BACKGROUND
birch trees grow in the temperate climate zone of the northern hemisphere and release large amounts of pollen during spring.
this pollen is a major cause of type i allergies.
the main birch allergen in northern europe is a pathogenesis-related class  <dig>  protein from the european white birch  termed bet v  <dig>  <cit> .
pollen of other fagales species contains pr- <dig> homologues that share epitopes with bet v  <dig>  <cit> , as do several fruits, nuts and vegetables  <cit> .
an ige-mediated cross-reaction to these food homologues causes the so-called oral allergy syndrome   <cit> .
pr- <dig> proteins constitute the largest group of aeroallergens and are among the four most common food allergens  <cit> .
the genus betula encompasses over  <dig> tree and shrub species that are found in diverse habitats in the boreal and temperate climate zone of the northern hemisphere.
the taxonomy of the betula genus is debated, as is the number of recognized species.
the genus is either divided into three, four or five groups or subgenera  <cit> .
b. pendula occurs in europe and is the only species whose relation to birch pollen allergy has been extensively investigated.
sensitization to birch pollen is also reported across asia and north america, where b. pendula is not present  <cit> .
other betula species occur in these areas, but their allergenic potency is unknown.
betula species may vary in their allergenicity as variation in allergenicity has been found among cultivars of apple  <cit> , peach and nectarine  <cit> , and among olive trees  <cit> .
pr- <dig> proteins are present as a multigene family in many higher plants, including gymnosperms as well as monocots and dicots  <cit> .
the classification as pr-proteins  <cit>  is based on the induced expression in response to pathogen infections by viruses, bacteria or fungi  <cit> , to wounding  <cit>  or to abiotic stress  <cit> .
some members of the pr- <dig> gene family are constitutively expressed during plant development  <cit>  or expressed in specific tissues  <cit> .
multiple pr- <dig> genes have been reported for b. pendula as well  <cit> .
mrnas of these genes have been detected in various birch tissues, including pollen  <cit> , roots, leaves  <cit> , and in cells that are grown in a liquid medium in the presence of microbial pathogens  <cit> .
pr- <dig> genes share a high sequence similarity and form a homogeneous group.
homogeneity is believed to be maintained by concerted evolution  <cit> .
arrangements of pr- <dig> genes into clusters, such as found for mal d  <dig> genes in apple, may facilitate concerted evolution  <cit> .
several bet v  <dig> isoforms have been described for b. pendula  <cit> , including both allergenic and hypoallergenic isoforms  <cit> .
individual b. pendula trees have the genetic background to produce a mixture of bet v  <dig> isoforms with varying ige-reactivity  <cit> .
the relative abundance of individual isoforms at the protein level will influence the allergenicity of the pollen.
molecular masses and sequences of tryptic peptides from bet v  <dig> can be determined by q-tof ms/ms  <cit> .
the recently developed q-tof lc-mse method enables peptide identification, but has the additional advantage of being able to determine relative abundances of peptides in a single run  <cit> .
by quantifying isoforms with a known ige-reactivity  <cit> , the allergenicity of particular birch trees can be predicted.
the existence of allergenic and hypoallergenic isoforms indicates that pr- <dig> isoforms vary in allergenicity, and some pr- <dig> isoforms do not bind ige at all.
this has already been demonstrated for two truncated bet v  <dig> isoforms  <cit> .
therefore, not all pr- <dig> isoforms are necessarily isoallergens.
knowledge on the allergenicity of birch species may facilitate selection and breeding of hypoallergenic birch trees.
to investigate the presence and abundance of bet v  <dig> isoforms in betula species that are potential crossing material, we:  cloned and sequenced pr- <dig> alleles from eight representative betula species to detect pr- <dig> genes at the genomic level,  applied q-tof lc-mse to identify the pollen-expressed bet v  <dig> genes,  determined relative abundances of isoforms in the pollen proteome, and  compared these isoforms to isoforms with a known ige-reactivity.
RESULTS
this study encompasses several experimental and analytical steps, involving both genomics and proteomics.
all main steps have been summarized in fig.
<dig>
pr- <dig> subfamilies
we examined eight betula species for the presence of pr- <dig> genes by sequencing  <dig> individual clones in both directions .
sequences that contained pcr artifacts were excluded by combining information from independent pcrs.
the open reading frames  of the sequences were highly conserved, making the alignment straightforward.
the consensus sequence of the exon had  <dig> positions excluding the  <dig> bps in the primer regions.
<dig> out of the  <dig> variable consensus positions were phylogenetically informative.
the sequences grouped into seven well-supported clusters in the neighbor joining  tree .
five clusters coincided with the division between subfamilies as found in b. pendula  <cit> .
two new subfamilies  were identified, which occurred only in two species, contrary to the previously described subfamilies  <dig> to  <dig> that were found in all species .
in all sequences, an intron was located between the first and second nucleotide of codon  <dig>  this intron was highly variable in length and composition, which was an additional characteristic for inferring the proper subfamily.
intron sequences were excluded from the phenetic/phylogenetic analysis because introns evolve at a different speed compared to exons.
species
sequenced
the number of clones sequenced in both directions and the number of identified sequences and genes are shown per species.
<dig> subfamily  <dig> to  <dig> were previously identified  <cit> , while subfamily  <dig> and  <dig> are new.
homology to mrna sequences suggests that subfamily  <dig> and  <dig> are expressed in pollen.
* <dig> species were diploid  as measured by flow cytometry.
the identification of alleles of a single gene is based on the criterion of having >  <dig> % sequence similarity, and by allowing maximally two alleles per gene.
* <dig> genes identified in b. pendula  <cit> .
pr- <dig> sequences and genes
we recovered  <dig> to  <dig> unique pr- <dig> sequences per species, adding up to  <dig> sequences in total .
out of the  <dig> unique sequences, over  <dig> sequences have never been described before.
b. pendula, b. plathyphylla and b. populifolia are closely related members of the subgenus betula and consequently had multiple alleles in common.
these species shared one allele with b. costata, which is another member of the subgenus betula.
we applied a predefined cut-off level of  <dig> % to attribute all sequences to different genes, while allowing maximally two alleles per gene per species.
these criteria coincided in the majority of cases, but several genes of b. chichibuensis in the large cluster in subfamily  <dig> and of b. lenta in subfamily  <dig>  and the genes 02a/02b and 03c/03d in b. pendula were more than  <dig> % similar.
table  <dig> shows the total number of identified pr- <dig> genes per species.
out of the  <dig> genes that have previously been identified in b. pendula ,  <dig> genes were recovered from the newly sequenced b. pendula cultivar 'youngii'.
this study identified no new genes in this cultivar.
this indicates that the majority of genes has been recovered by sequencing over  <dig> clones per species, and that only a small number of genes might be missing in the dataset.
homologues of the pr- <dig> genes of b. pendula were identified in b. populifolia and b. plathyphylla.
sequences from these species were labeled according to the procedure described by gao et al.
<cit>  that was previously used for b. pendula  <cit> .
these labels consist of the subfamily's number, followed by a letter for each distinct gene, then a number for each unique protein variant and an additional number referring to silent mutations.
when applicable, an additional letter indicates variations in the intron.
the pr- <dig> genes in b. costata displayed a considerable degree of homology to the genes in b. pendula, but differentiating homologues and paralogues was not always possible.
it was not possible to differentiate between homologues and paralogues of the pr- <dig> genes in b. lenta, b chichibuensis, b. nigra, and b. schmidtii.
rather than developing a separate denomination scheme for each species, we labeled sequences with the pr- <dig> subfamily number, followed by a number for each unique protein variant and an additional number referring to silent mutations.
this facilitates the protein analysis which distinguishes protein variants rather than separate alleles or genes.
the pr- <dig> gene copy number varied between different birch species.
this is caused by evolutionary processes such as duplication, extinction, and recombination.
the overall clustering pattern appears to reflect a combination of such events.
genes from the same species tend to group close to each other on several positions in the nj tree .
examples are the clusters of highly similar sequences from b. costata in subfamily  <dig> and from b. chichibuensis in subfamily  <dig>  which either reflect unequal crossing-over, gene conversion or duplication events.
the b. populifolia genome harbors two clear examples of unequal crossing-over.
allele 01e <dig>  is a recombination between the 01a gene and the 01b gene.
the first part matches exactly to allele 01a <dig> , while the second part differs by  <dig> snp from 01b <dig>  with position  <dig> of the orf as the point of recombination.
both original genes were also present.
similarly, allele 03e <dig>  is a recombination between the 03b gene and the 03d gene.
in this case, the recombination probably occurred without gene duplication, since the original 03b gene, as present in b. pendula, was absent.
pr- <dig> protein predictions
not all pr- <dig> alleles will be expressed as a full-sized protein.
<dig> unique sequences had an intact orf, while the remaining  <dig> sequences contain early stop codons or indels in the orf that result in frame shifts followed by an early stop codon.
the latter sequences were denoted as pseudogenes, although it cannot be excluded that these sequences produce truncated proteins.
we calculated ka/ks ratios within each subfamily.
the suspected pseudogenes displayed higher ka/ks ratios than the alleles with an intact orf in the subfamilies  <dig>   <dig> and  <dig> .
this points to an alleviated selection pressure in the pseudogenes.
the other pr- <dig> subfamilies do not contain sufficient numbers of both genes and pseudogenes to perform this comparison.
the majority of sequences had 5' splicing sites of ag:gt and 3' splicing sites of ag:gc, ag:gt or ag:ga, which is in concordance with known motifs for plant introns.
notable exceptions were: an ac:gt  and an ag:at  5' splicing site, an ac:gc  and a tg:gc  3' splicing site, and two deletions  at the 3' end of the intron.
except for the ag:at splicing site, all exceptions belonged to sequences that were denoted as pseudogenes, providing additional evidence for these designations.
n = number of unique sequences.
ka/ks ratio = ratio between non-synonymous and synonymous mutations.
range substitutions = minimum and maximum number of amino acid substitutions in pair wise comparisons between sequences of the same subfamilies.
n. d. = not determined.
depending on the subfamily, ka/ks ratios ranged from  <dig>  to  <dig>  for sequences with an intact orf , indicating strong purifying selection.
the pr- <dig> alleles in birch encode a putative protein that consists of  <dig> amino acids, yielding a relative molecular mass of approximately  <dig> kda.
the only exception is 01var <dig>  in b. chichibuensis, which contains an indel that results in the deletion of two amino acids.
the allelic variation is lower at the protein level than at the nucleic acid level, which is consistent with the low ka/ks ratios.
hence, the  <dig> unique genomic sequences encode  <dig> unique isoforms.
the pr- <dig>  gene is an extreme example for which only four putative isoforms are predicted, despite the presence of  <dig> allelic variants.
one of these isoforms is predicted in all species except b. nigra.
parts of the pr- <dig> protein sequences are highly conserved, as is demonstrated in the amino-acid alignment of five pr- <dig> isoforms  from b. pendula .
the most prominent region lies between glu <dig> and ile <dig> and contains only a single amino acid variation among all  <dig> isoforms.
a phosphate-binding loop with the sequence motive gxggxgx characterizes this region.
additional conserved glycine residues are present at positions  <dig>   <dig>   <dig>   <dig> and  <dig>
bet v  <dig> expression in pollen
the presence of bet v 1-like proteins was examined in pollen of b. nigra, b. chichibuensis, b. lenta, b. costata and b. pendula 'youngii'.
pollen proteins were solubilized in an aqueous buffer and analyzed by sds-page.
each sample displayed an intense protein band after cbb-staining at the expected molecular mass of bet v  <dig>  between 16– <dig> kda , while other intense bands were visible at  <dig> kda and  <dig> kda.
no 16– <dig> kda band was visible when the pellet that remained after extraction was separated by sds-page , indicating the efficiency of the extraction procedure with regard to bet v  <dig>
to establish the identity of the proteins in the 16– <dig> kda band, we cut out this band from the lane of b. pendula  and performed in-gel digestion with trypsin.
q-tof lc-ms/ms analysis of the tryptic peptides yielded multiple bet v  <dig> isoforms .
the bands just above and below the 16– <dig> kda band were also sequenced and checked for the presence of bet v  <dig>  the lower band at  <dig> kda contained birch profilin  and contained no bet v  <dig> fragments.
the higher band at  <dig> kda contained birch cyclophilin  and some minor traces of bet v  <dig> .
bollen et al.
<cit>  detected a band of ~ <dig> kda when purified bet v  <dig> was analyzed by sds-page, consisting of  bet v  <dig>  we identified the intense band at ~ <dig> kda in our b. pendula extract as isoflavone reductase  and detected no bet v  <dig> fragments in this band.
analysis of bet v  <dig> isoforms by q-tof lc-mse
the tryptic digests of the 16– <dig> kda bands were examined in detail to elucidate the expression of separate bet v  <dig> isoforms in pollen.
trypsin cleaves proteins exclusively at the c-terminus of arginine and lysine.
fig.
<dig> shows an example of the fragments i to xvii that are theoretically formed after tryptic digestion of isoforms from the subfamilies  <dig> to  <dig>  isoforms of different subfamilies can be discriminated by several fragments on the basis of peptide mass and sequence.
the number of discriminating fragments becomes lower for bet v  <dig> isoforms within a subfamily.
a new mass spectrometric technique called q-tof lc-mse allows simultaneous identification and quantification of peptides .
a distinct feature of the lc-mse procedure is that information is obtained for all peptides.
this contrasts ms/ms, in which a subset of peptides is selected for fragmentation.
a software program analyses the data, while using a search database for interpretation of the fragmentation spectra.
this database contained the sequence information of all pr- <dig> isoforms described in this paper and of previously described pr- <dig> isoforms from b. pendula  <cit> .
the lc-mse results indicated that pr- <dig> proteins of subfamily  <dig> and  <dig> are expressed in the pollen of the five examined birch species.
we found no evidence for the expression of genes from subfamilies  <dig> to  <dig> in pollen.
for example, we identified  <dig> bet v  <dig> peptide fragments in b. pendula , all of which were predicted from the gdna sequences.
eight detected peptides could distinguish between isoforms of subfamily  <dig> and  <dig>  the b. pendula genome contains seven genes from subfamily  <dig> and  <dig>  the expression of four of these  was confirmed .
sequence coverage of the expressed isoforms amounted to  <dig> to 79% .
four peptides were specific for isoform 01b <dig>  while one peptide was specific for isoform 02c <dig>  two peptides were specific for both isoforms of gene 01a, while two others were specific for both isoforms of gene 01c.
isoforms 02a <dig> and 02b <dig> could not be separated, so either one or both of them are expressed.
table  <dig> also shows the peptide fragments that were long enough to be detected in the tryptic digest, but were not observed.
information on absent fragments can be used to exclude expression of particular isoforms, such as isoform 01d <dig> in b. pendula.
each isoform is displayed on a separate line.
when isoforms are encoded by the same gene this is indicated in the third column.
note that gene labels in one species do not correspond to gene labels in other species.
peptide fragments are shown at the top of the table and are labelled with roman numbers as indicated in fig.
<dig>  each variant of these fragments is displayed in the table by a letter.
bold capital letters indicate that a fragment is unique for the isoforms of a particular gene.
bold italic letters indicate that a fragment is unique for the isoforms of a particular subfamily.
letters displayed between brackets indicate that a particular fragment was predicted, but was absent in the pr- <dig> mixture.
finally, the last column displays the coverage of the total protein sequence, including the fragments that were too small to be detected .
fig.
<dig> displays the representative amino acid sequences of the isoforms 01a <dig> and 02a <dig>
* <dig> the isoforms in subfamily  <dig> to  <dig> were summarized into a single row and not displayed for the other species, because specific peptides were not detected in any of the species.
* <dig> fragments xa and xg have exactly the same mass and cannot be distinguished.
the peak of peptide xc overlaps with the first isotope peak of peptide xa = g because they differ exactly  <dig> da in size and have the same charge.
as a consequence, xc cannot be identified separately.
* <dig> the xvi-peptides are not always detected because of their small size.
altogether, at least  <dig> to  <dig> isoforms were expressed in each of the five examined species.
in total, the presence of unique peptides confirmed the expression of  <dig> isoforms among the five species in total .
an additional  <dig> isoforms lacked one or more unique peptides to distinguish them from other isoforms or from each other, but several of these must be expressed.
the expression of five isoforms was ruled out, because multiple unique peptides from these variants were lacking from the peptide mixture.
two identified peptides in b. costata and one peptide from b. nigra did not match to any sequence that was recovered from these species.
these peptides belong to "unknown isoforms"  and this indicates that the sequences that encode these isoforms are missing from the dataset.
finally, conflicting evidence was found for expression of the isoforms 01var <dig> and 01var <dig> in b. lenta.
two peptides that were unique for these isoforms were detected, while three peptides that were expected if the isoforms would be expressed were lacking.
expression of an allele that is missing from our dataset is a more likely explanation than the expression of 01var <dig> or 01var <dig>
quantification by q-tof lc-mse
we determined the relative amounts of individual bet v  <dig> isoforms in pollen from b. pendula 'youngii' .
this information can be deduced from the peak intensities of bet v  <dig> peptides in the tryptic digest.
not all identified fragments can be used for quantification, because the peak detection algorithm groups peaks with highly similar masses and retention times together, also when they might belong to different fragments.
for example, fragment ia  and viia  have a retention time that is marginally different, causing a strong overlap in peak area.
the relative amounts of two isoforms could be estimated directly: peptide iiif is unique for isoform 02c <dig> and comprises 17% of all fragment iii-variants, while peptides iiib and xb are unique for 01b <dig> and comprise 18–19% of all fragment iii and x-variants.
the isoforms 02a <dig> and 02b <dig> could not be separated, but together they comprise 13% of the mixture based on fragment iiie.
the relative amounts of the other isoforms were estimated indirectly.
isoform 01a <dig> and 01b <dig> share fragment vb, which comprises 23% of all fragment v-variants.
01a <dig> is thus estimated to comprise 4–5% of the mixture.
the ratio between 01b <dig> and 01c <dig> plus 01c <dig> can be deduced from fragment ib.
01c <dig> plus 01c <dig> are thus estimated to comprise 6% of the mixture.
this leaves 40–41% of the total amount of bet v  <dig> for isoform 01a <dig>
numbers indicate the relative amount of fragment variants compared to the total amount of homologues fragments.
amounts were averaged over the two duplicates.
note that quantification was not possible for all peptide variants <dig>  and that the displayed abundances indicate the relative amounts among those variants that could be quantified.
n.q.
= not possible to quantify.
*  <dig> quantification was not possible for all the peptide variants, because ia  and viiia , and ij  and viiic  had a similar mass.
fragment viiik overlaps with a keratin peptide.
*  <dig> fragments xa and xg have exactly the same mass and cannot be distinguished.
the peptide peak of xc overlaps with the first isotope peak of peptide xa = g because they differ exactly 1da in size and have the same charge.
xc cannot be identified as a result.
isoform 01a <dig> is identical to isoform bet v 1a, which had the highest ige-reactivity in several tests performed by ferreira et al.
<cit> .
pollen of b. costata, b. nigra and b. chichibuensis contained isoforms that are highly similar to bet v 1a and differ by only 1– <dig> amino acids from this isoform.
we determined the expression of individual bet v  <dig> isoforms in a similar fashion as reported for b. pendula.
the bet v 1a-like isoforms were estimated to comprise 38% , 36–44%  and 36–41%  of the total amount of bet v  <dig>  b. lenta differed from the other species, because the isoform with the highest similarity to bet v 1a differed by seven amino acids.
this isoform was estimated to comprise 12–19% of the total amount of bet v  <dig>  the expression of subfamily  <dig> isoforms relative to subfamily  <dig> isoforms was another major difference between b. lenta and the other species.
in b. lenta, subfamily  <dig> accounted for 74–83% of the total amount of bet v  <dig>  compared to 25–40% in b. pendula, b. nigra and b. chichibuensis and 49–56% in b. costata.
discussion
pr- <dig> gene family organization and evolution
the presence and diversity of bet v  <dig> and closely related pr- <dig> genes in eight birch species was established by amplification and sequencing of more than  <dig> clones per species.
the eight species belong to four different subgenera/groups in the genus betula  <cit>  and thereby represent a large part of the existing variation within the genus.
each birch species contains pr- <dig> genes, as could be expected given the broad range of plant species in which pr- <dig> genes are found  <cit> .
the pr- <dig> genes grouped into subfamilies, as previously reported for b. pendula  <cit> .
five subfamilies were recovered from all species.
two new subfamilies were identified, but these were each restricted to two species and were mostly composed of pseudogenes.
the pr- <dig> subfamily has a complex genomic organization.
differentiating between paralogues and homologues was not possible beyond closely related species.
one likely explanation is concerted evolution, for which cladistic evidence was found .
concerted evolution causes genes to evolve as a single unit whose members  exchange genetic information through gene conversion or unequal crossing-over  <cit> .
tandemly arranged genes have increased conversion rates, while such an arrangement is a prerequisite for the occurrence of unequal crossing-over  <cit> .
most pr- <dig> genes in apple are arranged in a duplicated cluster  <cit> , thus facilitating the main mechanisms for concerted evolution.
we obtained two alleles that appear the direct result of unequal crossing-over between bet v  <dig> genes.
on a higher taxonomic level, cladistic evidence for concerted evolution is present in the overall gene tree of the pr- <dig> family  <cit> , as sequence divergence is generally smaller between different genes from the same species than between genes from different species.
nei and rooney  <cit>  suggested that a combination of recent gene duplications and purifying selection could also explain why tandem gene duplicates appear similar.
in their model of birth-and-death evolution of genes, new genes arise due to gene duplications, evolve independently while undergoing purifying selection, and go extinct after becoming non-functional.
pseudogenes are characteristic for this process.
the low ka/ks ratios clearly point to the occurrence of purifying selection.
pseudogenes are a common feature among the pr- <dig> genes in birch, since we recovered them from six out of eight species.
as much as one-third of the recovered alleles in b. nigra had an interrupted orf.
we did not determine the potential expression of these alleles, since truncated isoforms would have migrated outside the 16– <dig> kda band in the sds-page.
none were, however, detected in the  <dig> kda band.
basically, all ingredients for the "birth-and-death" model are present, except that independent evolution is questionable due to the presences of duplicates that resulted from unequal crossing-over.
moreover, the clustering of for example the b. chichibuensis alleles  would suggest an extremely high number of recent duplications.
both processes of "birth-and-death" and concerted evolution may, therefore, be active in the pr- <dig> gene family.
regardless of the evolutionary processes, its outcome is clear: pr- <dig> proteins are homogenous as a group and even stronger so within subfamilies.
the high homogeneity allowed us to use q-tof lc-mse to quantify the relative expression of separate bet v  <dig> isoforms, because large differences in amino acid composition would have distorted the quantification.
bet v  <dig> expression
which pr- <dig> genes are actually expressed in pollen and are thereby the true bet v  <dig> allergens?
we used q-tof analysis to investigate the expression of bet v  <dig> isoforms in pollen of five betula species.
isoforms from subfamily  <dig> and  <dig> were identified in birch pollen, confirming predictions based on mrna expression  <cit> .
the single gene in subfamily  <dig> that was present in all eight birch species, is homologous to ãpr10c, which has a high basal transcription level in roots and a relatively lower basal transcription level in leaves  <cit> .
its expression is induced by copper stress  <cit>  and during senescence in leaves  <cit> .
regarding subfamily  <dig>  the genes pr- <dig> c and 03d  in b. pendula become transcriptionally upregulated upon infection of the leaves with fungal pathogens  <cit> .
their transcription is induced by wounding or auxin treatment in roots  <cit> .
no data have been reported about the expression of the sequenced pr- <dig> genes in subfamilies  <dig>   <dig> and  <dig>
the pollen-expressed bet v  <dig> genes are transcribed during the late stages of anther development  <cit> , but which factors induce transcription is unknown.
bet v  <dig> is an abundant pollen protein that has been estimated to encompass 10% of the total protein in b. pendula pollen  <cit> .
the bet v  <dig> band was the most intense band in the sds-page gels of birch pollen extracts.
its exact abundance is difficult to estimate due to differences in extraction efficiency between different proteins.
however, given the low amount of residual protein in the pellet, our results suggest that the abundance of bet v  <dig> is higher than 10% of the total protein content and is likely to exceed 20%.
the occurrence of bet v  <dig> isoforms in b. pendula has previously been studied in a mixture of pollen from different trees by swoboda et al.
<cit> .
they analyzed tryptic digests of purified bet v  <dig> isoforms by plasma desorption mass spectrometry , a technique that only reveals peptide masses.
we examined pollen from individual trees and analyzed the tryptic digests by q-tof lc-mse, which reveals total masses of peptides and the underlying amino acid sequences, based on available sequence information.
the ability to determine the peptide sequences yields more accurate information on expression of individual isoforms.
we demonstrated that at least  <dig> to  <dig> isoforms were expressed in the pollen of one single tree of the birch species b. pendula, b. nigra, b. chichibuensis, b. lenta and b. costata.
the actual number is likely to be higher since we could not discriminate each individual isoform due to the high similarity between some isoforms.
q-tof lc-mse has the advantageous ability to simultaneously separate, identify and quantify peptide fragments.
a similar strategy has recently been followed by chassaigne et al.
<cit> .
they identified five peanut-specific peptide ions that were used as specific tags for the peanut allergenic proteins ara h  <dig>  ara h  <dig>  and ara h  <dig>  the relative intensity of the specific peptides even provided information on the processing history of the peanut material.
napoli et al.
<cit>  also used mass spectrometry to analyze an ole e  <dig> mixture of multiple isoforms and their post-translational modifications, which could not be separate completely by 2-dimension gel electrophoresis.
a disadvantage of using q-tof lc-mse instead of q-tof lc-msms in combination with 2d gel electrophoresis and western blotting – in which allergic sera and specific anti-ige antibodies are employed – is that our method does not distinguish ige-binding isoforms from non-ige-binding isoforms.
therefore, not all described pr- <dig> isoforms are necessarily true isoallergens.
we included no purification step in the extraction procedure apart from protein separation on sds-page.
this minimizes the chance that certain isoforms are lost during purification, but the bet v  <dig> protein band might be contaminated with other pollen proteins with a similar mass.
three peptides of the pollen allergen bet v  <dig> were detected in the 16– <dig> kda band, but the amount of bet v  <dig> was estimated to be less than 2% of the amount of bet v  <dig>  based on the peak intensities of these peptides.
all peptides with high peak intensities could be attributed to bet v  <dig> isoforms.
full sequence coverage of bet v  <dig> isoforms cannot be achieved by using only trypsin as a protease, as smaller peptides will be lost during peptide extraction from the sds-page gel.
proteases that cleave at other sites will yield peptides that cover part of the missing protein sequence.
coverage with q-tof lc-mse was 71–79% for the b. pendula isoforms, which is higher than the 57–60% coverage reported for q-tof ms/ms  <cit> .
swoboda et al.
<cit>  estimated that, based on pdms peak areas of peptides, the relative amount of bet v 1a  in the pollen mixture from several b. pendula trees was at least 50% of the total amount of bet v  <dig>  ferreira et al.
<cit>  estimated the relative amounts of different bet v  <dig> isoforms by nh2-terminal sequencing of purified natural bet v  <dig> and reported a ~2:2: <dig> ratio for isoforms that respectively contain ser, thr and ile at the 7th amino acid position.
this would correspond to expression of the isoforms 02a01+02b01+02c01: 01a01+01a06: 01b01+01c04+01c <dig> in pollen of b. pendula 'youngii'.
when we sum our results in this way, a ratio of 30%:45%:25% is obtained.
the similarity between our results and previously obtained estimates suggests that the quantities obtained from b. pendula 'youngii' are also representative for other b. pendula trees.
allergenicity
ferreira et al.
<cit>  distinguished bet v  <dig> isoforms with a low, intermediate and high ige-reactivity.
expression of the isoforms 01b <dig> , 02c <dig> , 01c <dig>  and 01a <dig>  in the pollen of b. pendula 'youngii' was confirmed by identification of unique peptides .
isoforms of all three levels of ige-reactivity were abundant and encompassed 35–38% , 22–24%  and 18–19%  of the total amount of bet v  <dig>  this leaves 17–22% of the total bet v  <dig> for isoforms with an unknown ige-reactivity.
we observed similar quantities in two other b. pendula cultivars as well .
since b. pendula is known to be highly allergenic, the presence of isoforms with a high ige-reactivity is apparently of determining influence on its allergenicity.
interestingly, people that are not yet sensitized to b. pendula pollen come into contact with several abundant isoforms.
however, the ability of these isoforms to provoke an ige-mediated response varies in patients that have become sensitized  <cit> .
the factors that cause one isoform to develop into having a high ige-reactivity and another isoform into having a low ige-reactivity are currently unknown.
the abundance of the isoforms may play a role, but as isoforms with intermediate or low ige-reactivity are also present in considerable quantities, this is unlikely to be the only factor.
recently, gao et al.
<cit> , investigated the association of allelic diversity of mal d  <dig> and allergenicity in ten pedigree-linked apple cultivars, and found that qualitative as well as quantitative factors were involved.
the opportunities for identifying birch trees that only express hypoallergenic isoforms are limited.
the isoforms bet v 1l and bet v1d  are currently known as hypoallergenic  <cit> .
the crystal-structure of bet v 1l has been determined  <cit>  and its allergenicity has recently been tested on a large group of patients  <cit> .
however, none of the examined species contained bet v 1l, despite thorough examination, and bet v 1l may represent a sequencing artifact or an unexpressed allele.
only b. pendula, b. populifolia and b. plathyphylla contained 01b <dig>  the most similar isoforms in the other species differed by at least five amino acids.
in contrast, the highly allergenic isoform 01a <dig> is expressed in pollen of b. pendula 'youngii', while the b. populifolia genome also contains the 01a <dig> sequence.
the other betula species do not harbor bet v 1a, but b. chichibuensis, b. costata and b. nigra contain isoforms that differ only by 1– <dig> amino acids from bet v 1a.
a high similarity between isoforms increases the chance that they share epitopes, although a few amino acid substitutions may influence the allergenicity drastically  <cit> .
in all species, these isoforms are abundant.
b. lenta forms an exception as the isoform most similar to bet v 1a has a sequence similarity of  <dig> % and encodes a protein that differs by seven amino acids.
CONCLUSIONS
we identified  <dig> to  <dig> unique pr- <dig> sequences in each of eight different birch species.
application of q-tof lc-mse revealed that genes from two large subfamilies  were expressed in birch pollen.
we showed that q-tof lc-mse allowed fast screening of bet v  <dig> isoforms in birch pollen by determining presence and relative abundances of individual isoforms.
the pollen of four birch species contained a mixture of bet v  <dig> isoforms, with abundant levels of isoforms that were similar to isoforms with a high ige-reactivity.
we predict that the allergenic potency of these species will be high.
b. lenta  lacked isoforms with a high similarity to isoforms with a high ige-reactivity.
this species and related species represent the most promising candidates for further screening of hypoallergenicity by for example skin prick tests or nasal challenges.

 birch trees grow in the temperate climate zone of the northern hemisphere and release large amounts of pollen during spring. this pollen is a major cause of type i allergies. the main birch allergen in northern europe is a pathogenesis-related class protein from the european white birch termed bet v. pollen of other fagales species contains pr- homologues that share epitopes with bet v, as do several fruits, nuts and vegetables. an ige-mediated cross-reaction to these food homologues causes the so-called oral allergy syndrome. pr- proteins constitute the largest group of aeroallergens and are among the four most common food allergens. the genus betula encompasses over tree and shrub species that are found in diverse habitats in the boreal and temperate climate zone of the northern hemisphere. the taxonomy of the betula genus is debated, as is the number of recognized species. the genus is either divided into three, four or five groups or subgenera. b. pendula occurs in europe and is the only species whose relation to birch pollen allergy has been extensively investigated. sensitization to birch pollen is also reported across asia and north america, where b. pendula is not present. other betula species occur in these areas, but their allergenic potency is unknown. betula species may vary in their allergenicity as variation in allergenicity has been found among cultivars of apple, peach and nectarine, and among olive trees. pr- proteins are present as a multigene family in many higher plants, including gymnosperms as well as monocots and dicots. the classification as pr-proteins is based on the induced expression in response to pathogen infections by viruses, bacteria or fungi, to wounding or to abiotic stress. some members of the pr- gene family are constitutively expressed during plant development or expressed in specific tissues. multiple pr- genes have been reported for b. pendula as well. mrnas of these genes have been detected in various birch tissues, including pollen, roots, leaves, and in cells that are grown in a liquid medium in the presence of microbial pathogens. pr- genes share a high sequence similarity and form a homogeneous group. homogeneity is believed to be maintained by concerted evolution. arrangements of pr- genes into clusters, such as found for mal d genes in apple, may facilitate concerted evolution. several bet v isoforms have been described for b. pendula, including both allergenic and hypoallergenic isoforms. individual b. pendula trees have the genetic background to produce a mixture of bet v isoforms with varying ige-reactivity. the relative abundance of individual isoforms at the protein level will influence the allergenicity of the pollen. molecular masses and sequences of tryptic peptides from bet v can be determined by q-tof ms/ms. the recently developed q-tof lc-mse method enables peptide identification, but has the additional advantage of being able to determine relative abundances of peptides in a single run. by quantifying isoforms with a known ige-reactivity, the allergenicity of particular birch trees can be predicted. the existence of allergenic and hypoallergenic isoforms indicates that pr- isoforms vary in allergenicity, and some pr- isoforms do not bind ige at all. this has already been demonstrated for two truncated bet v isoforms. therefore, not all pr- isoforms are necessarily isoallergens. knowledge on the allergenicity of birch species may facilitate selection and breeding of hypoallergenic birch trees. to investigate the presence and abundance of bet v isoforms in betula species that are potential crossing material, we: cloned and sequenced pr- alleles from eight representative betula species to detect pr- genes at the genomic level, applied q-tof lc-mse to identify the pollen-expressed bet v genes, determined relative abundances of isoforms in the pollen proteome, and compared these isoforms to isoforms with a known ige-reactivity.  this study encompasses several experimental and analytical steps, involving both genomics and proteomics. all main steps have been summarized in fig. pr- subfamilies we examined eight betula species for the presence of pr- genes by sequencing individual clones in both directions. sequences that contained pcr artifacts were excluded by combining information from independent pcrs. the open reading frames of the sequences were highly conserved, making the alignment straightforward. the consensus sequence of the exon had positions excluding the bps in the primer regions. out of the variable consensus positions were phylogenetically informative. the sequences grouped into seven well-supported clusters in the neighbor joining tree. five clusters coincided with the division between subfamilies as found in b. pendula. two new subfamilies were identified, which occurred only in two species, contrary to the previously described subfamilies to that were found in all species. in all sequences, an intron was located between the first and second nucleotide of codon this intron was highly variable in length and composition, which was an additional characteristic for inferring the proper subfamily. intron sequences were excluded from the phenetic/phylogenetic analysis because introns evolve at a different speed compared to exons. species sequenced the number of clones sequenced in both directions and the number of identified sequences and genes are shown per species. subfamily to were previously identified, while subfamily and are new. homology to mrna sequences suggests that subfamily and are expressed in pollen. * species were diploid as measured by flow cytometry. the identification of alleles of a single gene is based on the criterion of having > % sequence similarity, and by allowing maximally two alleles per gene. * genes identified in b. pendula. pr- sequences and genes we recovered to unique pr- sequences per species, adding up to sequences in total. out of the unique sequences, over sequences have never been described before. b. pendula, b. plathyphylla and b. populifolia are closely related members of the subgenus betula and consequently had multiple alleles in common. these species shared one allele with b. costata, which is another member of the subgenus betula. we applied a predefined cut-off level of % to attribute all sequences to different genes, while allowing maximally two alleles per gene per species. these criteria coincided in the majority of cases, but several genes of b. chichibuensis in the large cluster in subfamily and of b. lenta in subfamily and the genes 02a/02b and 03c/03d in b. pendula were more than % similar. table shows the total number of identified pr- genes per species. out of the genes that have previously been identified in b. pendula, genes were recovered from the newly sequenced b. pendula cultivar'youngii'. this study identified no new genes in this cultivar. this indicates that the majority of genes has been recovered by sequencing over clones per species, and that only a small number of genes might be missing in the dataset. homologues of the pr- genes of b. pendula were identified in b. populifolia and b. plathyphylla. sequences from these species were labeled according to the procedure described by gao et al. that was previously used for b. pendula. these labels consist of the subfamily's number, followed by a letter for each distinct gene, then a number for each unique protein variant and an additional number referring to silent mutations. when applicable, an additional letter indicates variations in the intron. the pr- genes in b. costata displayed a considerable degree of homology to the genes in b. pendula, but differentiating homologues and paralogues was not always possible. it was not possible to differentiate between homologues and paralogues of the pr- genes in b. lenta, b chichibuensis, b. nigra, and b. schmidtii. rather than developing a separate denomination scheme for each species, we labeled sequences with the pr- subfamily number, followed by a number for each unique protein variant and an additional number referring to silent mutations. this facilitates the protein analysis which distinguishes protein variants rather than separate alleles or genes. the pr- gene copy number varied between different birch species. this is caused by evolutionary processes such as duplication, extinction, and recombination. the overall clustering pattern appears to reflect a combination of such events. genes from the same species tend to group close to each other on several positions in the nj tree. examples are the clusters of highly similar sequences from b. costata in subfamily and from b. chichibuensis in subfamily which either reflect unequal crossing-over, gene conversion or duplication events. the b. populifolia genome harbors two clear examples of unequal crossing-over. allele 01e is a recombination between the 01a gene and the 01b gene. the first part matches exactly to allele 01a, while the second part differs by snp from 01b with position of the orf as the point of recombination. both original genes were also present. similarly, allele 03e is a recombination between the 03b gene and the 03d gene. in this case, the recombination probably occurred without gene duplication, since the original 03b gene, as present in b. pendula, was absent. pr- protein predictions not all pr- alleles will be expressed as a full-sized protein. unique sequences had an intact orf, while the remaining sequences contain early stop codons or indels in the orf that result in frame shifts followed by an early stop codon. the latter sequences were denoted as pseudogenes, although it cannot be excluded that these sequences produce truncated proteins. we calculated ka/ks ratios within each subfamily. the suspected pseudogenes displayed higher ka/ks ratios than the alleles with an intact orf in the subfamilies and. this points to an alleviated selection pressure in the pseudogenes. the other pr- subfamilies do not contain sufficient numbers of both genes and pseudogenes to perform this comparison. the majority of sequences had 5' splicing sites of ag:gt and 3' splicing sites of ag:gc, ag:gt or ag:ga, which is in concordance with known motifs for plant introns. notable exceptions were: an ac:gt and an ag:at 5' splicing site, an ac:gc and a tg:gc 3' splicing site, and two deletions at the 3' end of the intron. except for the ag:at splicing site, all exceptions belonged to sequences that were denoted as pseudogenes, providing additional evidence for these designations. n = number of unique sequences. ka/ks ratio = ratio between non-synonymous and synonymous mutations. range substitutions = minimum and maximum number of amino acid substitutions in pair wise comparisons between sequences of the same subfamilies. n. d. = not determined. depending on the subfamily, ka/ks ratios ranged from to for sequences with an intact orf, indicating strong purifying selection. the pr- alleles in birch encode a putative protein that consists of amino acids, yielding a relative molecular mass of approximately kda. the only exception is 01var in b. chichibuensis, which contains an indel that results in the deletion of two amino acids. the allelic variation is lower at the protein level than at the nucleic acid level, which is consistent with the low ka/ks ratios. hence, the unique genomic sequences encode unique isoforms. the pr- gene is an extreme example for which only four putative isoforms are predicted, despite the presence of allelic variants. one of these isoforms is predicted in all species except b. nigra. parts of the pr- protein sequences are highly conserved, as is demonstrated in the amino-acid alignment of five pr- isoforms from b. pendula. the most prominent region lies between glu and ile and contains only a single amino acid variation among all isoforms. a phosphate-binding loop with the sequence motive gxggxgx characterizes this region. additional conserved glycine residues are present at positions and bet v expression in pollen the presence of bet v 1-like proteins was examined in pollen of b. nigra, b. chichibuensis, b. lenta, b. costata and b. pendula'youngii'. pollen proteins were solubilized in an aqueous buffer and analyzed by sds-page. each sample displayed an intense protein band after cbb-staining at the expected molecular mass of bet v between 16– kda, while other intense bands were visible at kda and kda. no 16– kda band was visible when the pellet that remained after extraction was separated by sds-page, indicating the efficiency of the extraction procedure with regard to bet v to establish the identity of the proteins in the 16– kda band, we cut out this band from the lane of b. pendula and performed in-gel digestion with trypsin. q-tof lc-ms/ms analysis of the tryptic peptides yielded multiple bet v isoforms. the bands just above and below the 16– kda band were also sequenced and checked for the presence of bet v the lower band at kda contained birch profilin and contained no bet v fragments. the higher band at kda contained birch cyclophilin and some minor traces of bet v. bollen et al. detected a band of ~ kda when purified bet v was analyzed by sds-page, consisting of bet v we identified the intense band at ~ kda in our b. pendula extract as isoflavone reductase and detected no bet v fragments in this band. analysis of bet v isoforms by q-tof lc-mse the tryptic digests of the 16– kda bands were examined in detail to elucidate the expression of separate bet v isoforms in pollen. trypsin cleaves proteins exclusively at the c-terminus of arginine and lysine. fig. shows an example of the fragments i to xvii that are theoretically formed after tryptic digestion of isoforms from the subfamilies to isoforms of different subfamilies can be discriminated by several fragments on the basis of peptide mass and sequence. the number of discriminating fragments becomes lower for bet v isoforms within a subfamily. a new mass spectrometric technique called q-tof lc-mse allows simultaneous identification and quantification of peptides. a distinct feature of the lc-mse procedure is that information is obtained for all peptides. this contrasts ms/ms, in which a subset of peptides is selected for fragmentation. a software program analyses the data, while using a search database for interpretation of the fragmentation spectra. this database contained the sequence information of all pr- isoforms described in this paper and of previously described pr- isoforms from b. pendula. the lc-mse results indicated that pr- proteins of subfamily and are expressed in the pollen of the five examined birch species. we found no evidence for the expression of genes from subfamilies to in pollen. for example, we identified bet v peptide fragments in b. pendula, all of which were predicted from the gdna sequences. eight detected peptides could distinguish between isoforms of subfamily and the b. pendula genome contains seven genes from subfamily and the expression of four of these was confirmed. sequence coverage of the expressed isoforms amounted to to 79%. four peptides were specific for isoform 01b while one peptide was specific for isoform 02c two peptides were specific for both isoforms of gene 01a, while two others were specific for both isoforms of gene 01c. isoforms 02a and 02b could not be separated, so either one or both of them are expressed. table also shows the peptide fragments that were long enough to be detected in the tryptic digest, but were not observed. information on absent fragments can be used to exclude expression of particular isoforms, such as isoform 01d in b. pendula. each isoform is displayed on a separate line. when isoforms are encoded by the same gene this is indicated in the third column. note that gene labels in one species do not correspond to gene labels in other species. peptide fragments are shown at the top of the table and are labelled with roman numbers as indicated in fig. each variant of these fragments is displayed in the table by a letter. bold capital letters indicate that a fragment is unique for the isoforms of a particular gene. bold italic letters indicate that a fragment is unique for the isoforms of a particular subfamily. letters displayed between brackets indicate that a particular fragment was predicted, but was absent in the pr- mixture. finally, the last column displays the coverage of the total protein sequence, including the fragments that were too small to be detected. fig. displays the representative amino acid sequences of the isoforms 01a and 02a * the isoforms in subfamily to were summarized into a single row and not displayed for the other species, because specific peptides were not detected in any of the species. * fragments xa and xg have exactly the same mass and cannot be distinguished. the peak of peptide xc overlaps with the first isotope peak of peptide xa = g because they differ exactly da in size and have the same charge. as a consequence, xc cannot be identified separately. * the xvi-peptides are not always detected because of their small size. altogether, at least to isoforms were expressed in each of the five examined species. in total, the presence of unique peptides confirmed the expression of isoforms among the five species in total. an additional isoforms lacked one or more unique peptides to distinguish them from other isoforms or from each other, but several of these must be expressed. the expression of five isoforms was ruled out, because multiple unique peptides from these variants were lacking from the peptide mixture. two identified peptides in b. costata and one peptide from b. nigra did not match to any sequence that was recovered from these species. these peptides belong to "unknown isoforms" and this indicates that the sequences that encode these isoforms are missing from the dataset. finally, conflicting evidence was found for expression of the isoforms 01var and 01var in b. lenta. two peptides that were unique for these isoforms were detected, while three peptides that were expected if the isoforms would be expressed were lacking. expression of an allele that is missing from our dataset is a more likely explanation than the expression of 01var or 01var quantification by q-tof lc-mse we determined the relative amounts of individual bet v isoforms in pollen from b. pendula'youngii'. this information can be deduced from the peak intensities of bet v peptides in the tryptic digest. not all identified fragments can be used for quantification, because the peak detection algorithm groups peaks with highly similar masses and retention times together, also when they might belong to different fragments. for example, fragment ia and viia have a retention time that is marginally different, causing a strong overlap in peak area. the relative amounts of two isoforms could be estimated directly: peptide iiif is unique for isoform 02c and comprises 17% of all fragment iii-variants, while peptides iiib and xb are unique for 01b and comprise 18–19% of all fragment iii and x-variants. the isoforms 02a and 02b could not be separated, but together they comprise 13% of the mixture based on fragment iiie. the relative amounts of the other isoforms were estimated indirectly. isoform 01a and 01b share fragment vb, which comprises 23% of all fragment v-variants. 01a is thus estimated to comprise 4–5% of the mixture. the ratio between 01b and 01c plus 01c can be deduced from fragment ib. 01c plus 01c are thus estimated to comprise 6% of the mixture. this leaves 40–41% of the total amount of bet v for isoform 01a numbers indicate the relative amount of fragment variants compared to the total amount of homologues fragments. amounts were averaged over the two duplicates. note that quantification was not possible for all peptide variants and that the displayed abundances indicate the relative amounts among those variants that could be quantified. n.q. = not possible to quantify. * quantification was not possible for all the peptide variants, because ia and viiia, and ij and viiic had a similar mass. fragment viiik overlaps with a keratin peptide. * fragments xa and xg have exactly the same mass and cannot be distinguished. the peptide peak of xc overlaps with the first isotope peak of peptide xa = g because they differ exactly 1da in size and have the same charge. xc cannot be identified as a result. isoform 01a is identical to isoform bet v 1a, which had the highest ige-reactivity in several tests performed by ferreira et al.. pollen of b. costata, b. nigra and b. chichibuensis contained isoforms that are highly similar to bet v 1a and differ by only 1– amino acids from this isoform. we determined the expression of individual bet v isoforms in a similar fashion as reported for b. pendula. the bet v 1a-like isoforms were estimated to comprise 38%, 36–44% and 36–41% of the total amount of bet v b. lenta differed from the other species, because the isoform with the highest similarity to bet v 1a differed by seven amino acids. this isoform was estimated to comprise 12–19% of the total amount of bet v the expression of subfamily isoforms relative to subfamily isoforms was another major difference between b. lenta and the other species. in b. lenta, subfamily accounted for 74–83% of the total amount of bet v compared to 25–40% in b. pendula, b. nigra and b. chichibuensis and 49–56% in b. costata. discussion pr- gene family organization and evolution the presence and diversity of bet v and closely related pr- genes in eight birch species was established by amplification and sequencing of more than clones per species. the eight species belong to four different subgenera/groups in the genus betula and thereby represent a large part of the existing variation within the genus. each birch species contains pr- genes, as could be expected given the broad range of plant species in which pr- genes are found. the pr- genes grouped into subfamilies, as previously reported for b. pendula. five subfamilies were recovered from all species. two new subfamilies were identified, but these were each restricted to two species and were mostly composed of pseudogenes. the pr- subfamily has a complex genomic organization. differentiating between paralogues and homologues was not possible beyond closely related species. one likely explanation is concerted evolution, for which cladistic evidence was found. concerted evolution causes genes to evolve as a single unit whose members exchange genetic information through gene conversion or unequal crossing-over. tandemly arranged genes have increased conversion rates, while such an arrangement is a prerequisite for the occurrence of unequal crossing-over. most pr- genes in apple are arranged in a duplicated cluster, thus facilitating the main mechanisms for concerted evolution. we obtained two alleles that appear the direct result of unequal crossing-over between bet v genes. on a higher taxonomic level, cladistic evidence for concerted evolution is present in the overall gene tree of the pr- family, as sequence divergence is generally smaller between different genes from the same species than between genes from different species. nei and rooney suggested that a combination of recent gene duplications and purifying selection could also explain why tandem gene duplicates appear similar. in their model of birth-and-death evolution of genes, new genes arise due to gene duplications, evolve independently while undergoing purifying selection, and go extinct after becoming non-functional. pseudogenes are characteristic for this process. the low ka/ks ratios clearly point to the occurrence of purifying selection. pseudogenes are a common feature among the pr- genes in birch, since we recovered them from six out of eight species. as much as one-third of the recovered alleles in b. nigra had an interrupted orf. we did not determine the potential expression of these alleles, since truncated isoforms would have migrated outside the 16– kda band in the sds-page. none were, however, detected in the kda band. basically, all ingredients for the "birth-and-death" model are present, except that independent evolution is questionable due to the presences of duplicates that resulted from unequal crossing-over. moreover, the clustering of for example the b. chichibuensis alleles would suggest an extremely high number of recent duplications. both processes of "birth-and-death" and concerted evolution may, therefore, be active in the pr- gene family. regardless of the evolutionary processes, its outcome is clear: pr- proteins are homogenous as a group and even stronger so within subfamilies. the high homogeneity allowed us to use q-tof lc-mse to quantify the relative expression of separate bet v isoforms, because large differences in amino acid composition would have distorted the quantification. bet v expression which pr- genes are actually expressed in pollen and are thereby the true bet v allergens? we used q-tof analysis to investigate the expression of bet v isoforms in pollen of five betula species. isoforms from subfamily and were identified in birch pollen, confirming predictions based on mrna expression. the single gene in subfamily that was present in all eight birch species, is homologous to ãpr10c, which has a high basal transcription level in roots and a relatively lower basal transcription level in leaves. its expression is induced by copper stress and during senescence in leaves. regarding subfamily the genes pr- c and 03d in b. pendula become transcriptionally upregulated upon infection of the leaves with fungal pathogens. their transcription is induced by wounding or auxin treatment in roots. no data have been reported about the expression of the sequenced pr- genes in subfamilies and the pollen-expressed bet v genes are transcribed during the late stages of anther development, but which factors induce transcription is unknown. bet v is an abundant pollen protein that has been estimated to encompass 10% of the total protein in b. pendula pollen. the bet v band was the most intense band in the sds-page gels of birch pollen extracts. its exact abundance is difficult to estimate due to differences in extraction efficiency between different proteins. however, given the low amount of residual protein in the pellet, our results suggest that the abundance of bet v is higher than 10% of the total protein content and is likely to exceed 20%. the occurrence of bet v isoforms in b. pendula has previously been studied in a mixture of pollen from different trees by swoboda et al.. they analyzed tryptic digests of purified bet v isoforms by plasma desorption mass spectrometry, a technique that only reveals peptide masses. we examined pollen from individual trees and analyzed the tryptic digests by q-tof lc-mse, which reveals total masses of peptides and the underlying amino acid sequences, based on available sequence information. the ability to determine the peptide sequences yields more accurate information on expression of individual isoforms. we demonstrated that at least to isoforms were expressed in the pollen of one single tree of the birch species b. pendula, b. nigra, b. chichibuensis, b. lenta and b. costata. the actual number is likely to be higher since we could not discriminate each individual isoform due to the high similarity between some isoforms. q-tof lc-mse has the advantageous ability to simultaneously separate, identify and quantify peptide fragments. a similar strategy has recently been followed by chassaigne et al.. they identified five peanut-specific peptide ions that were used as specific tags for the peanut allergenic proteins ara h ara h and ara h the relative intensity of the specific peptides even provided information on the processing history of the peanut material. napoli et al. also used mass spectrometry to analyze an ole e mixture of multiple isoforms and their post-translational modifications, which could not be separate completely by 2-dimension gel electrophoresis. a disadvantage of using q-tof lc-mse instead of q-tof lc-msms in combination with 2d gel electrophoresis and western blotting – in which allergic sera and specific anti-ige antibodies are employed – is that our method does not distinguish ige-binding isoforms from non-ige-binding isoforms. therefore, not all described pr- isoforms are necessarily true isoallergens. we included no purification step in the extraction procedure apart from protein separation on sds-page. this minimizes the chance that certain isoforms are lost during purification, but the bet v protein band might be contaminated with other pollen proteins with a similar mass. three peptides of the pollen allergen bet v were detected in the 16– kda band, but the amount of bet v was estimated to be less than 2% of the amount of bet v based on the peak intensities of these peptides. all peptides with high peak intensities could be attributed to bet v isoforms. full sequence coverage of bet v isoforms cannot be achieved by using only trypsin as a protease, as smaller peptides will be lost during peptide extraction from the sds-page gel. proteases that cleave at other sites will yield peptides that cover part of the missing protein sequence. coverage with q-tof lc-mse was 71–79% for the b. pendula isoforms, which is higher than the 57–60% coverage reported for q-tof ms/ms. swoboda et al. estimated that, based on pdms peak areas of peptides, the relative amount of bet v 1a in the pollen mixture from several b. pendula trees was at least 50% of the total amount of bet v ferreira et al. estimated the relative amounts of different bet v isoforms by nh2-terminal sequencing of purified natural bet v and reported a ~2:2: ratio for isoforms that respectively contain ser, thr and ile at the 7th amino acid position. this would correspond to expression of the isoforms 02a01+02b01+02c01: 01a01+01a06: 01b01+01c04+01c in pollen of b. pendula'youngii'. when we sum our results in this way, a ratio of 30%:45%:25% is obtained. the similarity between our results and previously obtained estimates suggests that the quantities obtained from b. pendula'youngii' are also representative for other b. pendula trees. allergenicity ferreira et al. distinguished bet v isoforms with a low, intermediate and high ige-reactivity. expression of the isoforms 01b, 02c, 01c and 01a in the pollen of b. pendula'youngii' was confirmed by identification of unique peptides. isoforms of all three levels of ige-reactivity were abundant and encompassed 35–38%, 22–24% and 18–19% of the total amount of bet v this leaves 17–22% of the total bet v for isoforms with an unknown ige-reactivity. we observed similar quantities in two other b. pendula cultivars as well. since b. pendula is known to be highly allergenic, the presence of isoforms with a high ige-reactivity is apparently of determining influence on its allergenicity. interestingly, people that are not yet sensitized to b. pendula pollen come into contact with several abundant isoforms. however, the ability of these isoforms to provoke an ige-mediated response varies in patients that have become sensitized. the factors that cause one isoform to develop into having a high ige-reactivity and another isoform into having a low ige-reactivity are currently unknown. the abundance of the isoforms may play a role, but as isoforms with intermediate or low ige-reactivity are also present in considerable quantities, this is unlikely to be the only factor. recently, gao et al., investigated the association of allelic diversity of mal d and allergenicity in ten pedigree-linked apple cultivars, and found that qualitative as well as quantitative factors were involved. the opportunities for identifying birch trees that only express hypoallergenic isoforms are limited. the isoforms bet v 1l and bet v1d are currently known as hypoallergenic. the crystal-structure of bet v 1l has been determined and its allergenicity has recently been tested on a large group of patients. however, none of the examined species contained bet v 1l, despite thorough examination, and bet v 1l may represent a sequencing artifact or an unexpressed allele. only b. pendula, b. populifolia and b. plathyphylla contained 01b the most similar isoforms in the other species differed by at least five amino acids. in contrast, the highly allergenic isoform 01a is expressed in pollen of b. pendula'youngii', while the b. populifolia genome also contains the 01a sequence. the other betula species do not harbor bet v 1a, but b. chichibuensis, b. costata and b. nigra contain isoforms that differ only by 1– amino acids from bet v 1a. a high similarity between isoforms increases the chance that they share epitopes, although a few amino acid substitutions may influence the allergenicity drastically. in all species, these isoforms are abundant. b. lenta forms an exception as the isoform most similar to bet v 1a has a sequence similarity of % and encodes a protein that differs by seven amino acids.  we identified to unique pr- sequences in each of eight different birch species. application of q-tof lc-mse revealed that genes from two large subfamilies were expressed in birch pollen. we showed that q-tof lc-mse allowed fast screening of bet v isoforms in birch pollen by determining presence and relative abundances of individual isoforms. the pollen of four birch species contained a mixture of bet v isoforms, with abundant levels of isoforms that were similar to isoforms with a high ige-reactivity. we predict that the allergenic potency of these species will be high. b. lenta lacked isoforms with a high similarity to isoforms with a high ige-reactivity. this species and related species represent the most promising candidates for further screening of hypoallergenicity by for example skin prick tests or nasal challenges. 
bet v isoforms from the european white birch have been investigated extensively, but the allergenic potency of other birch species is unknown. q-tof lc-mse was applied to identify which pr-10/bet v genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. isoforms that were similar to isoforms with a high ige-reactivity were abundant in all species except b. lenta, while the hypoallergenic isoform bet v 1d was only found in b. pendula and its closest relatives. with the possible exception of b. lenta, isoforms identical or very similar to those with a high ige-reactivity were found in the pollen proteome of all examined birch species. bet v is an important cause of hay fever in northern europe. the presence of bet v and closely related pr- genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus betula. all examined birch species contained several pr- genes. five subfamilies were common to all birch species. genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. in total, unique sequences were recovered. q-tof lc-mse allows efficient screening of bet v isoforms by determining the presence and relative abundance of these isoforms in pollen. b. pendula contains a bet v 1-mixture in which isoforms with a high and low ige-reactivity are both abundant. sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies.
 bet v is an important cause of hay fever in northern europe. bet v isoforms from the european white birch have been investigated extensively, but the allergenic potency of other birch species is unknown. the presence of bet v and closely related pr- genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus betula. q-tof lc-mse was applied to identify which pr-10/bet v genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.  all examined birch species contained several pr- genes. in total, unique sequences were recovered. sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. five subfamilies were common to all birch species. genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. isoforms that were similar to isoforms with a high ige-reactivity were abundant in all species except b. lenta, while the hypoallergenic isoform bet v 1d was only found in b. pendula and its closest relatives.  q-tof lc-mse allows efficient screening of bet v isoforms by determining the presence and relative abundance of these isoforms in pollen. b. pendula contains a bet v 1-mixture in which isoforms with a high and low ige-reactivity are both abundant. with the possible exception of b. lenta, isoforms identical or very similar to those with a high ige-reactivity were found in the pollen proteome of all examined birch species. consequently, these species are also predicted to be allergenic with regard to bet v related allergies. 