BACKGROUND
carotenoids represent a diverse group of pigments found in bacteria, fungi and plants  <cit> .
in plants, carotenoids have several important functions, essential for plant development as:  they serve as accessory pigments to harvest light for photosynthesis and constitute the basic structural units of photosynthetic apparatus and  they also act as photoprotectors for plants to adapt to high light stress  <cit> .
in addition, oxidative cleavage of carotenoids produces apocarotenoids considered as  signals in plant development,  antifungal agents,  contributors to flower and fruit flavours and aromas  <cit> .
the phyto-hormone abscisic acid, the most well known apocarotenoid, plays an important role in plant response to stress conditions  <cit> .
in addition, carotenoids have long been considered as essential nutrients as well as important health beneficial compounds.
in animals including humans, unable to synthesize carotenoids de novo, dietary carotenoids are essential precursors of vitamin a and retinoid compounds needed in development  <cit> .
plant carotenogenesis occurs in the membrane of chloroplasts, chromoplasts and amyloplasts, plastids having different internal membrane architectures.
the enzymes involved in the carotenoid biosynthetic pathway are nuclear encoded and targeted to the plastids.
carotenoid biosynthesis begins with the formation of the 40-carbon backbone, phytoene, a step mediated by phytoene synthase    <cit> .
psy, which is known to control carotenoid flux in seeds and to catalyze the first committed step in carotenogenesis, is thought to be a rate-limiting enzyme.
in “golden rice”  <cit> , for example, psy activity was crucial to obtain increased carotenoid levels  <cit> , whereas bacterial carotene desaturase crti had no effect  <cit> .
psy is coded by a small gene family, which had been already shown to exist throughout the grasses  <cit>  and which consists of three paralogous genes that have been identified and characterized in rice, maize and sorghum.
the three rice psy genes share the same structure, with six exons and five introns, and they exhibit a tissue-specific expression.
rice endosperm is carotenoid free and transcripts for all three genes are absent.
in photosynthetic tissues, all three psy mrnas are present, but with different levels of expression, ospsy <dig> and ospsy <dig> playing predominant roles with similar expression patterns and regulation in response to light.
ospsy <dig> plays a specialized role as it is not regulated by light but is strongly inducible in roots by high salt concentrations and/or drought.
ospsy <dig> and ospsy <dig> contain cis-acting elements involved in light regulation which are absent from the promoter region of ospsy <dig>  explaining why psy <dig> does not respond to light  <cit> .
li et al.
<cit>  described the psy gene family and their tissue-specific transcript patterns in maize.
they showed that psy <dig> is the only gene family member for which transcript level increased during maize endosperm development and was correlated with accumulation of endosperm carotenoids.
transcript changes of psys were monitored in maize leaves during de-etiolation.
in dark-grown plants, psy <dig> represented the major transcript whereas psy <dig> was the only paralog for which transcript levels increased in response to illumination, suggesting then that psy <dig> plays an important role in controlling leaf carotenogenesis during greening.
the abundance of psy <dig> in roots suggests that it plays a unique role in roots carotenogenesis when induced by drought, salt and exogenous aba treatment.
the increase of psy <dig> transcripts observed was associated with an induced level of carotenoid intermediates, and elevation of other downstream carotenogenic genes, followed by aba accumulation  <cit> .
psy genes are of primary importance in wheat due to their impact on flour colour, so that numerous genetics studies have been conducted on yellow pigment  content in wheat grain  <cit> .
a high yp concentration is a desirable trait in durum wheat and is the target of breeding programs worldwide  <cit> .
however, low yp is a breeding target in hexaploid wheat, for which bright white flour is preferred.
the inheritance of yp is complex, controlled by additive gene effects and highly heritable  <cit> .
several qtls for yp have been identified in both durum and bread wheat on chromosomes 1a  <cit> , 1b  <cit> , 3a  <cit> , 3b  <cit> , 4a and 5a  <cit> , 2a, 4b and 6b  <cit> , 5b  <cit> , and 6a  <cit> .
however, the majority of studies agreed that chromosome group  <dig> has the greatest impact in controlling yp content in bread wheat  <cit> .
elouafi et al.
<cit>  identified two minor qtls on chromosome 7a and a third major qtl on chromosome 7bl.
this last qtl has been corroborated in two other durum wheat populations  <cit>  and is ortholog to the one identified in hexaploid wheat on the same chromosome  <cit> .
a qtl for yp in durum wheat was identified by patil et al.
<cit>  on chromosome 7a that accounted for up to 55% of the trait variation.
mares and campbell  <cit>  identified qtls on chromosome 7a in two hexaploid wheat populations that had a large impact on flour yellowness, and correspond to loci also associated with xanthophyll concentration.
cloning of genes controlling flour pigmentation would be of great help in breeding for genotypes with appropriate levels of yellow pigments in durum and bread wheat.
using a pcr-based approach cenci et al.
<cit>  identified and mapped clones from a durum wheat bac library thought to contain genes coding for important enzymes involved in carotenogenesis.
in their study, putative phytoene synthase, phytoene desaturase and ζ-carotene desaturase clones were assigned to the chromosome groups  <dig>   <dig> and  <dig>  respectively.
to date, four psy genes have been identified in durum wheat, forming two paralogous series on chromosome groups  <dig>  and  <dig>   <cit> .
one of these, psy1-b <dig>  co-segregates with a qtl for yp on 7bl  <cit> .
psy1-a <dig> was localized on the distal end of the chromosome 7al in one hexaploid wheat mapping population, associated with variation for flour colour  <cit> .
wheat psy <dig> genomic sequences show similar exon-intron characteristic to psy <dig> known in other grass species, with six exons separated by five introns  <cit> .
a partial sequence of psy <dig> was also obtained and it was mapped on the short arm of the chromosome group  <dig>  <cit> , with no clear data regarding the impact of psy <dig> on carotenoid accumulation.
in this article, we report evidence that the wheat psy gene family also consists of three paralogous genes and that psy <dig> is characterized by a novel gene structure, consequence of intron loss events.
since the wheat psy <dig> promoter region is characterized by cis-acting elements involved in plant response to abiotic stress, we studied changes in wheat psy expression levels in leaves and roots during exogenous treatment with aba.
finally, after comparative genomic investigation of psy loci, we reconstructed the paleo-history for this gene family in grasses identifying evolutionary events that have shaped psy gene copies in modern species.
RESULTS
psy <dig> gene sequencing and mapping in bread wheat
we recently reassessed the syntenic relationships within monocots  and developed tools to identify precisely chromosome-to-chromosome orthologous relationships and derived conserved orthologous set  markers,  <cit> .
rice , sorghum  and brachypodium  psy <dig> orthologs, available from the literature, respectively mapped on chromosomes  <dig>   <dig> and  <dig> were used to clone wheat psy genes.
on the basis of the known rice/sorghum/wheat/brachypodium syntenic blocks covering psy <dig> orthologs, psy <dig> was expected to be located on chromosome group  <dig> in wheat .
the blast search did not result in significant orthologous wheat sequences from public databanks .
primers were therefore developed from conserved genic regions characterized between rice and brachypodium psy <dig> coding sequences to screen a bread wheat bac library .
this library is organised in 384-well plates of bac pools with a specific screening strategy to reduce the number of pcr reactions necessary to identify a single clone of interest .
primer pairs psy_4g <dig> _bra_f2/psy_ <dig> _bra_r <dig>  designed on the fourth exon of brachypodium psy <dig>  were used for library screening.
positive pools were sequenced and three homoeoforms were obtained and assigned to the wheat chromosomes using nullisomic-tetrasomic lines  <cit> .
by crossing the pool coordinates  we identified the bac clones harbouring psy <dig> gene homoeologs.
three bac clones spanning the psy <dig> gene of a, b and d sub-genomes of t. aestivum were screened with pcr-specific markers targeting each of the three psy <dig> homoeologs  and then sequenced.
sequencing resulted in  <dig>  <dig>   <dig>  <dig> and  <dig> 149 bp of gapped contigs for, respectively, the a, b and d sub-genomes.
each homoeologous copy was then precisely assigned to chromosome bins using genome-specific primer pairs  and dna from deletion lines: psy3s were located precisely on the chromosome bins 5al10- <dig> - <dig> , 5bl14- <dig> - <dig>  and 5dl1- <dig> - <dig> , respectively, for a, b and d genome copies.
identification and characterization of psy <dig> loci
bac sequencing and annotation  made it possible to identify psy <dig> orthologous regions.
we selected ungapped contigs of  <dig> 000 bp,  <dig> 411 bp and  <dig> 803 bp harbouring, respectively, psy3a, psy3b and psy3d genes .
figure 2b illustrates bac annotation results as well as micro-colinearity with rice, brachypodium, maize and sorghum genome sequences, confirming that psy <dig> and bradi4g <dig>  genes are at orthologous positions.
in the psy <dig> loci we found the insertion of a non-syntenic gene .
according to paleogenomic data in monocots  <cit> , this gene is located on the syntenic chromosomes  <dig>   <dig> and  <dig> of, rice, sorghum and brachypodium genomes respectively.
in wheat, this gene would be expected to map on the orthologous chromosome group  <dig> and not group  <dig>  where it was found based on the psy sequenced region assignation.
we can thus suppose that this gene has been specifically transposed in wheat from an ancestral donor region  to the modern current acceptor region .
alignment with brachypodium psy <dig> cds clarified wheat psy3s exon-intron boundaries.
wheat psy3s showed a structure characterized by four exons spaced by three introns that were numbered according to their position within the genes, with the first intron closest to the 5′ and the third intron closest to the 3′ end .
this structure and exon-intron sizes were conserved between the a, b and d homoeologous copies.
rice and maize psy <dig> were the only genes with six exons spaced by five introns.
in brachypodium and sorghum, the loss of the third intron determined the appearance of a different structure.
in rice and maize, this intron showed at its donor site, the t/c transition, starting with the gc motif instead of gt.
we also found in rice psy <dig> the presence of miniature inverted transposable elements , in the first intron: nt628-nt <dig> and nt953-nt <dig>  and in the third intron  the same mite was identified in inverted orientation.
wheat psy <dig> was found to include the largest first exon of 466 bp, 433 bp and 436 bp, respectively, for psy3a, b and d, resulting from fusion of the first and second exons after an intron loss event.
the second exon had 412 bp and resulted from the fusion of the third and fourth exons.
it was characterized by a close sequence homology with the third exon of the brachypodium ortholog.
figure 3a shows psy <dig> gene structure evolution throughout grasses.
rice and maize share the same psy <dig> gene structure, while sorghum and brachypodium gene structures are characterized, as in wheat, by the loss of the third intron and the subsequent fusion of the two flanking exons.
we suggest that, during evolution, different and independent intron loss events occurred.
at the deletion breakpoints, we identified repeated inversed motifs, tgg|cca and cgg|ccg  in the first and the second exon respectively.
it is therefore possible to assume that these motifs have facilitated intron loss events through illegitimate recombination.
the first intron lost was at phase  <dig>  while the second was at phase  <dig>  the tssp database  defined the promoter region of psy <dig>  around  <dig> kb upstream from the start codon and polya signal miner software identified the terminator signal 865 bp, 867 bp and 828 bp, respectively for a, b and d genomes.
sequence analysis allowed us to identify a duplication of the fourth exon in the psy3d locus, 1729 bp downstream from the stop codon.
this repeat did not start with the trinucleotide gtg, as expected based on the exon # <dig> sequence , and was interrupted by a 154 bp deletion flanked at each extremity by the tactgg motif.
we can thus suppose that a novel illegitimate recombination event mediated the duplication of the fourth exon and its insertion in the sequence.
despite intron/exon shuffling events reported previously, it was possible to predict protein structure.
psy3a encodes a protein of  <dig> amino acids, longer than those coded by psy3b and psy3d homoeologs  .
the ncbi conserved domain search software allowed us to identify the trans-isoprenyl diphosphate synthase  domain which includes squalene and phytoene synthases.
according to the previous domain characterization, wheat psy <dig> was classed in the ipss superfamily and class i terpene cyclise.
the conserved trans-ipps domain was identified at positions: aa123- <dig>  aa112- <dig> and aa113- <dig>  respectively, for psy3a, b and d copies.
protein motifs identified based on scanprosite are available in additional file 1: figure s <dig>  chlorop <dig>   <cit>  identified transit peptides with  <dig> and  <dig> residues for psy3b and psy3d, but only 6-residues for psy3a and a signal peptide of two amino acids for brachypodium psy <dig>  however, according to protcomp  <dig>   <cit>  prediction, wheat psy <dig> may have a putative chloroplast localization.
we also analyzed sequence homology between wheat psy <dig> proteins: psy3a and psy3b showed 91%, psy3a and psy3d 90% and psy3b and psy3d 96% amino acid sequence identity.
evolutionary history of psy gene family
throughout monocots, the psy gene family is characterized by three paralogous genes, annotated as psy <dig>  psy <dig> and psy <dig>  while, in eudicots, the presence of psy <dig> and psy <dig> homologs have been reported  <cit> .
the presence of only two copies in eudicots was our starting point to explore evolution of the psy gene family to explain the presence of a third copy in monocots.
we proposed that the angiosperm ancestor was characterized by the presence of the two psy genes, psy <dig> and psy <dig> , and a specific evolutionary event must have occurred in the monocots, duplicating psy <dig> or psy <dig> to give third copy psy <dig>  to test this hypothesis, we analyzed homology between the three psy genes of.
brachypodium, rice and sorghum and observed that psy <dig> was most closely related to psy <dig> in terms of sequence similarity .
however, according to the precise characterization of duplicated chromosomes in monocots reported by murat et al.
<cit> , we concluded that psy1- <dig> genes are not located on chromosomes that have been duplicated during the ancestral shared whole genome duplication  dating back to 50– <dig> mya.
psy <dig> probably originated as a duplication of psy <dig> during a more ancient and specific wgd event in monocot evolution that determined its modern localization in monocot genomes .
this duplicated region corresponds to a more ancient wgd  that predate the 50– <dig> million years old ancestral paleotetraploidization in monocots  <cit> .
psy genes expression profile and function investigation in bread wheat
since there is no evidence of psy <dig> expression in the literature, we used the public wheat affymetrix chip data  <cit>  based on array hybridization with rna extracted from wheat grains at different development stages , 200dd, 250dd, 300dd, 500dd), from cellular division to starch filling, to unravel the expression of psy genes in wheat.
we blast-searched the psy <dig> sequences obtained against affymetrix chip sequences and found two probes, ta.
<dig> .s1_at and ta.
<dig> .s1_at, homoeologs of psy <dig> and psy <dig>  respectively.
the expression level of psy <dig> in grains was twice that of psy <dig> during all developmental stages except at 500dd when psy <dig> levels decreased .
psy <dig> expression was constant during grain development, whereas psy <dig> expression exhibited significant differences between 350dd and 500dd .
our data showed that psy <dig> increased at 200dd while its level remained constant at 300dd and then decreased at 500dd.
no evidence was found for psy <dig> expression in grain at these developmental stages.
to gain insight into psy gene expression during grain development, we used single-strand conformation polymorphism  analysis  <cit>  to screen wheat rnas  extracted from leaves, roots and grains at different development stages.
sscp offered in this experiment the opportunity to  detect minor sequence changes in polymerase chain reaction-amplified samples and  evaluate the expression level of three homoeologous copies of each psy gene in the same running time  <cit> .
in the same pcr reaction, we amplified specifically one paralogous gene and its homoeologous copies that were successfully separated, by capillary electrophoresis according to their sequence differences and assigned to each genome, using amplification profiling approaches on nullitetrasomic lines as references  <cit> .
psy <dig> expression was evaluated based on the primer pair psy1_f1r <dig>  designed on the fourth exon.
its expression in developing grain  was constant and psy1-b showed the highest transcript level.
psy <dig> expression profile did not change in leaves and roots where psy1-b was always the copy most expressed.
we also amplified psy <dig> in each tissue using the primer pair psy2f1r <dig>  designed on the fourth exon .
psy <dig> showed a constant expression pattern in developing grains, the b and d copies showing the highest transcript levels.
in leaves, all three homoeolog copies presented the same expression profiles; whereas, in roots, the d transcript decreased compared to b and a, whose levels remain unchanged.
using this sscp approach we were able to detect psy <dig> expression using the primer pair psy3f2r <dig>  designed on the third exon .
during grain development at 100dd the d copy showed the highest transcript level, while, at 200dd, amplification intensity of the b copy increased in contrast with the two other homoeologs, which decreased.
for the remaining stages, expression levels of the three homoeologous copies were constant.
in leaves, a and d copies had the highest expression levels, while in roots, the d copy showed a greater expression than the a and b copies .
we exploited the wheat psy <dig> promoter sequence to search for regulatory elements.
toucan  <dig>  workbench software revealed the presence of conserved cis-regulatory elements such as, aba-responsive element , g-box and a coupling element , involved in the response to salt and drought stress and application of aba, and also identified in rice and sorghum psy <dig> promoters  <cit> .
in order to confirm this hypothesis, real-time pcr was used to investigate the expression levels of all three psys in leaves and roots during the application of exogenous abscisic acid .
three concentrations of aba  were tested and samples from each tissue were collected after  <dig>   <dig>   <dig>   <dig> and 8 hours  of treatment .
in photosynthetically non-stressed tissues, psy <dig> and psy <dig> gave the major transcripts, which were three times greater from psy <dig> than from psy <dig> .
in non-stressed roots, psy <dig> expression was low and psy <dig> expression levels were at least more than  <dig> times lower than in leaves .
this result agrees with the hypothesis that, in wheat, psy <dig> is directly involved in carotenoid accumulation in grain, associated with major qtls for yp  <cit>  whereas psy <dig> plays a major role in carotenoids accumulation in leaves.
to investigate changes in psy expression during stress, we determined transcript levels in leaf and root samples after stress treatment.
psy responses to the different aba concentrations tested were constant.
psy <dig> mrna levels, in stressed leaves, underwent to a minimum reduction during the first 2 h to then returned to the initial level at 5 h. psy <dig> transcripts slightly decreased in stressed leaves after 2 h and then remained constant.
psy <dig> expression levels increased about 4-fold after 5 h of stress treatment and then returned to initial levels.
in stressed roots, differences in expression levels became more obvious: psy <dig> mrna levels doubled after 5 h of treatment, while psy <dig> transcript levels doubled after only 2 h of stress and then decreased gradually after  <dig> and 8 h of treatments to initial expression levels .
psy <dig> expression levels started to increase just 1 h after the aba application to 18-fold after 2 h and then returned to lower levels .
overall, figure 5f clearly shows the effectiveness of aba treatment on psy <dig> up-regulation in roots.
normalized expression data of psy genes obtained in this experiment are available as additional file 1: figure s <dig>
discussion
carotenoids are widely recognized as essential pigments in plants because of their role in development, photosynthesis, photoprotection and in response to stress since they are essential precursors of apocarotenoids, such as the abscisic acid .
the psy gene family, playing a central role in carotenoid biosynthesis, is characterized by duplicated gene copies.
their characterization in grasses and the recent identification of a third copy in rice, maize and sorghum raises questions about psy gene family organization in wheat.
the role and function of psy <dig> have been largely investigated because of its association with major qtls for carotenoid accumulation in wheat grain.
partial sequences of psy <dig> from several wheat varieties are available but little is known about its function and, up to now, there was no evidence of a psy <dig> homolog and associated activity in wheat.
our current analysis has demonstrated that wheat psy gene family is characterized by the existence of psy <dig> to  <dig> gene copies.
we report here the precise nature of gene family structure, evolution and function.
because of the polyploid nature of wheat and the lack of a whole genome sequence, we identified orthologous regions in reference genomes such as brachypodium and developed cos markers to isolate wheat psy <dig> from a bac library.
wheat psy <dig> is characterized by a novel gene structure due to intron loss events leading to four exons and three introns.
rice and maize psy <dig>  whose gene structure is highly conserved, are characterized by a third intron starting with a non-canonical 5′splicing site gc instead of gt.
this type of intron is deleted by the same spliceosome mechanism that removes gt-ag introns, mainly involved in alternative splicing rather than in introns loss events  <cit> .
brachypodium and sorghum show differences in psy gene structure, characterized by five exons and four introns.
the third intron has been deleted but the causal mechanism is not yet clear.
the classical model to explain intron loss is based on recombination of a genomic copy of a gene with a reverse-transcribed copy of a spliced mrna transcript, deleting one or more adjacent introns.
a key aspect of this theory is that only multicellular eukaryotes that have genes transcribed in germ cells would be susceptible to mrna-mediated intron loss  <cit> .
we identified inverted and repeated short motifs at deletion breakpoints in the wheat psy <dig> sequence, corresponding to the 5′ and the 3′ splicing sites, which may have driven illegitimate recombination leading to the observed intron loss events.
brachypodium and sorghum psy <dig> sequences are not characterized by such repeated motifs at deletion breakpoint, suggesting that intron loss occurred by simple genomic deletion.
maize psy <dig> intron  <dig> and rice intron  <dig> and maize intron  <dig> and rice intron  <dig> are similar in size, leading to the hypothesis that these introns may have been inverted but this could not be confirmed due to limited sequence similarities between introns.
howitt et al.
<cit>  described the presence within the intron # <dig> in wheat psy-d <dig> of an insertion of 1200 bp of an inverted repeat motif.
the characterization of either repeated motifs and/or tes in this intron in grasses may have driven intron loss or shuffling events leading to the distinct psy <dig> gene structures observed.
psy duplicated genes exist throughout monocots and eudicots.
gallagher et al.
<cit>  proposed an evolutionary model for the grass psy gene family in which monocots and eudicots share the same ancestral gene and the same duplication event, giving rise to genes psy <dig> and psy <dig>  welsch and collaborators  <cit>  proposed that rice psy <dig> originated from psy <dig> duplication or vice versa, because of their close similarity in the 5′utr region.
we analyzed homology between psy genes in reference genomes and showed that psy <dig> had greater homology with psy <dig> sequence than psy <dig>  suggesting that psy <dig> originated from a duplication of psy <dig>  furthermore, public paleogenomics data in grasses indicated that psy genes are not located on known duplicated chromosomes originating from the characterized ancestral wgd event reported by murat et al.
<cit> .
we suggest, according to jaillon et al.
and tang et al.
<cit> , that a more ancient wgd event, specific to monocots has shaped the psy <dig> copy as a duplication of psy <dig>
based on a semi-quantitative approach, such as sscp strategy, we observed psy expression in wheat grains, leaves and roots.
psy <dig> and psy <dig> homoeologous copies showed stable expression profiles in the different tissues.
psy-b <dig> was the copy most expressed, while, psy-b <dig> and psy-d <dig> showed the largest transcript levels in developing grains and, in leaves and roots, psy-a <dig> transcripts increased.
psy <dig> homoeologous copies showed a variable pattern of expression during the first stage of cellular division during early grain development and we found psy-a <dig> and psy-d <dig> most expressed in leaves and psy-d <dig> in roots.
however, with this approach, a statistically basis comparison was not possible between the expression levels of the three paralogous genes because it is semi-quantitative and only the broad transcript levels of homoeologous copies of each gene can be compared when amplified with the same primer pairs and in the same conditions.
access to recently published affymetrix data  <cit>  provided the opportunity to investigate psy gene expression pattern and showed that psy <dig> expression level was higher than psy <dig> in developing grains.
psy <dig> transcripts increased at 200dd to remain constant until 500dd, when they were reduced.
in developing maize endosperm, li et al.
<cit>  showed that during endosperm carotenogenesis, only psy <dig> transcript increased significantly, starting after 12dap until 22dap.
basis on this evidence, we can speculate that wheat psy <dig> in the endosperm follows the same kinetics as the maize psy <dig> ortholog and that apparently there is a correspondence in timing of expression between wheat and maize psy <dig> orthologs.
our results are consistent with the evidence that psy <dig> plays an important role in carotenoid accumulation in the endosperm and with its association with qtls for yp on chromosome group  <dig> in wheat  <cit> .
gene expression is closely associated with cis-regulatory dna sequences  located in its upstream regions  <cit> .
in rice and maize, psy <dig> expression is suggested to be strongly inducible in roots by drought and salt stress and application of aba, since their promoter regions harbour cis-elements involved in plant response to abiotic stress  <cit> .
we identified several regulatory elements in the wheat psy <dig> promoter region,  the aba-responsive element  whose core is represented by the g-box, and  a coupling element  that is necessary to obtain abre functional  <cit> .
through a real-time pcr approach, we quantified wheat psy transcript levels in leaves and roots before and after the exogenous application of aba, observing that psy <dig> represents the most abundant transcript in unstressed leaves, while psy <dig> transcript levels were 3-fold lower than in carotenoid accumulation in grains  <cit> .
wheat psy <dig> may thus play an important and complementary role in yp accumulation in photosynthetic tissues.
psy <dig> expression levels were low in non-stressed leaves compared to psy <dig> and psy <dig> while its levels increased in non-stressed roots.
psy <dig> and psy <dig> transcript levels did not show significant changes in leaves after aba treatment.
in stressed roots, psy <dig> and psy <dig> levels doubled after 5 h and 2 h of treatment respectively.
psy <dig> showed the most intense response to aba as in leaves, after 5 h of aba application, psy <dig> transcript was 4-fold higher, while in stressed roots after 2 h its transcript levels increase about 18-fold.
psy <dig> was the only gene that showed significant changes of transcript levels in the studied conditions.
carotenoids provide precursors for aba which is the main apocarotenoid that plants can accumulate under drought conditions, aba applications having been shown to affect plant growth and development, mimicking the effect of water stress and improving capacity to escape or tolerate such stress  <cit> .
since psy genes are considered as rate-limiting in carotenogenesis, we can explain the up-regulation of psy <dig> by aba as the necessity of plants to control carotenoid flux to roots during stress conditions to provide aba precursors and thus activate stress responses.
in a previous study, singh et al.
<cit> , studying the association of psy1-a <dig> allelic variation with yp colour in durum wheat, speculated about the possible existence of a third psy on the chromosome 7al to explain the presence of a second qtl near the marker xwmc <dig>  we identified the chromosomal bin localization for each homoeolog and the expected syntenic relationships were confirmed assigning psy <dig> to chromosome group  <dig> which has the highest concentration of qtls and major loci controlling plant adaptation to environment, particularly, those controlling heading date, frost and salt tolerance, whereas a region with a crucial role in drought tolerance is located on chromosome group  <dig>  multiple-stress qtls and linked markers have also been detected, suggesting the existence of common mechanisms for different stresses or of gene clusters controlling different stress tolerance processes  <cit> .
for example, in wheat a significant qtl for aba accumulation in droughted leaves was found on chromosome 5a co-located with the major vernalization responsive gene, vrn <dig>  <cit> , and in the same chromosomal bin we mapped psy-3a.
aprile et al.
<cit>  studied different physiological reactions to water stress and the substantially different molecular responses of bread and durum wheat genotypes demonstrating that there is a difference between durum and bread wheat response to water stress and that deletion of bin 5al- <dig> from chinese spring determined a lower response to such stresses.
they also showed that the different response by durum and bread wheat can be associated with the absence of d genome, where genes or factors that modulate expression of cluster of genes may be localized.
psy <dig> localization and function in wheat are consistent with these data, especially the psy3d genomic structure, function and expression reported in the current study.
finally, we also showed here that psy3s are characterized by intron losses, inverted repeats and up-regulation by abiotic stresses.
it is important to note that biotic/abiotic stresses could also activate transposons and retrotransposons  <cit>  whose expression may have played a role in psy <dig> gene structure modification.
CONCLUSIONS
psy gene family plays an important role in plant development  as well as is a key contributor to flower and fruit flavours and aromas.
the current analysis provides relevant structural , functional  and evolutionary  informations that can be used as applied tools in wheat breeding programs.

 carotenoids represent a diverse group of pigments found in bacteria, fungi and plants. in plants, carotenoids have several important functions, essential for plant development as: they serve as accessory pigments to harvest light for photosynthesis and constitute the basic structural units of photosynthetic apparatus and they also act as photoprotectors for plants to adapt to high light stress. in addition, oxidative cleavage of carotenoids produces apocarotenoids considered as signals in plant development, antifungal agents, contributors to flower and fruit flavours and aromas. the phyto-hormone abscisic acid, the most well known apocarotenoid, plays an important role in plant response to stress conditions. in addition, carotenoids have long been considered as essential nutrients as well as important health beneficial compounds. in animals including humans, unable to synthesize carotenoids de novo, dietary carotenoids are essential precursors of vitamin a and retinoid compounds needed in development. plant carotenogenesis occurs in the membrane of chloroplasts, chromoplasts and amyloplasts, plastids having different internal membrane architectures. the enzymes involved in the carotenoid biosynthetic pathway are nuclear encoded and targeted to the plastids. carotenoid biosynthesis begins with the formation of the 40-carbon backbone, phytoene, a step mediated by phytoene synthase. psy, which is known to control carotenoid flux in seeds and to catalyze the first committed step in carotenogenesis, is thought to be a rate-limiting enzyme. in “golden rice”, for example, psy activity was crucial to obtain increased carotenoid levels, whereas bacterial carotene desaturase crti had no effect. psy is coded by a small gene family, which had been already shown to exist throughout the grasses and which consists of three paralogous genes that have been identified and characterized in rice, maize and sorghum. the three rice psy genes share the same structure, with six exons and five introns, and they exhibit a tissue-specific expression. rice endosperm is carotenoid free and transcripts for all three genes are absent. in photosynthetic tissues, all three psy mrnas are present, but with different levels of expression, ospsy and ospsy playing predominant roles with similar expression patterns and regulation in response to light. ospsy plays a specialized role as it is not regulated by light but is strongly inducible in roots by high salt concentrations and/or drought. ospsy and ospsy contain cis-acting elements involved in light regulation which are absent from the promoter region of ospsy explaining why psy does not respond to light. li et al. described the psy gene family and their tissue-specific transcript patterns in maize. they showed that psy is the only gene family member for which transcript level increased during maize endosperm development and was correlated with accumulation of endosperm carotenoids. transcript changes of psys were monitored in maize leaves during de-etiolation. in dark-grown plants, psy represented the major transcript whereas psy was the only paralog for which transcript levels increased in response to illumination, suggesting then that psy plays an important role in controlling leaf carotenogenesis during greening. the abundance of psy in roots suggests that it plays a unique role in roots carotenogenesis when induced by drought, salt and exogenous aba treatment. the increase of psy transcripts observed was associated with an induced level of carotenoid intermediates, and elevation of other downstream carotenogenic genes, followed by aba accumulation. psy genes are of primary importance in wheat due to their impact on flour colour, so that numerous genetics studies have been conducted on yellow pigment content in wheat grain. a high yp concentration is a desirable trait in durum wheat and is the target of breeding programs worldwide. however, low yp is a breeding target in hexaploid wheat, for which bright white flour is preferred. the inheritance of yp is complex, controlled by additive gene effects and highly heritable. several qtls for yp have been identified in both durum and bread wheat on chromosomes 1a, 1b, 3a, 3b, 4a and 5a, 2a, 4b and 6b, 5b, and 6a. however, the majority of studies agreed that chromosome group has the greatest impact in controlling yp content in bread wheat. elouafi et al. identified two minor qtls on chromosome 7a and a third major qtl on chromosome 7bl. this last qtl has been corroborated in two other durum wheat populations and is ortholog to the one identified in hexaploid wheat on the same chromosome. a qtl for yp in durum wheat was identified by patil et al. on chromosome 7a that accounted for up to 55% of the trait variation. mares and campbell identified qtls on chromosome 7a in two hexaploid wheat populations that had a large impact on flour yellowness, and correspond to loci also associated with xanthophyll concentration. cloning of genes controlling flour pigmentation would be of great help in breeding for genotypes with appropriate levels of yellow pigments in durum and bread wheat. using a pcr-based approach cenci et al. identified and mapped clones from a durum wheat bac library thought to contain genes coding for important enzymes involved in carotenogenesis. in their study, putative phytoene synthase, phytoene desaturase and ζ-carotene desaturase clones were assigned to the chromosome groups and respectively. to date, four psy genes have been identified in durum wheat, forming two paralogous series on chromosome groups and. one of these, psy1-b co-segregates with a qtl for yp on 7bl. psy1-a was localized on the distal end of the chromosome 7al in one hexaploid wheat mapping population, associated with variation for flour colour. wheat psy genomic sequences show similar exon-intron characteristic to psy known in other grass species, with six exons separated by five introns. a partial sequence of psy was also obtained and it was mapped on the short arm of the chromosome group, with no clear data regarding the impact of psy on carotenoid accumulation. in this article, we report evidence that the wheat psy gene family also consists of three paralogous genes and that psy is characterized by a novel gene structure, consequence of intron loss events. since the wheat psy promoter region is characterized by cis-acting elements involved in plant response to abiotic stress, we studied changes in wheat psy expression levels in leaves and roots during exogenous treatment with aba. finally, after comparative genomic investigation of psy loci, we reconstructed the paleo-history for this gene family in grasses identifying evolutionary events that have shaped psy gene copies in modern species.  psy gene sequencing and mapping in bread wheat we recently reassessed the syntenic relationships within monocots and developed tools to identify precisely chromosome-to-chromosome orthologous relationships and derived conserved orthologous set markers,. rice, sorghum and brachypodium psy orthologs, available from the literature, respectively mapped on chromosomes and were used to clone wheat psy genes. on the basis of the known rice/sorghum/wheat/brachypodium syntenic blocks covering psy orthologs, psy was expected to be located on chromosome group in wheat. the blast search did not result in significant orthologous wheat sequences from public databanks. primers were therefore developed from conserved genic regions characterized between rice and brachypodium psy coding sequences to screen a bread wheat bac library. this library is organised in 384-well plates of bac pools with a specific screening strategy to reduce the number of pcr reactions necessary to identify a single clone of interest. primer pairs psy_4g _bra_f2/psy_ _bra_r designed on the fourth exon of brachypodium psy were used for library screening. positive pools were sequenced and three homoeoforms were obtained and assigned to the wheat chromosomes using nullisomic-tetrasomic lines. by crossing the pool coordinates we identified the bac clones harbouring psy gene homoeologs. three bac clones spanning the psy gene of a, b and d sub-genomes of t. aestivum were screened with pcr-specific markers targeting each of the three psy homoeologs and then sequenced. sequencing resulted in and 149 bp of gapped contigs for, respectively, the a, b and d sub-genomes. each homoeologous copy was then precisely assigned to chromosome bins using genome-specific primer pairs and dna from deletion lines: psy3s were located precisely on the chromosome bins 5al10- -, 5bl14- - and 5dl1- -, respectively, for a, b and d genome copies. identification and characterization of psy loci bac sequencing and annotation made it possible to identify psy orthologous regions. we selected ungapped contigs of 000 bp, 411 bp and 803 bp harbouring, respectively, psy3a, psy3b and psy3d genes. figure 2b illustrates bac annotation results as well as micro-colinearity with rice, brachypodium, maize and sorghum genome sequences, confirming that psy and bradi4g genes are at orthologous positions. in the psy loci we found the insertion of a non-syntenic gene. according to paleogenomic data in monocots, this gene is located on the syntenic chromosomes and of, rice, sorghum and brachypodium genomes respectively. in wheat, this gene would be expected to map on the orthologous chromosome group and not group where it was found based on the psy sequenced region assignation. we can thus suppose that this gene has been specifically transposed in wheat from an ancestral donor region to the modern current acceptor region. alignment with brachypodium psy cds clarified wheat psy3s exon-intron boundaries. wheat psy3s showed a structure characterized by four exons spaced by three introns that were numbered according to their position within the genes, with the first intron closest to the 5′ and the third intron closest to the 3′ end. this structure and exon-intron sizes were conserved between the a, b and d homoeologous copies. rice and maize psy were the only genes with six exons spaced by five introns. in brachypodium and sorghum, the loss of the third intron determined the appearance of a different structure. in rice and maize, this intron showed at its donor site, the t/c transition, starting with the gc motif instead of gt. we also found in rice psy the presence of miniature inverted transposable elements, in the first intron: nt628-nt and nt953-nt and in the third intron the same mite was identified in inverted orientation. wheat psy was found to include the largest first exon of 466 bp, 433 bp and 436 bp, respectively, for psy3a, b and d, resulting from fusion of the first and second exons after an intron loss event. the second exon had 412 bp and resulted from the fusion of the third and fourth exons. it was characterized by a close sequence homology with the third exon of the brachypodium ortholog. figure 3a shows psy gene structure evolution throughout grasses. rice and maize share the same psy gene structure, while sorghum and brachypodium gene structures are characterized, as in wheat, by the loss of the third intron and the subsequent fusion of the two flanking exons. we suggest that, during evolution, different and independent intron loss events occurred. at the deletion breakpoints, we identified repeated inversed motifs, tgg|cca and cgg|ccg in the first and the second exon respectively. it is therefore possible to assume that these motifs have facilitated intron loss events through illegitimate recombination. the first intron lost was at phase while the second was at phase the tssp database defined the promoter region of psy around kb upstream from the start codon and polya signal miner software identified the terminator signal 865 bp, 867 bp and 828 bp, respectively for a, b and d genomes. sequence analysis allowed us to identify a duplication of the fourth exon in the psy3d locus, 1729 bp downstream from the stop codon. this repeat did not start with the trinucleotide gtg, as expected based on the exon # sequence, and was interrupted by a 154 bp deletion flanked at each extremity by the tactgg motif. we can thus suppose that a novel illegitimate recombination event mediated the duplication of the fourth exon and its insertion in the sequence. despite intron/exon shuffling events reported previously, it was possible to predict protein structure. psy3a encodes a protein of amino acids, longer than those coded by psy3b and psy3d homoeologs. the ncbi conserved domain search software allowed us to identify the trans-isoprenyl diphosphate synthase domain which includes squalene and phytoene synthases. according to the previous domain characterization, wheat psy was classed in the ipss superfamily and class i terpene cyclise. the conserved trans-ipps domain was identified at positions: aa123- aa112- and aa113- respectively, for psy3a, b and d copies. protein motifs identified based on scanprosite are available in additional file 1: figure s chlorop identified transit peptides with and residues for psy3b and psy3d, but only 6-residues for psy3a and a signal peptide of two amino acids for brachypodium psy however, according to protcomp prediction, wheat psy may have a putative chloroplast localization. we also analyzed sequence homology between wheat psy proteins: psy3a and psy3b showed 91%, psy3a and psy3d 90% and psy3b and psy3d 96% amino acid sequence identity. evolutionary history of psy gene family throughout monocots, the psy gene family is characterized by three paralogous genes, annotated as psy psy and psy while, in eudicots, the presence of psy and psy homologs have been reported. the presence of only two copies in eudicots was our starting point to explore evolution of the psy gene family to explain the presence of a third copy in monocots. we proposed that the angiosperm ancestor was characterized by the presence of the two psy genes, psy and psy, and a specific evolutionary event must have occurred in the monocots, duplicating psy or psy to give third copy psy to test this hypothesis, we analyzed homology between the three psy genes of. brachypodium, rice and sorghum and observed that psy was most closely related to psy in terms of sequence similarity. however, according to the precise characterization of duplicated chromosomes in monocots reported by murat et al., we concluded that psy1- genes are not located on chromosomes that have been duplicated during the ancestral shared whole genome duplication dating back to 50– mya. psy probably originated as a duplication of psy during a more ancient and specific wgd event in monocot evolution that determined its modern localization in monocot genomes. this duplicated region corresponds to a more ancient wgd that predate the 50– million years old ancestral paleotetraploidization in monocots. psy genes expression profile and function investigation in bread wheat since there is no evidence of psy expression in the literature, we used the public wheat affymetrix chip data based on array hybridization with rna extracted from wheat grains at different development stages, 200dd, 250dd, 300dd, 500dd), from cellular division to starch filling, to unravel the expression of psy genes in wheat. we blast-searched the psy sequences obtained against affymetrix chip sequences and found two probes, ta..s1_at and ta..s1_at, homoeologs of psy and psy respectively. the expression level of psy in grains was twice that of psy during all developmental stages except at 500dd when psy levels decreased. psy expression was constant during grain development, whereas psy expression exhibited significant differences between 350dd and 500dd. our data showed that psy increased at 200dd while its level remained constant at 300dd and then decreased at 500dd. no evidence was found for psy expression in grain at these developmental stages. to gain insight into psy gene expression during grain development, we used single-strand conformation polymorphism analysis to screen wheat rnas extracted from leaves, roots and grains at different development stages. sscp offered in this experiment the opportunity to detect minor sequence changes in polymerase chain reaction-amplified samples and evaluate the expression level of three homoeologous copies of each psy gene in the same running time. in the same pcr reaction, we amplified specifically one paralogous gene and its homoeologous copies that were successfully separated, by capillary electrophoresis according to their sequence differences and assigned to each genome, using amplification profiling approaches on nullitetrasomic lines as references. psy expression was evaluated based on the primer pair psy1_f1r designed on the fourth exon. its expression in developing grain was constant and psy1-b showed the highest transcript level. psy expression profile did not change in leaves and roots where psy1-b was always the copy most expressed. we also amplified psy in each tissue using the primer pair psy2f1r designed on the fourth exon. psy showed a constant expression pattern in developing grains, the b and d copies showing the highest transcript levels. in leaves, all three homoeolog copies presented the same expression profiles; whereas, in roots, the d transcript decreased compared to b and a, whose levels remain unchanged. using this sscp approach we were able to detect psy expression using the primer pair psy3f2r designed on the third exon. during grain development at 100dd the d copy showed the highest transcript level, while, at 200dd, amplification intensity of the b copy increased in contrast with the two other homoeologs, which decreased. for the remaining stages, expression levels of the three homoeologous copies were constant. in leaves, a and d copies had the highest expression levels, while in roots, the d copy showed a greater expression than the a and b copies. we exploited the wheat psy promoter sequence to search for regulatory elements. toucan workbench software revealed the presence of conserved cis-regulatory elements such as, aba-responsive element, g-box and a coupling element, involved in the response to salt and drought stress and application of aba, and also identified in rice and sorghum psy promoters. in order to confirm this hypothesis, real-time pcr was used to investigate the expression levels of all three psys in leaves and roots during the application of exogenous abscisic acid. three concentrations of aba were tested and samples from each tissue were collected after and 8 hours of treatment. in photosynthetically non-stressed tissues, psy and psy gave the major transcripts, which were three times greater from psy than from psy. in non-stressed roots, psy expression was low and psy expression levels were at least more than times lower than in leaves. this result agrees with the hypothesis that, in wheat, psy is directly involved in carotenoid accumulation in grain, associated with major qtls for yp whereas psy plays a major role in carotenoids accumulation in leaves. to investigate changes in psy expression during stress, we determined transcript levels in leaf and root samples after stress treatment. psy responses to the different aba concentrations tested were constant. psy mrna levels, in stressed leaves, underwent to a minimum reduction during the first 2 h to then returned to the initial level at 5 h. psy transcripts slightly decreased in stressed leaves after 2 h and then remained constant. psy expression levels increased about 4-fold after 5 h of stress treatment and then returned to initial levels. in stressed roots, differences in expression levels became more obvious: psy mrna levels doubled after 5 h of treatment, while psy transcript levels doubled after only 2 h of stress and then decreased gradually after and 8 h of treatments to initial expression levels. psy expression levels started to increase just 1 h after the aba application to 18-fold after 2 h and then returned to lower levels. overall, figure 5f clearly shows the effectiveness of aba treatment on psy up-regulation in roots. normalized expression data of psy genes obtained in this experiment are available as additional file 1: figure s discussion carotenoids are widely recognized as essential pigments in plants because of their role in development, photosynthesis, photoprotection and in response to stress since they are essential precursors of apocarotenoids, such as the abscisic acid. the psy gene family, playing a central role in carotenoid biosynthesis, is characterized by duplicated gene copies. their characterization in grasses and the recent identification of a third copy in rice, maize and sorghum raises questions about psy gene family organization in wheat. the role and function of psy have been largely investigated because of its association with major qtls for carotenoid accumulation in wheat grain. partial sequences of psy from several wheat varieties are available but little is known about its function and, up to now, there was no evidence of a psy homolog and associated activity in wheat. our current analysis has demonstrated that wheat psy gene family is characterized by the existence of psy to gene copies. we report here the precise nature of gene family structure, evolution and function. because of the polyploid nature of wheat and the lack of a whole genome sequence, we identified orthologous regions in reference genomes such as brachypodium and developed cos markers to isolate wheat psy from a bac library. wheat psy is characterized by a novel gene structure due to intron loss events leading to four exons and three introns. rice and maize psy whose gene structure is highly conserved, are characterized by a third intron starting with a non-canonical 5′splicing site gc instead of gt. this type of intron is deleted by the same spliceosome mechanism that removes gt-ag introns, mainly involved in alternative splicing rather than in introns loss events. brachypodium and sorghum show differences in psy gene structure, characterized by five exons and four introns. the third intron has been deleted but the causal mechanism is not yet clear. the classical model to explain intron loss is based on recombination of a genomic copy of a gene with a reverse-transcribed copy of a spliced mrna transcript, deleting one or more adjacent introns. a key aspect of this theory is that only multicellular eukaryotes that have genes transcribed in germ cells would be susceptible to mrna-mediated intron loss. we identified inverted and repeated short motifs at deletion breakpoints in the wheat psy sequence, corresponding to the 5′ and the 3′ splicing sites, which may have driven illegitimate recombination leading to the observed intron loss events. brachypodium and sorghum psy sequences are not characterized by such repeated motifs at deletion breakpoint, suggesting that intron loss occurred by simple genomic deletion. maize psy intron and rice intron and maize intron and rice intron are similar in size, leading to the hypothesis that these introns may have been inverted but this could not be confirmed due to limited sequence similarities between introns. howitt et al. described the presence within the intron # in wheat psy-d of an insertion of 1200 bp of an inverted repeat motif. the characterization of either repeated motifs and/or tes in this intron in grasses may have driven intron loss or shuffling events leading to the distinct psy gene structures observed. psy duplicated genes exist throughout monocots and eudicots. gallagher et al. proposed an evolutionary model for the grass psy gene family in which monocots and eudicots share the same ancestral gene and the same duplication event, giving rise to genes psy and psy welsch and collaborators proposed that rice psy originated from psy duplication or vice versa, because of their close similarity in the 5′utr region. we analyzed homology between psy genes in reference genomes and showed that psy had greater homology with psy sequence than psy suggesting that psy originated from a duplication of psy furthermore, public paleogenomics data in grasses indicated that psy genes are not located on known duplicated chromosomes originating from the characterized ancestral wgd event reported by murat et al.. we suggest, according to jaillon et al. and tang et al., that a more ancient wgd event, specific to monocots has shaped the psy copy as a duplication of psy based on a semi-quantitative approach, such as sscp strategy, we observed psy expression in wheat grains, leaves and roots. psy and psy homoeologous copies showed stable expression profiles in the different tissues. psy-b was the copy most expressed, while, psy-b and psy-d showed the largest transcript levels in developing grains and, in leaves and roots, psy-a transcripts increased. psy homoeologous copies showed a variable pattern of expression during the first stage of cellular division during early grain development and we found psy-a and psy-d most expressed in leaves and psy-d in roots. however, with this approach, a statistically basis comparison was not possible between the expression levels of the three paralogous genes because it is semi-quantitative and only the broad transcript levels of homoeologous copies of each gene can be compared when amplified with the same primer pairs and in the same conditions. access to recently published affymetrix data provided the opportunity to investigate psy gene expression pattern and showed that psy expression level was higher than psy in developing grains. psy transcripts increased at 200dd to remain constant until 500dd, when they were reduced. in developing maize endosperm, li et al. showed that during endosperm carotenogenesis, only psy transcript increased significantly, starting after 12dap until 22dap. basis on this evidence, we can speculate that wheat psy in the endosperm follows the same kinetics as the maize psy ortholog and that apparently there is a correspondence in timing of expression between wheat and maize psy orthologs. our results are consistent with the evidence that psy plays an important role in carotenoid accumulation in the endosperm and with its association with qtls for yp on chromosome group in wheat. gene expression is closely associated with cis-regulatory dna sequences located in its upstream regions. in rice and maize, psy expression is suggested to be strongly inducible in roots by drought and salt stress and application of aba, since their promoter regions harbour cis-elements involved in plant response to abiotic stress. we identified several regulatory elements in the wheat psy promoter region, the aba-responsive element whose core is represented by the g-box, and a coupling element that is necessary to obtain abre functional. through a real-time pcr approach, we quantified wheat psy transcript levels in leaves and roots before and after the exogenous application of aba, observing that psy represents the most abundant transcript in unstressed leaves, while psy transcript levels were 3-fold lower than in carotenoid accumulation in grains. wheat psy may thus play an important and complementary role in yp accumulation in photosynthetic tissues. psy expression levels were low in non-stressed leaves compared to psy and psy while its levels increased in non-stressed roots. psy and psy transcript levels did not show significant changes in leaves after aba treatment. in stressed roots, psy and psy levels doubled after 5 h and 2 h of treatment respectively. psy showed the most intense response to aba as in leaves, after 5 h of aba application, psy transcript was 4-fold higher, while in stressed roots after 2 h its transcript levels increase about 18-fold. psy was the only gene that showed significant changes of transcript levels in the studied conditions. carotenoids provide precursors for aba which is the main apocarotenoid that plants can accumulate under drought conditions, aba applications having been shown to affect plant growth and development, mimicking the effect of water stress and improving capacity to escape or tolerate such stress. since psy genes are considered as rate-limiting in carotenogenesis, we can explain the up-regulation of psy by aba as the necessity of plants to control carotenoid flux to roots during stress conditions to provide aba precursors and thus activate stress responses. in a previous study, singh et al., studying the association of psy1-a allelic variation with yp colour in durum wheat, speculated about the possible existence of a third psy on the chromosome 7al to explain the presence of a second qtl near the marker xwmc we identified the chromosomal bin localization for each homoeolog and the expected syntenic relationships were confirmed assigning psy to chromosome group which has the highest concentration of qtls and major loci controlling plant adaptation to environment, particularly, those controlling heading date, frost and salt tolerance, whereas a region with a crucial role in drought tolerance is located on chromosome group multiple-stress qtls and linked markers have also been detected, suggesting the existence of common mechanisms for different stresses or of gene clusters controlling different stress tolerance processes. for example, in wheat a significant qtl for aba accumulation in droughted leaves was found on chromosome 5a co-located with the major vernalization responsive gene, vrn, and in the same chromosomal bin we mapped psy-3a. aprile et al. studied different physiological reactions to water stress and the substantially different molecular responses of bread and durum wheat genotypes demonstrating that there is a difference between durum and bread wheat response to water stress and that deletion of bin 5al- from chinese spring determined a lower response to such stresses. they also showed that the different response by durum and bread wheat can be associated with the absence of d genome, where genes or factors that modulate expression of cluster of genes may be localized. psy localization and function in wheat are consistent with these data, especially the psy3d genomic structure, function and expression reported in the current study. finally, we also showed here that psy3s are characterized by intron losses, inverted repeats and up-regulation by abiotic stresses. it is important to note that biotic/abiotic stresses could also activate transposons and retrotransposons whose expression may have played a role in psy gene structure modification.  psy gene family plays an important role in plant development as well as is a key contributor to flower and fruit flavours and aromas. the current analysis provides relevant structural, functional and evolutionary informations that can be used as applied tools in wheat breeding programs. 
a wheat psy promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid, since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. psy are nuclear enzymes encoded by a small gene family consisting of three paralogous genes that have been widely characterized in rice, maize and sorghum. in wheat, for which yellow pigment content is extremely important for flour colour, only psy has been extensively studied because of its association with qtls reported for yellow pigment whereas psy has been partially characterized. expression of wheat psys in leaves and roots was investigated during aba treatment to confirm the up-regulation of psy during abiotic stress. we investigated the structural and functional determinisms of psy genes in wheat. more generally, among eudicots and monocots, the psy gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire psy gene family in grasses.
 carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. the enzyme phytoene synthase plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. psy are nuclear enzymes encoded by a small gene family consisting of three paralogous genes that have been widely characterized in rice, maize and sorghum.  in wheat, for which yellow pigment content is extremely important for flour colour, only psy has been extensively studied because of its association with qtls reported for yellow pigment whereas psy has been partially characterized. here, we report the isolation of bread wheat psy genes from a renan bac library using brachypodium as a model genome for the triticeae to develop conserved orthologous set markers prior to gene cloning and sequencing. wheat psy homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. a wheat psy promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid, since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. expression of wheat psys in leaves and roots was investigated during aba treatment to confirm the up-regulation of psy during abiotic stress.  we investigated the structural and functional determinisms of psy genes in wheat. more generally, among eudicots and monocots, the psy gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire psy gene family in grasses. carotenoidsphytoene synthasewheatintron lossabiotic stressevolution 