single pmm gene for arabidopsis, rice and b. distachyon .
a search of recently accumulated genomic data deposited in the phytozome website  reveals that medicago truncatula, vitis vinifera, sorghum bicolor and zea mays each possess a single pmm gene.
however, both glycine max and populus trichocarpa harbor two pmm copies in their genomes .
two functional pmm copies have also been found present in several mammals   <cit> .
biochemically active pmm members in bread wheat and its progenitors
from the presence of intact orf, the results of yeast complementation experiments, and the data of biochemical assays using recombinant proteins, we deduce that bread wheat expresses four biochemically active pmms, tapmm-a <dig>  b <dig>  d <dig> and d <dig>  the primary structure and basic biochemical function of the four pmms are similar to their orthologs in human, s. cerevisiae and arabidopsis.
in contrast to tapmm-a <dig>  b <dig>  d <dig> and d <dig>  tapmm-b <dig> is probably not active in vivo because it did not complement pmm deficiency of yeast sec53- <dig> cells efficiently, and was not catalytically competent in the in vitro biochemical assay.
the lack of detectable biochemical activity by tapmm-b <dig> correlates with the presence of unusual amino acid substitutions in its deduced protein .
further work is needed to determine the specific amino acid change responsible for decreasing the biochemical activity of tapmm-b <dig>
owing to the possession of identical amino acid sequences , it is likely that tupmm-a <dig>  behaves like tapmm-a <dig>  and aetpmm-d <dig> and d <dig> of ae.
tauschii may act like tapmm-d <dig> and d <dig>  respectively.
contrary to the above scenario, it is not possible to deduce the biochemical activity of ttpmm-b <dig> and b <dig> based on the data gathered for tapmm-b <dig> and b <dig>  because complete amino acid sequence identity did not exist between the two b <dig> or b <dig> members .
main features of pmm evolution in diploid and polyploid triticeae species
despite its vital importance in eukaryotes, the molecular evolution of pmm has not been investigated in higher plants previously, and has been studied only recently in animals.
several investigations suggest that the presence of two functional pmm copies in human and murine cells may be caused by a gene duplication event that occurred  <dig> to  <dig> mya  <cit> .
from the data presented in this work, the main features in the evolution of pmm genes in diploid and polyploid triticeae species may be summarized below.
first, a major event in pmm evolution in the diploid triticeae species carrying aa, amam, bb, cc, dd, hh, mm, nn, rr, ss, sbsb, slsl, ssss, sshssh, stst or uu genomes is the duplication of pmm.
this duplication event gave rise to two different pmm genes, pmm- <dig> and  <dig>  located on groups  <dig> and  <dig> chromosomes, respectively.
out of the two pmm genes in diploid triticeae species, pmm- <dig> is likely more ancestral.
this possibility is mainly supported by 1) the phylogenetic tree shown in figure  <dig>  2) the existence of a single pmm gene in rice, and 3) the established syntenic relationship between triticeae group  <dig> chromosomes  and rice chromosome  <dig>   <cit> .
second, the pmm duplication, as discussed above, occurred after the divergence between b. distachyon and wheat, but before the radiation of diploid triticeae genomes.
this is supported by not only the presence of two different pmm gene members in many diploid triticeae genomes examined in this work, but also the estimated divergence time between tapmm- <dig> and tapmm- <dig> genes .
the two estimates, whether  <dig>  ±  <dig>  or  <dig>  ±  <dig>  my, are both much larger than the divergence time between wheat and barley  reported in past studies  <cit> .
but they are substantially smaller than the divergence time between b. distachyon and wheat  calculated by previous investigations  <cit> .
third, substantial differences exist in the evolutionary patterns of pmm- <dig> and  <dig> genes.
pmm- <dig> genes generally had intact orf, their transcript levels in the vegetative and reproductive organs were comparatively higher , and their protein products were all biochemically active .
by contrast, multiple types of defects occurred to pmm- <dig> members.
the pmm-a <dig> copies in bread wheat and its diploid and tetraploid progenitors and hvpmm- <dig> in barley possessed mutated orf .
tapmm-b <dig> encoded a protein with undetectable biochemical activity .
the recombinant protein of tapmm-d <dig> was significantly less active than that of the product of tapmm-d <dig> .
moreover, the three pmm- <dig> members in bread wheat all exhibited lower transcript levels relative to their pmm- <dig> paralogs .
fourth, the differences of pmm- <dig> and  <dig> genes established at the diploid level  are basically retained in polyploid wheats.
thus, pmm-a <dig> is active in diploid, tetraploid and hexaploid levels whereas the orf of pmm-a <dig> is disrupted at all three ploidy levels.
the two pmm genes encoded in d genome are all active in both ae.
tauschii and bread wheat.
in contrast to the pmm genes in a and d genomes, the origin of the contrasting biochemical features of the two b genome pmm members in bread wheat is unclear at present, because the absence of a diploid species containing the b genome made it difficult to study the evolution of pmm-b <dig> and b <dig> members at the diploid level.
finally, from the data described in additional file  <dig>  the duplicated pmm genes in the g, c, n, m and u genomes are also likely retained in the tetraploid species possessing aagg, ddcc, ddmm, ddnn or uumm genomes, although further work is needed to study the pmm genes in these species in more detail.
because pmm- <dig> and  <dig> genes were located on groups  <dig> and  <dig> chromosomes, respectively, in wheat and progenitors and barley, the duplication event that led to the formation of two pmm copies in the ancestral triticeae species must have occurred interchromosomally.
strong evidence for the occurrence of interchromosomal duplications in wheat genome evolution has been obtained in recent years  <cit> .
importantly, it has been suggested that these duplications are largely conserved across different triticeae species  <cit> .
however, it is still not well understood how these events might be accomplished in triticeae genomes.
thus, the pmm duplication reported here may serve as a useful model for studying the molecular mechanisms underlying the interchromosomal duplications in triticeae in the future.
the average nucleotide substitution rates of grass pmm- <dig> or  <dig> genes estimated based on the  <dig> my divergence time between rice and wheat by this work are both lower than the commonly adopted mutation rate  for grass genes  <cit> .
this may not be unusual because evolutionary rates frequently differ among different grass genes  <cit> .
for example, the mutation rates of ten randomly selected grass genes have been found to vary from  <dig>  to  <dig>  × 10- <dig> per site per year  <cit> .
furthermore, we estimated that the divergence time between bdpmm and wheat pmm- <dig> genes was about  <dig>  ±  <dig>  my  using the average nucleotide substitution rate calculated for grass pmm- <dig> genes.
this estimate is comparable to the previously reported timeline  for the divergence of the two species  <cit> .
possible mechanisms involved in the evolution of pmm- <dig> and  <dig> genes
past studies have suggested several fates in the evolution of duplicated genes  <cit> .
neofunctionalization leads to the acquisition of new function by one copy, whereas subfunctionalization renders the progenitor and the duplicated copy acquiring non-overlapping but complementary expression patterns.
non-functionalization results in pseudogenization of one of the duplicates.
in barley, the duplicate copy of hvhox <dig>  acquired a new function in controlling spikelet fertility  <cit> .
in rice, evidence for subfunctionalization has been obtained for two c-class mads box genes   <cit> .
the evolutionary process of duplicated genes may be divided into two stages  <cit> .
during the early stage, the duplicates generally evolve under purifying selection, with a phase of relaxed selective constraint immediately after the duplication  <cit> .
during the later stage, the great majority of the duplicates become non-functional pseudogenes that are no longer expressed, with only a few of them being involved in neofunctionalization or subfunctionalization  <cit> .
from the data in table  <dig> and the main evolutionary features of pmm- <dig> and  <dig> genes discussed above, we suggest that 1) purifying selection, but not positive selection, may have dominated the evolution of pmm gene family in wheat and relatives, and 2) despite the prevalence of purifying selection, the function of pmm- <dig> copies is not as strongly maintained as that of pmm- <dig> members.
the first point is consistent with the findings that the vast majority of duplicated genes in cellular organisms experience purifying selection  <cit> , even though some of them are in the process of becoming pseudogenes  <cit> .
owing to purifying selection, the pmm- <dig> genes are generally and relatively stable.
by contrast, tapmm-a <dig> is close to becoming a non-functional pseudogene, because its orf was mutated and its transcript level was decreased relative to pmm- <dig> genes .
this scenario may also apply to pmm-a <dig> copies in the diploid and tetraploid progenitors of bread wheat and hvpmm- <dig> in barley although further work is needed to check if the transcript levels of these members may be lowered than those of pmm- <dig> genes.
tapmm-b <dig> and d <dig>  although possessing intact orf, also show signs of degenerative evolution, because their transcript levels were lower than corresponding pmm- <dig> members, and the pmm enzyme activity of their recombinant proteins was either undetectable or reduced .
the degeneration of pmm- <dig> genes and the comparatively high mean evolutionary rate of these members indicate the occurrence of relaxed purifying selection in the evolution of pmm gene family in wheat and relatives, which may have affected pmm- <dig> copies more strongly than pmm- <dig> paralogs.
however, further work is needed to verify this hypothesis.
in contrast to the degenerative evolution of the duplicated pmm- <dig> copies in diploid triticeae species, the two mammalian pmm members, products of a duplication event occurred before the radiation of mammals , all encode highly active enzymes  <cit> .
however, unlike pmm <dig> whose mutation leads to hypoglycosylation of cellular proteins and severe disease  <cit> , the deletion of pmm <dig> in mouse does not cause obvious abnormalities in either growth or development under normal conditions  <cit> .
moreover, despite their overlapping expression profiles, pmm <dig> does not compensate for the functional loss of pmm <dig>  <cit> .
these findings have led to the suggestion that pmm <dig> may have diverged from pmm <dig> in physiological function  <cit> .
this possibility has been confirmed with the recent discovery that pmm <dig> but not pmm <dig> acts as a glucose- <dig> -bisphosphatase in the brain tissues  <cit> .
clearly, the unique pmm duplication event in triticeae plants revealed in this work differs from the one in mammals in multiple aspects including the timing of the event, the evolutionary fates of pmm members after the duplication, and possibly the main mechanism involved in the evolution.
the evolutionary knowledge generated in this work increases our understanding of pmm evolution in eukaryotes, and may be useful for future studies of pmm evolution in additional plant species such as g. max and p. trichocarpa, both of which have been found to contain two pmm genes .
the main active pmm isozymes in bread wheat tissues
among the four biochemically active tapmms, functional similarity may be limited to only tapmm-a <dig> and b <dig> because their deduced proteins were identical, and the transcriptional patterns of their coding genes were very similar .
by contrast, functional differences are likely to exist among a <dig>  d <dig> and d <dig> members because of dissimilarities in their deduced amino acid sequences and transcriptional patterns.
for further analysis of the physiological function of pmm genes in bread wheat, it is necessary to find out the main active pmm isozymes in vivo.
considering the fundamental importance of pmm in the temperature adaptability of eukaryotes  <cit> , the differences in the temperature-activity profiles of the four enzymatically active tapmms are of particular interest.
from the data shown in figure  <dig>  it is suggested that tapmm-d <dig> may be highly active in a wider range of temperatures .
the activity of tapmm-a <dig> is less than that of tapmm-d <dig>  but it is unique in exhibiting the highest activity at 37°c .
the activity of tapmm-d <dig> is generally lower than that of tapmm-d <dig> and a <dig>  especially at 37°c .
this finding and the observation that the transcript levels of tapmm-d <dig> were considerably lower than those of tapmm-a <dig>  b <dig> and d <dig> in multiple organs  indicate that tapmm-a <dig>  b <dig> and d <dig> are likely to be the main active pmm isozymes in bread wheat tissues.
moreover, judging from figure  <dig>  at least two main pmm isozymes are expressed in each of the five organs  examined in this work.
as displayed in figure  <dig>  rice, a tropical cereal and adaptable to both tropical and temperate regions  <cit> , possesses a pmm showing very high activity levels at elevated test temperatures .
by contrast, b. distachyon, barley and bread wheat, originated from temperate regions  <cit> , have the pmms exhibiting relatively high activity levels at  <dig> or 30°c .
a general decrease in the activities of the pmms of b. distachyon, barley and bread wheat at 37°c seems to correlate with the temperate origin of these species.
however, compared with bdpmm and hvpmm- <dig>  at 37°c, the magnitude of activity decrease was much smaller for tapmm-d <dig> and d <dig>  and the activity of tapmm-a <dig> even increased relative to its performance at  <dig> and 30°c.
this indicates that bread wheat pmm isozymes may be generally more stable than their counterparts in b. distachyon and barley at elevated test temperatures , which correlates well with the wide temperature adaptability of the bread wheat crop  <cit> .
CONCLUSIONS
this work has generated new information on pmm genes and their evolution in bread wheat and related triticeae species.
the main active pmm isozymes  in bread wheat tissues are revealed.
the three isozymes may be more tolerant to elevated temperatures than their counterparts in barley and b. distachyon.
the insights gained in this work have broadened the understanding of pmm evolution in eukaryotic organisms.
the resources produced here may aid future investigations of the physiological function of this important gene in the temperature adaptability of bread wheat.

 phosphomannomutase, catalyzing the interconversion between mannose-6-phosphate and mannose-1-phosphate, is an essential and conserved enzyme in eukaryotic organisms. mannose-1-phosphate is necessary for synthesizing the vital cellular metabolite gdp-mannose, which plays a crucial role in the formation of polysaccharide chains required for the glycosylation of protein and lipid molecules. in higher plants, gdp-mannose also acts as an important precursor for the biosynthesis of the key antioxidant ascorbic acid, and the mannose containing polysaccharides are essential for the development of functional cell walls. from available information, the copy number of functional pmm in diploid eukaryotic species varies from one to two. pmm proteins from different eukaryotic species are highly similar in primary structure, with more than 50% amino acid sequence identities found among the pmms from s. cerevisiae, arabidopsis and human. the crystal structure and catalytic mechanism of mammalian pmms are well understood, and there is also a wealth of information on human pmm whose point mutations lead to a genetically inherited disease cdg-ia. two recent molecular genetic studies in arabidopsis show that defect in pmm function reduces asa biosynthesis and protein glycosylation, causing enhanced susceptibility to oxidation stress and plant death at high growth temperature. the requirement of a functional pmm protein for arabidopsis to grow at 28°c is consistent with the pioneering finding of temperature sensitive pmm mutations that arrest s. cerevisiae growth at 37°c, suggesting that pmm may be generally important for the temperature adaptability of eukaryotes. compared to the above progress, little is known about pmm gene and function in crop plants, although broad temperature adaptability is vitally important for crops to achieve high yield potential under diverse environmental conditions. to improve our understanding on pmm in crop plants, we have embarked on a systematic molecular and biochemical analysis of pmm genes in bread wheat, a polyploid crop species cultivated in both temperate and tropical regions and with harvest possible under a wide range of temperature conditions. bread wheat belongs to the tribe triticeae, which is composed of approximately species, and contains both diploid and polyploid crop plants. the diploid genomes in triticeae species are homoeologous, and may be combined to form polyploids through natural and artificial hybridizations. bread wheat was evolved through natural hybridization between tetraploid wheat and aegilops tauschii about years ago. t. turgidum was formed about million years ago through spontaneous hybridization between an unidentified triticeae species containing the b genome and the wild einkorn wheat t. urartu. the a, b and d genomes diverged from each other around to mya, whereas the divergence of wheat and barley occurred around mya. although bread wheat and related triticeae species generally contain large and complex genomes, the advent of structural and functional genomics research in model cereals has made it possible to conduct relatively detailed molecular genetic and biochemical studies of important genes both within triticeae and among rice, brachypodium and triticeae species. the genomic resources of rice and b. distachyon have both been proved to be very useful in the investigations of important triticeae genes, such as ph and lr34/yr in common wheat and ppd-h and cly in barley. from the information presented above, the main objectives of this work were to 1) determine pmm genes and loci in bread wheat and related triticeae species, and 2) investigate the evolution of triticeae pmm genes and the main active pmm isozymes in bread wheat tissues, with the aid of the genomic knowledge of rice and b. distachyon.  isolation and characterization of pmm genes two approaches were used to identify and isolate the pmm genes in b. distachyon, bread wheat and related triticeae species. in total, distinct pmm coding sequences were isolated. a single pmm gene was found for both rice and b. distachyon. the cdna of ospmm was reported previously, whereas that of bdpmm was isolated in this work using rt-pcr. brachypodium distachyon rice a the genomic dna and cdna sequences include start and stop codons. b plant materials from which pmm sequences were isolated. c orf disrupted by premature stop codon. d orf interrupted by frameshift mutation. e the chromosomal locus containing ospmm genomic sequence is os04g f the genbank accession number for ospmm cdna is dq six different pmm genes were isolated from each of the two bread wheat varieties, xiaoyan and chinese spring. t. urartu, ae. tauschii and barley were each found to contain two distinct pmm genes, while four different pmm genes were cloned from t. turgidum. based on the data on exon and intron pattern, nucleotide and deduced protein identities, chromosomal location, and phylogenetic clustering analysis of genomic coding sequences, the a, b and d genomes were each found to contribute two members to the six pmm genes in bread wheat, which were thus named as tapmm-a b d a b and d respectively. the pmm- genes in bread wheat and its diploid and tetraploid progenitor species and barley were homoeologous. likewisely, homoeologous relationship existed among the pmm- genes from these species. the intron and exon patterns of the pmm genes from rice, b. distachyon, and wheat and relatives were highly similar. except for ospmm and bdpmm, both of which had exons owing to the split of the first exon into two parts, the remaining genes all had exons and introns. apart from pmm-a homoeologs and hvpmm- the remaining triticeae pmm genes cloned in this work all contained intact orf. the coding region of hvpmm- was disrupted by premature stop codon based on analyzing the pcr products amplified from two barley varieties. consistent with our earlier study, the proteins deduced from the pmm genes of common wheat and progenitor species, barley and b. distachyon were more than 50% identical to the pmms of human and s. cerevisiae. the identities among the grass pmms were generally above 90%, with bdpmm showing higher identity to its homologs from bread wheat and related triticeae species than to ospmm. the identities among the deduced pmm- proteins of bread wheat and its diploid and tetraploid ancestors were generally 98% or higher, with complete identity found among the deduced tapmm-a tupmm-a ttpmm-a and tapmm-b proteins. the identities among the deduced pmm-b and d proteins of bread wheat and its diploid and tetraploid ancestors were generally 97%. by contrast, the identities between the deduced pmm- and proteins were comparatively lower, being 95% between b and b proteins, and 96% between d and d proteins. the deduced pmm-b proteins of tetraploid and bread wheats were 99% identical. interestingly, tapmm-b and ttpmm-b displayed unusual amino acid substitutions. the first one, involving the substitution of a broadly conserved glycine residue by alanine, was shared between tapmm-b and ttpmm-b. the second one was unique to tapmm-b involving the replacement of a highly conserved arginine residue by cysteine. the four structural and active site motifs, identified by analyzing the crystal structure of hspmm-, were generally conserved among the compared plant pmms. determination of pmm copy number and chromosomal locations to investigate the copy number of pmm genes, we made use of intron length polymorphisms and designed a pair of intron flanking primers, which enabled the amplification of pcr fragments containing introns iii to v. by carefully analyzing the products amplified with fluorescently labeled if primers, six distinct fragments were found for bread wheat, and their size equaled to that calculated from the six cloned tapmm genomic sequences in all cases. using the same strategy, two pmm fragments were found for t. urartu, ae. tauschii and barley, respectively, and for all three species, the size of the amplified fragments corresponded exactly to the one deduced from the cloned pmm members. four fragments were amplified from t. turgidum, whose size again showed specific correspondence to that derived from each of the four cloned ttpmm members. finally, a single fragment was amplified from b. distachyon and its size agreed with the one calculated from the cloned bdpmm genomic sequence. the copy number of pmm genes in an additional set of materials was also investigated by fragment analysis using the if primers. the results showed that the diploid triticeae species carrying the amam, cc, mm, nn, rr, ss, sbsb, slsl, ssss, sshssh, stst or uu genomes generally had two different pmm fragments, whereas the tetraploid triticeae species containing the aagg, ddcc, ddmm, ddnn or uumm genomes generally possessed four pmm specific fragments. the fluorescently labeled if primers were further employed for investigating the chromosomal locations of wheat and barley pmm members by pcr amplifications. the fragments derived from pmm-a b and d of bread wheat were not amplified from the nt lines lacking 2a, 2b and 2d chromosomes, respectively, indicating that tapmm- homoeologs were located on group chromosomes. this finding was confirmed by the fragment analysis data obtained with ldn substitution lines. the fragments derived from pmm-a and d members of bread wheat were not amplified from the nt lines lacking 4a and 4d chromosomes, respectively. moreover, the fragment from ttpmm-b was absent in the ldn substitution line lacking the 4b chromosome. together, these results indicated that tapmm- homoeologs were located on group chromosomes. using a similar strategy, the two barley pmm genes were mapped to the 2h and 4h chromosomes, respectively, which are homoeologous to wheat groups and chromosomes, respectively. the finding of six pmm genes located on groups and chromosomes in bread wheat by this work is consistent with the data generated in the wheat expressed sequence tag mapping project http://wheat.pw.usda.gov/nsf/progress_ mapping.html. the est be a bp cdna exhibiting higher than 95% nucleotide sequence identity to the 5' half of the mrnas of the six bread wheat pmm genes, has been used as a probe to hybridize with the genomic dna samples of cs and associated nt lines digested with the restriction enzyme ecori. the hybridization yields positive bands, which are mapped to groups or chromosomes. our bioinformatic analysis suggests the presence of three to five ecori sites in the genomic orf of bread wheat pmm genes, with the last ecori site located in the 8th exon. because the first seven exons give rise to the 5' half of pmm mrnas upon post transcriptional processing, the dna blot hybridization with be probe is expected to show about to hybridizing bands from six tapmm gene members. the hybridization with another pmm est probe yields approximately seven positive bands, which are mapped mainly to groups or chromosomes. consequently, it is suggested that the number of homoeologous pmm genes in bread wheat is likely to be around six. however, we can not exclude the existence of additional and divergent pmm copies or fragments in bread wheat genome, because they may not be amplified by our pcr strategy, or be distinguishable from one or more of the six completely sequenced copies during fragment analysis. duplication of pmm in diploid triticeae species the foregoing experiments indicated that there existed two pmm genes in diploid triticeae species, and a single pmm gene in either b. distachyon or rice. these findings raised an important question on how and when the two pmm gene members in diploid triticeae species were evolved. to investigate this question, phylogenetic analysis was conducted using the genomic dna sequences of pmm genes. two distinct clades, one composed of triticeae pmm- homoeologs and the other of the pmm- homoeologs from common wheat and progenitors, were consistently found in the phylogenetic trees constructed using different tree building methods. hvpmm- bdpmm and ospmm fell outside of the two clades, with hvpmm- showing a closer association with both clades than bdpmm and ospmm. these observations, plus the established synteny between barley chromosome and rice chromosome, indicated that the triticeae pmm- genes were most likely the orthologs of bdpmm and ospmm, and that the triticeae pmm- member may be derived from pmm- through duplication. trees with topology highly similar to that displayed in figure were also obtained when the deduced amino acid sequences of the pmm genes were used for phylogenetic analysis, but they could not reveal the phylogenetic relationships of pmm-a homoeologs to other pmm genes because tapmm-a ttpmm-a and tupmm-a could not yield intact proteins upon conceptual translation. to find the approximate timing at which the duplication of triticeae pmm occurred, we first calculated the number of substitutions per synonymous site for the grass pmm genes characterized in this work. the values of synonymous substitutions calculated with pmm- genes were generally lower compared to those obtained with pmm- members. this prompted us to calculate the molecular clocks for grass pmm- and genes, respectively. based on the averaged synonymous substitutions between ospmm and wheat pmm- or genes, and assuming that rice and wheat diverged about mya, the average nucleotide substitution rate for pmm- genes was estimated to be × 10- per site per year, whereas that for pmm- genes was × 10- per site per year. bdpmm and wheat pmm- genes diverged about ± mya. the divergence time between wheat pmm- and genes was estimated to be ± or ± mya, depending on whether the clock rates of pmm- or genes were used for the calculation. a the ds values and their standard deviations were calculated using pmm cdna coding sequences. tapmm-a was not included in the calculation owing to the occurrence of mutation in its coding region. a specific molecular clock used for the estimation: × 10- per site per year. b specific molecular clock: × 10- per site per year. analysis of evolutionary rate and potential positive selection the cdna sequence of pmm genes were aligned, and the resultant alignment was used to construct a nj tree. the non-synonymous/synonymous substitution rate ratios were calculated for analyzing evolution rate and potential positive selection on amino acid sites by appropriate models. the analysis with one-ratio model produced an averaged ω value of, indicating that purifying selection dominated the evolution of the examined pmm gene members. the analysis with two-ratio model revealed that the mean ω value of pmm- genes was larger than that of pmm- members, though this difference was not statistically significant by the likelihood-ratio test comparing one-ratio with two-ratio models. the analysis with six site specific models, m1a, m2a, m, m, m and m suggested that more than 90% of codon sites were under strong purifying selection. two candidate sites were found to be under positive selection based on naïve empirical bayes posterior probability, one of which was significant. however, the positive selection on site was not significant according to bayes empirical bayes posterior probability, and neither was it supported by lrt. a ω values are dn/ds ratios. p p and p indicate the proportion of sites in categories or b the codon sites were numbered based on tapmm-d cdna. asterisk denotes statistical significance level at or above. neb, naïve empirical bayes; beb, bayes empirical bayes; na, not applicable. transcriptional patterns of six tapmm genes semiquantitative rt-pcr experiments, using member specific primers, were conducted to compare the transcriptional patterns of six pmm genes in xiaoyan in general, the transcripts of the six pmm members were detectable in both vegetative and reproductive organs, with relatively higher transcript levels detected in the seedling and flag leaves. the transcript levels of tapmm- members were generally and relatively higher than those of tapmm- members. the transcriptional profiles of the three tapmm- genes were basically similar, except that the transcript level of d in the immature spikes was much lower relative to those of a or b. among the three tapmm- members, b and d showed a highly similar transcriptional profile. the transcript levels of a in the roots, culms and immature spikes were substantially lower than those of b and d in the same set of organs. biochemical activity of tapmm proteins first, yeast complementation assays were carried out to test if the proteins expressed from tapmm-a b d b or d may complement the temperature sensitivity of the sec53- mutant of s. cerevisiae. tapmm-a was not included because it did not possess an intact orf. from figure it is clear that the expression of tapmm-a b d or d restored the growth of sec53- cells at or 37°c, and to a similar degree. by contrast, the expression of tapmm-b complemented pmm deficiency of sec53- only weakly at 30°c, with no complementation observed at 37°c. subsequently, tapmm-a b b d and d were individually expressed in the bacterial cells, and the resultant recombinant proteins were purified using nickel affinity chromatography. the recombinant proteins of a b d and d were examined for catalytic activity using mannose-1-phosphate as the substrate. the b recombinant protein was not included in this analysis because its deduced amino acid sequence was identical to that of a. in three independent experiments, b did not show detectable catalytic activity. by contrast, the recombinant a d and d proteins were all catalytically active. the average km values calculated for recombinant a d and d proteins were ±, ± and ±, respectively. temperature-activity profiles of the recombinant pmms of rice, b. distachyon, barley and bread wheat ospmm, bdpmm and hvpmm- were expressed in the bacterial cells, and the resultant recombinant proteins were purified as described above. the main temperature-activity characteristics of the three pmms and tapmm-a d and d were compared using mm mannose-1-phosphate as the substrate and at three test temperatures. from figure it is evident that the activity level of ospmm was significantly higher at and 37°c than at 24°c. moreover, increasing the test temperature from and 37°c did not decrease ospmm activity significantly. bdpmm was most active at 30°c, but its activity level was reduced by nearly 95% as the temperature increased from to 37°c. hvpmm- was most active at 24°c, and its activity level also decreased sharply as the temperature shifted from to 37°c. the three bread wheat pmms exhibited different temperature-activity characteristics. tapmm-a was less active than bdpmm and hvpmm- at and 30°c, but its activity level was substantially higher than those of bdpmm and hvpmm- at 37°c, owing to a 51% increase of its activity as the temperature changed from to 37°c. tapmm-d although less active than bdpmm and hvpmm- at and 30°c, displayed substantially higher activity levels than bdpmm and hvpmm- at 37°c, because the change of test temperature from to 37°c did not reduce tapmm-d activity as strongly as that occurred to bdpmm and hvpmm-. the activity level of tapmm-d was lower than those of bdpmm and hvpmm- but higher than those of tapmm-a and d at 24°c. however, tapmm-d was highly active at 30°c, with an activity level statistically comparable to that of ospmm. the shift of assay temperature from to 37°c reduced the activity of tapmm-d by about 40%, but the scale of this reduction was much lower compared to that observed for bdpmm, hvpmm- or tapmm-d discussion pmm genes and chromosomal loci in triticeae species through the molecular cloning experiments in this work, the following suggestions may be made regarding pmm genes and chromosome loci in triticeae species. first, there exist two distinct pmm gene members in the progenitor diploid genomes of bread wheat. there are four and six pmm genes in tetraploid and hexaploid wheats, respectively. second, in bread wheat and its diploid and tetraploid progenitors, the loci containing pmm- or genes are located on homoeologous groups and chromosomes, respectively. third, two pmm genes exist in diploid barley. the loci harboring hvpmm- or are located on the 2h and 4h chromosomes, respectively. finally, judging from the fragment analysis data of pmm genes described in additional file it is probable that the basic organization of pmm gene family, as defined for the a, b, d and h genomes, is conserved in the diploid triticeae species possessing amam, cc, mm, nn, rr, ss, sbsb, slsl, ssss, sshssh, stst or uu genomes. however, further work is needed to verify if in the triticeae genomes the two pmm genes may also be located on homoeologous group and chromosomes. the existence of two pmm genes in diploid triticeae species contrasts with the finding of a single pmm gene for arabidopsis, rice and b. distachyon. a search of recently accumulated genomic data deposited in the phytozome website reveals that medicago truncatula, vitis vinifera, sorghum bicolor and zea mays each possess a single pmm gene. however, both glycine max and populus trichocarpa harbor two pmm copies in their genomes. two functional pmm copies have also been found present in several mammals. biochemically active pmm members in bread wheat and its progenitors from the presence of intact orf, the results of yeast complementation experiments, and the data of biochemical assays using recombinant proteins, we deduce that bread wheat expresses four biochemically active pmms, tapmm-a b d and d the primary structure and basic biochemical function of the four pmms are similar to their orthologs in human, s. cerevisiae and arabidopsis. in contrast to tapmm-a b d and d tapmm-b is probably not active in vivo because it did not complement pmm deficiency of yeast sec53- cells efficiently, and was not catalytically competent in the in vitro biochemical assay. the lack of detectable biochemical activity by tapmm-b correlates with the presence of unusual amino acid substitutions in its deduced protein. further work is needed to determine the specific amino acid change responsible for decreasing the biochemical activity of tapmm-b owing to the possession of identical amino acid sequences, it is likely that tupmm-a behaves like tapmm-a and aetpmm-d and d of ae. tauschii may act like tapmm-d and d respectively. contrary to the above scenario, it is not possible to deduce the biochemical activity of ttpmm-b and b based on the data gathered for tapmm-b and b because complete amino acid sequence identity did not exist between the two b or b members. main features of pmm evolution in diploid and polyploid triticeae species despite its vital importance in eukaryotes, the molecular evolution of pmm has not been investigated in higher plants previously, and has been studied only recently in animals. several investigations suggest that the presence of two functional pmm copies in human and murine cells may be caused by a gene duplication event that occurred to mya. from the data presented in this work, the main features in the evolution of pmm genes in diploid and polyploid triticeae species may be summarized below. first, a major event in pmm evolution in the diploid triticeae species carrying aa, amam, bb, cc, dd, hh, mm, nn, rr, ss, sbsb, slsl, ssss, sshssh, stst or uu genomes is the duplication of pmm. this duplication event gave rise to two different pmm genes, pmm- and located on groups and chromosomes, respectively. out of the two pmm genes in diploid triticeae species, pmm- is likely more ancestral. this possibility is mainly supported by 1) the phylogenetic tree shown in figure 2) the existence of a single pmm gene in rice, and 3) the established syntenic relationship between triticeae group chromosomes and rice chromosome. second, the pmm duplication, as discussed above, occurred after the divergence between b. distachyon and wheat, but before the radiation of diploid triticeae genomes. this is supported by not only the presence of two different pmm gene members in many diploid triticeae genomes examined in this work, but also the estimated divergence time between tapmm- and tapmm- genes. the two estimates, whether ± or ± my, are both much larger than the divergence time between wheat and barley reported in past studies. but they are substantially smaller than the divergence time between b. distachyon and wheat calculated by previous investigations. third, substantial differences exist in the evolutionary patterns of pmm- and genes. pmm- genes generally had intact orf, their transcript levels in the vegetative and reproductive organs were comparatively higher, and their protein products were all biochemically active. by contrast, multiple types of defects occurred to pmm- members. the pmm-a copies in bread wheat and its diploid and tetraploid progenitors and hvpmm- in barley possessed mutated orf. tapmm-b encoded a protein with undetectable biochemical activity. the recombinant protein of tapmm-d was significantly less active than that of the product of tapmm-d. moreover, the three pmm- members in bread wheat all exhibited lower transcript levels relative to their pmm- paralogs. fourth, the differences of pmm- and genes established at the diploid level are basically retained in polyploid wheats. thus, pmm-a is active in diploid, tetraploid and hexaploid levels whereas the orf of pmm-a is disrupted at all three ploidy levels. the two pmm genes encoded in d genome are all active in both ae. tauschii and bread wheat. in contrast to the pmm genes in a and d genomes, the origin of the contrasting biochemical features of the two b genome pmm members in bread wheat is unclear at present, because the absence of a diploid species containing the b genome made it difficult to study the evolution of pmm-b and b members at the diploid level. finally, from the data described in additional file the duplicated pmm genes in the g, c, n, m and u genomes are also likely retained in the tetraploid species possessing aagg, ddcc, ddmm, ddnn or uumm genomes, although further work is needed to study the pmm genes in these species in more detail. because pmm- and genes were located on groups and chromosomes, respectively, in wheat and progenitors and barley, the duplication event that led to the formation of two pmm copies in the ancestral triticeae species must have occurred interchromosomally. strong evidence for the occurrence of interchromosomal duplications in wheat genome evolution has been obtained in recent years. importantly, it has been suggested that these duplications are largely conserved across different triticeae species. however, it is still not well understood how these events might be accomplished in triticeae genomes. thus, the pmm duplication reported here may serve as a useful model for studying the molecular mechanisms underlying the interchromosomal duplications in triticeae in the future. the average nucleotide substitution rates of grass pmm- or genes estimated based on the my divergence time between rice and wheat by this work are both lower than the commonly adopted mutation rate for grass genes. this may not be unusual because evolutionary rates frequently differ among different grass genes. for example, the mutation rates of ten randomly selected grass genes have been found to vary from to × 10- per site per year. furthermore, we estimated that the divergence time between bdpmm and wheat pmm- genes was about ± my using the average nucleotide substitution rate calculated for grass pmm- genes. this estimate is comparable to the previously reported timeline for the divergence of the two species. possible mechanisms involved in the evolution of pmm- and genes past studies have suggested several fates in the evolution of duplicated genes. neofunctionalization leads to the acquisition of new function by one copy, whereas subfunctionalization renders the progenitor and the duplicated copy acquiring non-overlapping but complementary expression patterns. non-functionalization results in pseudogenization of one of the duplicates. in barley, the duplicate copy of hvhox acquired a new function in controlling spikelet fertility. in rice, evidence for subfunctionalization has been obtained for two c-class mads box genes. the evolutionary process of duplicated genes may be divided into two stages. during the early stage, the duplicates generally evolve under purifying selection, with a phase of relaxed selective constraint immediately after the duplication. during the later stage, the great majority of the duplicates become non-functional pseudogenes that are no longer expressed, with only a few of them being involved in neofunctionalization or subfunctionalization. from the data in table and the main evolutionary features of pmm- and genes discussed above, we suggest that 1) purifying selection, but not positive selection, may have dominated the evolution of pmm gene family in wheat and relatives, and 2) despite the prevalence of purifying selection, the function of pmm- copies is not as strongly maintained as that of pmm- members. the first point is consistent with the findings that the vast majority of duplicated genes in cellular organisms experience purifying selection, even though some of them are in the process of becoming pseudogenes. owing to purifying selection, the pmm- genes are generally and relatively stable. by contrast, tapmm-a is close to becoming a non-functional pseudogene, because its orf was mutated and its transcript level was decreased relative to pmm- genes. this scenario may also apply to pmm-a copies in the diploid and tetraploid progenitors of bread wheat and hvpmm- in barley although further work is needed to check if the transcript levels of these members may be lowered than those of pmm- genes. tapmm-b and d although possessing intact orf, also show signs of degenerative evolution, because their transcript levels were lower than corresponding pmm- members, and the pmm enzyme activity of their recombinant proteins was either undetectable or reduced. the degeneration of pmm- genes and the comparatively high mean evolutionary rate of these members indicate the occurrence of relaxed purifying selection in the evolution of pmm gene family in wheat and relatives, which may have affected pmm- copies more strongly than pmm- paralogs. however, further work is needed to verify this hypothesis. in contrast to the degenerative evolution of the duplicated pmm- copies in diploid triticeae species, the two mammalian pmm members, products of a duplication event occurred before the radiation of mammals, all encode highly active enzymes. however, unlike pmm whose mutation leads to hypoglycosylation of cellular proteins and severe disease, the deletion of pmm in mouse does not cause obvious abnormalities in either growth or development under normal conditions. moreover, despite their overlapping expression profiles, pmm does not compensate for the functional loss of pmm. these findings have led to the suggestion that pmm may have diverged from pmm in physiological function. this possibility has been confirmed with the recent discovery that pmm but not pmm acts as a glucose- -bisphosphatase in the brain tissues. clearly, the unique pmm duplication event in triticeae plants revealed in this work differs from the one in mammals in multiple aspects including the timing of the event, the evolutionary fates of pmm members after the duplication, and possibly the main mechanism involved in the evolution. the evolutionary knowledge generated in this work increases our understanding of pmm evolution in eukaryotes, and may be useful for future studies of pmm evolution in additional plant species such as g. max and p. trichocarpa, both of which have been found to contain two pmm genes. the main active pmm isozymes in bread wheat tissues among the four biochemically active tapmms, functional similarity may be limited to only tapmm-a and b because their deduced proteins were identical, and the transcriptional patterns of their coding genes were very similar. by contrast, functional differences are likely to exist among a d and d members because of dissimilarities in their deduced amino acid sequences and transcriptional patterns. for further analysis of the physiological function of pmm genes in bread wheat, it is necessary to find out the main active pmm isozymes in vivo. considering the fundamental importance of pmm in the temperature adaptability of eukaryotes, the differences in the temperature-activity profiles of the four enzymatically active tapmms are of particular interest. from the data shown in figure it is suggested that tapmm-d may be highly active in a wider range of temperatures. the activity of tapmm-a is less than that of tapmm-d but it is unique in exhibiting the highest activity at 37°c. the activity of tapmm-d is generally lower than that of tapmm-d and a especially at 37°c. this finding and the observation that the transcript levels of tapmm-d were considerably lower than those of tapmm-a b and d in multiple organs indicate that tapmm-a b and d are likely to be the main active pmm isozymes in bread wheat tissues. moreover, judging from figure at least two main pmm isozymes are expressed in each of the five organs examined in this work. as displayed in figure rice, a tropical cereal and adaptable to both tropical and temperate regions, possesses a pmm showing very high activity levels at elevated test temperatures. by contrast, b. distachyon, barley and bread wheat, originated from temperate regions, have the pmms exhibiting relatively high activity levels at or 30°c. a general decrease in the activities of the pmms of b. distachyon, barley and bread wheat at 37°c seems to correlate with the temperate origin of these species. however, compared with bdpmm and hvpmm- at 37°c, the magnitude of activity decrease was much smaller for tapmm-d and d and the activity of tapmm-a even increased relative to its performance at and 30°c. this indicates that bread wheat pmm isozymes may be generally more stable than their counterparts in b. distachyon and barley at elevated test temperatures, which correlates well with the wide temperature adaptability of the bread wheat crop.  this work has generated new information on pmm genes and their evolution in bread wheat and related triticeae species. the main active pmm isozymes in bread wheat tissues are revealed. the three isozymes may be more tolerant to elevated temperatures than their counterparts in barley and b. distachyon. the insights gained in this work have broadened the understanding of pmm evolution in eukaryotic organisms. the resources produced here may aid future investigations of the physiological function of this important gene in the temperature adaptability of bread wheat. 
the duplication event yielding pmm- and occurred before the radiation of diploid triticeae genomes. among the six tapmm genes, the transcript levels of pmm- members were comparatively high and their recombinant proteins were all enzymatically active. two sets of homoeologous pmm genes were found in bread wheat, and two corresponding pmm genes were identified in the diploid progenitors of bread wheat and many other diploid triticeae species. the pmm gene family in wheat and relatives may evolve largely under purifying selection. however, little is known about pmm gene and function in crop plants. our work identified the genes encoding pmm isozymes in bread wheat and relatives, uncovered a unique pmm duplication event in diverse triticeae species, and revealed the main active pmm isozymes in bread wheat tissues. the knowledge obtained here improves the understanding of pmm evolution in eukaryotic organisms, and may facilitate further investigations of pmm function in the temperature adaptability of bread wheat. here, we report molecular evolutionary and biochemical analysis of pmm genes in bread wheat and related triticeae species. however, pmm- homoeologs exhibited lower transcript levels, two of which were also inactive. tapmm-a b and d were probably the main active isozymes in bread wheat tissues. the three isozymes differed from their counterparts in barley and brachypodium distachyon in being more tolerant to elevated test temperatures.
 phosphomannomutase is an essential enzyme in eukaryotes. however, little is known about pmm gene and function in crop plants. here, we report molecular evolutionary and biochemical analysis of pmm genes in bread wheat and related triticeae species.  two sets of homoeologous pmm genes were found in bread wheat, and two corresponding pmm genes were identified in the diploid progenitors of bread wheat and many other diploid triticeae species. the duplication event yielding pmm- and occurred before the radiation of diploid triticeae genomes. the pmm gene family in wheat and relatives may evolve largely under purifying selection. among the six tapmm genes, the transcript levels of pmm- members were comparatively high and their recombinant proteins were all enzymatically active. however, pmm- homoeologs exhibited lower transcript levels, two of which were also inactive. tapmm-a b and d were probably the main active isozymes in bread wheat tissues. the three isozymes differed from their counterparts in barley and brachypodium distachyon in being more tolerant to elevated test temperatures.  our work identified the genes encoding pmm isozymes in bread wheat and relatives, uncovered a unique pmm duplication event in diverse triticeae species, and revealed the main active pmm isozymes in bread wheat tissues. the knowledge obtained here improves the understanding of pmm evolution in eukaryotic organisms, and may facilitate further investigations of pmm function in the temperature adaptability of bread wheat. 