BACKGROUND
characteristic fragrance in aromatic rice leads to its popularity worldwide  <cit>  and the aromatic traits have been extensively incorporated into commercial and hybrid rice breeding programs  <cit> .
the fragrance occurring in many aromatic rice varieties has been shown to be associated with the presence of 2-acetyl-1-pyrroline  <cit> , which has also been identified in a great variety of food products  <cit> .
in the thailand fragrant rice variety khao dawk mali  <dig>  2-acetyl-1-pyrroline formation and strong aroma emission has been found to be positively correlated with an accumulation of proline  <cit> .
although the biochemical pathway leading to the fragrance is largely unknown, l-proline has been demonstrated to be a possible precursor in the production of 2-acetyl-1-pyrroline in rice plants  <cit> .
the inheritance of fragrant trait in rice has been well documented.
genetic investigations implicated that the fragrant trait of rice was controlled by a single recessive locus  <cit> .
a number of genetic analyses using reciprocal crosses repetitively showed the aromatic trait is characteristics of recessive monogenic inheritance without impact from cytoplasmic genes  <cit> .
nevertheless, tsuzuki & shimokawa  <cit>  reported that two genes were responsible for the construction of the aromatic trait.
in addition, several investigations by using different aromatic rice cultivars also showed that two recessive genes were involved in the segregation of aromatic and non-aromatic traits  <cit> .
furthermore, genetic studies revealed that the underlying gene responsible for the aroma production was located on chromosome  <dig>  <cit> .
by using translocation and trisomics lines derived from non-fragrant rice cv.
ir <dig>  aromatic trait was also mapped to the chromosome  <dig>  <cit> .
in a recent study involved eight aromatic hybrid rice maintainer lines, a single recessive locus spanning ssr  markers rm <dig> and rm <dig> on chromosome  <dig> was identified to tightly link with the fragrant trait  <cit> .
physical mapping revealed that several candidate genes including a rice betaine aldehyde dehydrogenase  homolog on chromosome  <dig> was co-segregated with aroma production  <cit> .
meanwhile, a delicate study demonstrated that the aroma production in fragrant genotypes was well correlated with the multiple mutations in the fragrance rice  locus that is identical to betaine aldehyde dehydrogenase  on chromosome  <dig>  <cit> .
however, direct evidence proving the function of osbadh <dig> in 2-acetyl-1-pyrroline production is lacking.
furthermore, traditional aromatic cultivars have often undesirable agronomic performance, such as poor yield, susceptibility to pests and diseases, and strong shedding  <cit> .
the molecular mechanism of this major weakness occurred in the aromatic rice cultivars is largely unknown.
in present experiments, we sequenced the osbadh <dig> locus derived from a number of aromatic rice cultivars across china and uncovered that multiple mutations identical to fgr allele also occurred in all the tested fragrant accessions.
by using rna interference  technique combined with agrobacterium tumefaciens-mediated t-dna transfer, we show that the directed degradation of osbadh <dig> transcripts results in a significantly elevated fragrance emission and 2-acetyl-1-pyrroline accumulation, unambiguously suggesting that osbadh <dig> locus is responsible for aroma production in fragrant rice varieties.
in addition, disrupted osbadh <dig> leads to a detectable reduction of crop productivity, implying its multiple functions in secondary metabolism and agronomic performance.
a strategy to compromise the favorable aspects and to avoid the unfavorable effect during osbadh <dig> manipulation and breeding is discussed as well.
RESULTS
expression and nucleotide variation at the osbadh <dig> locus
the cv.
nipponbare was used to determine the expression patterns of osbadh <dig>  total rnas were extracted from roots, stems, leaves and flowers of mature plant, as well as embryos, seedling roots, seedling stems, seedling leaves.
messenger rna levels were analyzed by semi-quantitative rt-pcr.
the results show osbadh <dig> is expressed constitutively, with less expression abundance in mature roots .
we examined genetic variation in a  <dig> bp portion of osbadh <dig> gene in  <dig> fragrant and six non-fragrant rice accessions that are representatives of cultivated varieties across china .
multiple mutations identical to fgr allele identified by bradbury et al.
<cit>  were also observed in all the  <dig> fragrant accessions .
these mutations contain a total of six snps and eight deletions within a  <dig> bp region of the exon  <dig> as compared to the wild type allele in the tested non-fragrant varieties.
deficient in osbadh <dig> expression resulting in apparently sensory aroma production
to dissect the physiological role of osbadh <dig>  we generated a large number of transgenic rice plants expressing osbadh2-rnai .
construct was introduced into non-fragrant rice cv.
nipponbare by agrobacterium tumefaciens-mediated t-dna transfer.
the majority of osbadh2-rnai primary transgenics  resulted in pcr amplification with primers designed to the hpt  marker.
preliminary aroma evaluation revealed that  <dig> out of  <dig> hpt-positive t <dig> plants showed abundant fragrance emission.
ten t <dig> segregating populations derived from independent t <dig> plants with obvious aroma production were selected and cultivated in the university's farm.
leaves from the first and second crops and grains from the first crop of individual t <dig> plants were used for sensory evaluation of aroma production.
as a result, the presence and absence of fragrance is unambiguously segregated within t <dig> populations and well consistently expressed in leaves and grains from the same plants.
in all the  <dig> t1-segregating populations, we observed a correlation of osbadh2-rnai transgene integration with apparently sensory aroma production.
to investigate property of the emitted aromatic compound from the transgenic lines, a gas chromatography-mass spectrometry  analysis was conducted for thai hom mali  <dig> , transgenic line  and the wild type control.
as shown in figure  <dig>  a considerable aroma production of 2-acetyl-1-pyrroline  was detected in both thai hom mali  <dig> and transgenic line , by contrast to the undetectable wild type control.
in order to test if the aroma production is co-segregated with the presence of t-dna insertion in the transgenic segregating progeny, t <dig> mature leaves from five osbadh2-rnai repression plants and four non-fragrant wild-type plants segregated out from the same transgenic line  were collected and subjected to the gc-ms analysis.
as a result, 2ap accumulation was apparently enhanced in all the downregulation plants, by contrast to the undetectable wild-type plants without fragrance .
to ensure that the observed phenotypes are correlated with reduced endogenous transcript, total rna was extracted from leaves of wild type plants and four independent osbadh2-rnai repression lines.
analysis of semi-quantitative rt-pcr and real-time quantitative rt-pcr revealed a considerable reduction in endogenous osbadh <dig> transcript levels in osbadh2-rnai repression lines compared to that of wild-type plants .
to determine if the expression of osbadh <dig> gene is affected in the down-regulated osbadh <dig> transgenic lines, we conducted real-time pcr analysis to detect the osbadh <dig> cdna levels in the osbadh2-rnai repression lines.
the osbadh <dig> gene is the closest available homolog of osbadh <dig> and is almost unaffected in the down-regulated osbadh <dig> lines .
disrupted osbadh <dig> influencing crop productivity
to investigate the effects of down-regulated osbadh <dig> on crop productivity, the plant height and 1000-grain weight of osbadh2-deficient and wild type plants derived from four independent t <dig> transgenic lines were measured.
the results showed the reduction in plant height as well as 1000-grain weight in the osbadh2-deficient plants is differentially detectable when compared to the wild-type plants segregated out from the transgenic progenies .
* significant at p <  <dig>  compared to non-fragrant wild-type plants segregated out from the same transgenic line.
to further address the physiological functions of the osbadh <dig> gene, wild type seeds and t <dig> seeds derived from four independent transgenic lines were germinated in various salt-stressed conditions.
no significant difference in sprouting index and germination rates between wild type and osbadh <dig> repression lines was observed .
nevertheless, the seedling growth rates  in different salt concentrations were more or less inhibited in osbadh2-deficient lines as compared to wild type .
discussion
by using rna interference  technique in this experiment we have generated a number of transgenic plants expressing camv35s-osbadh2-rnai construct.
molecular analyses combined with panel sensory evaluation and gas chromatography-mass spectrometry demonstrates downregulation of osbadh <dig> transcripts in the transgenic plants results in significant elevation of 2-acetyl-1-pyrroline production.
this result is well consistent with the previous fine mapping data  <cit> .
interestingly, extensive sequence analysis in this and previous studies indicates that both the traditional and modern fragrant rice varieties with diverse origins possess the same mutant allele, suggesting the donor mutation leading to fragrance probably has a single evolutionary origin  <cit> .
distinctly, this spontaneous mutant allele prevalently present in all the tested fragrant rice varieties probably represents the capacity for plants to evolve phenotypic modifications in response to local cultural preferences.
the mutant may occur even before rice domestication and disperse worldwide over the course of the domestication.
rice badh <dig>  belongs to aldehyde dehydrogenases  superfamily comprises a group of divergently related enzymes that catalyze the irreversible nad+-dependent oxidation of a wide variety of aliphatic and aromatic aldehydes to their corresponding carboxylic acids and occur in most well-studied organisms  <cit> .
a distinct function of this gene family is involved in environmental stresses responses and tolerance  <cit> .
indeed, there are two closely related betaine aldehyde dehydrogenase  homologs  present in rice genome  <cit> .
several studies showed that glycine betaine accumulation in rice  plants was undetectable, indicating a possible functional defect resulting from an unusual post-transcriptional processing at choline monooxygenase  and/or betaine aldehyde dehydrogenase  loci specifically for glycine betaine biosynthesis  <cit> .
the current transgenic experiment demonstrates down-regulated osbadh <dig> expression leads to reduced crop productivity, indicating a bona fide activity of the gene product encoded by osbadh <dig> functioning in crop performance.
actually, the aldehyde dehydrogenase activity of osbadh <dig> has been recently demonstrated in an independent study  <cit> .
intriguingly, a unique function of rice badh <dig> involved in aroma production has been apparently implicated by rnai-mediated expression repression.
this result has been recently confirmed in an independent experiment  <cit> .
at present, the biochemical pathway leading to aroma production in rice has not been established.
the recessive nature of mutant fragrant allele suggests that a loss of function is responsible for the accumulation of fragrant compound  <cit> .
in present experiment, down regulation of osbadh <dig> mrna level resulting in elevated aroma production further suggests the multiple mutations in the fragrant allele most likely give rise to a loss-of-function version of osbadh <dig> and thereafter account for the aroma production.
accordingly, we are able to speculate the functional osbadh <dig> protein encoded by the wild allele may catalyze a reaction that consumes either 2-acetyl-1-pyrroline or an upstream precursor of 2-acetyl-1-pyrroline in a competing pathway.
obviously, to further elucidate the biochemical pathway of 2-acetyl-1-pyrroline accumulation, additional gene loci involved in aroma metabolism need to be identified.
to harness the positive effects of osbadh <dig> gene suppression in aromatic rice production without the collateral negative effects on crop performance, we could inhibit accumulation of the functional osbadh <dig> mrna either by rna interference  specifically in rice grain using osbadh2-derived inverted-repeat constructs driven by endosperms-specific promoters, or by incorporating the loss-of-function spontaneous mutant aromatic allele into one of the parental lines in hybrid rice.
due to the recessive nature of spontaneous mutant aromatic allele, the vegetative growth of the heterozygous f <dig> plants of the hybrid rice varieties will not be adversely affected, while the endosperm homozygous for the mutant allele derived from the heterozygous f <dig> hybrids will produce and accumulate fragrant compound.
CONCLUSIONS
this study indicates that down-regulated expression level of osbadh <dig> gene favorably enhances the accumulation of aromatic compound, 2-acetyl-1-pyrroline.
in addition, the sequencing data suggests the prevalent aromatic allele of spontaneous mutation has a single evolutionary origin.

 characteristic fragrance in aromatic rice leads to its popularity worldwide and the aromatic traits have been extensively incorporated into commercial and hybrid rice breeding programs. the fragrance occurring in many aromatic rice varieties has been shown to be associated with the presence of 2-acetyl-1-pyrroline, which has also been identified in a great variety of food products. in the thailand fragrant rice variety khao dawk mali 2-acetyl-1-pyrroline formation and strong aroma emission has been found to be positively correlated with an accumulation of proline. although the biochemical pathway leading to the fragrance is largely unknown, l-proline has been demonstrated to be a possible precursor in the production of 2-acetyl-1-pyrroline in rice plants. the inheritance of fragrant trait in rice has been well documented. genetic investigations implicated that the fragrant trait of rice was controlled by a single recessive locus. a number of genetic analyses using reciprocal crosses repetitively showed the aromatic trait is characteristics of recessive monogenic inheritance without impact from cytoplasmic genes. nevertheless, tsuzuki & shimokawa reported that two genes were responsible for the construction of the aromatic trait. in addition, several investigations by using different aromatic rice cultivars also showed that two recessive genes were involved in the segregation of aromatic and non-aromatic traits. furthermore, genetic studies revealed that the underlying gene responsible for the aroma production was located on chromosome. by using translocation and trisomics lines derived from non-fragrant rice cv. ir aromatic trait was also mapped to the chromosome. in a recent study involved eight aromatic hybrid rice maintainer lines, a single recessive locus spanning ssr markers rm and rm on chromosome was identified to tightly link with the fragrant trait. physical mapping revealed that several candidate genes including a rice betaine aldehyde dehydrogenase homolog on chromosome was co-segregated with aroma production. meanwhile, a delicate study demonstrated that the aroma production in fragrant genotypes was well correlated with the multiple mutations in the fragrance rice locus that is identical to betaine aldehyde dehydrogenase on chromosome. however, direct evidence proving the function of osbadh in 2-acetyl-1-pyrroline production is lacking. furthermore, traditional aromatic cultivars have often undesirable agronomic performance, such as poor yield, susceptibility to pests and diseases, and strong shedding. the molecular mechanism of this major weakness occurred in the aromatic rice cultivars is largely unknown. in present experiments, we sequenced the osbadh locus derived from a number of aromatic rice cultivars across china and uncovered that multiple mutations identical to fgr allele also occurred in all the tested fragrant accessions. by using rna interference technique combined with agrobacterium tumefaciens-mediated t-dna transfer, we show that the directed degradation of osbadh transcripts results in a significantly elevated fragrance emission and 2-acetyl-1-pyrroline accumulation, unambiguously suggesting that osbadh locus is responsible for aroma production in fragrant rice varieties. in addition, disrupted osbadh leads to a detectable reduction of crop productivity, implying its multiple functions in secondary metabolism and agronomic performance. a strategy to compromise the favorable aspects and to avoid the unfavorable effect during osbadh manipulation and breeding is discussed as well.  expression and nucleotide variation at the osbadh locus the cv. nipponbare was used to determine the expression patterns of osbadh total rnas were extracted from roots, stems, leaves and flowers of mature plant, as well as embryos, seedling roots, seedling stems, seedling leaves. messenger rna levels were analyzed by semi-quantitative rt-pcr. the results show osbadh is expressed constitutively, with less expression abundance in mature roots. we examined genetic variation in a bp portion of osbadh gene in fragrant and six non-fragrant rice accessions that are representatives of cultivated varieties across china. multiple mutations identical to fgr allele identified by bradbury et al. were also observed in all the fragrant accessions. these mutations contain a total of six snps and eight deletions within a bp region of the exon as compared to the wild type allele in the tested non-fragrant varieties. deficient in osbadh expression resulting in apparently sensory aroma production to dissect the physiological role of osbadh we generated a large number of transgenic rice plants expressing osbadh2-rnai. construct was introduced into non-fragrant rice cv. nipponbare by agrobacterium tumefaciens-mediated t-dna transfer. the majority of osbadh2-rnai primary transgenics resulted in pcr amplification with primers designed to the hpt marker. preliminary aroma evaluation revealed that out of hpt-positive t plants showed abundant fragrance emission. ten t segregating populations derived from independent t plants with obvious aroma production were selected and cultivated in the university's farm. leaves from the first and second crops and grains from the first crop of individual t plants were used for sensory evaluation of aroma production. as a result, the presence and absence of fragrance is unambiguously segregated within t populations and well consistently expressed in leaves and grains from the same plants. in all the t1-segregating populations, we observed a correlation of osbadh2-rnai transgene integration with apparently sensory aroma production. to investigate property of the emitted aromatic compound from the transgenic lines, a gas chromatography-mass spectrometry analysis was conducted for thai hom mali, transgenic line and the wild type control. as shown in figure a considerable aroma production of 2-acetyl-1-pyrroline was detected in both thai hom mali and transgenic line, by contrast to the undetectable wild type control. in order to test if the aroma production is co-segregated with the presence of t-dna insertion in the transgenic segregating progeny, t mature leaves from five osbadh2-rnai repression plants and four non-fragrant wild-type plants segregated out from the same transgenic line were collected and subjected to the gc-ms analysis. as a result, 2ap accumulation was apparently enhanced in all the downregulation plants, by contrast to the undetectable wild-type plants without fragrance. to ensure that the observed phenotypes are correlated with reduced endogenous transcript, total rna was extracted from leaves of wild type plants and four independent osbadh2-rnai repression lines. analysis of semi-quantitative rt-pcr and real-time quantitative rt-pcr revealed a considerable reduction in endogenous osbadh transcript levels in osbadh2-rnai repression lines compared to that of wild-type plants. to determine if the expression of osbadh gene is affected in the down-regulated osbadh transgenic lines, we conducted real-time pcr analysis to detect the osbadh cdna levels in the osbadh2-rnai repression lines. the osbadh gene is the closest available homolog of osbadh and is almost unaffected in the down-regulated osbadh lines. disrupted osbadh influencing crop productivity to investigate the effects of down-regulated osbadh on crop productivity, the plant height and 1000-grain weight of osbadh2-deficient and wild type plants derived from four independent t transgenic lines were measured. the results showed the reduction in plant height as well as 1000-grain weight in the osbadh2-deficient plants is differentially detectable when compared to the wild-type plants segregated out from the transgenic progenies. * significant at p < compared to non-fragrant wild-type plants segregated out from the same transgenic line. to further address the physiological functions of the osbadh gene, wild type seeds and t seeds derived from four independent transgenic lines were germinated in various salt-stressed conditions. no significant difference in sprouting index and germination rates between wild type and osbadh repression lines was observed. nevertheless, the seedling growth rates in different salt concentrations were more or less inhibited in osbadh2-deficient lines as compared to wild type. discussion by using rna interference technique in this experiment we have generated a number of transgenic plants expressing camv35s-osbadh2-rnai construct. molecular analyses combined with panel sensory evaluation and gas chromatography-mass spectrometry demonstrates downregulation of osbadh transcripts in the transgenic plants results in significant elevation of 2-acetyl-1-pyrroline production. this result is well consistent with the previous fine mapping data. interestingly, extensive sequence analysis in this and previous studies indicates that both the traditional and modern fragrant rice varieties with diverse origins possess the same mutant allele, suggesting the donor mutation leading to fragrance probably has a single evolutionary origin. distinctly, this spontaneous mutant allele prevalently present in all the tested fragrant rice varieties probably represents the capacity for plants to evolve phenotypic modifications in response to local cultural preferences. the mutant may occur even before rice domestication and disperse worldwide over the course of the domestication. rice badh belongs to aldehyde dehydrogenases superfamily comprises a group of divergently related enzymes that catalyze the irreversible nad+-dependent oxidation of a wide variety of aliphatic and aromatic aldehydes to their corresponding carboxylic acids and occur in most well-studied organisms. a distinct function of this gene family is involved in environmental stresses responses and tolerance. indeed, there are two closely related betaine aldehyde dehydrogenase homologs present in rice genome. several studies showed that glycine betaine accumulation in rice plants was undetectable, indicating a possible functional defect resulting from an unusual post-transcriptional processing at choline monooxygenase and/or betaine aldehyde dehydrogenase loci specifically for glycine betaine biosynthesis. the current transgenic experiment demonstrates down-regulated osbadh expression leads to reduced crop productivity, indicating a bona fide activity of the gene product encoded by osbadh functioning in crop performance. actually, the aldehyde dehydrogenase activity of osbadh has been recently demonstrated in an independent study. intriguingly, a unique function of rice badh involved in aroma production has been apparently implicated by rnai-mediated expression repression. this result has been recently confirmed in an independent experiment. at present, the biochemical pathway leading to aroma production in rice has not been established. the recessive nature of mutant fragrant allele suggests that a loss of function is responsible for the accumulation of fragrant compound. in present experiment, down regulation of osbadh mrna level resulting in elevated aroma production further suggests the multiple mutations in the fragrant allele most likely give rise to a loss-of-function version of osbadh and thereafter account for the aroma production. accordingly, we are able to speculate the functional osbadh protein encoded by the wild allele may catalyze a reaction that consumes either 2-acetyl-1-pyrroline or an upstream precursor of 2-acetyl-1-pyrroline in a competing pathway. obviously, to further elucidate the biochemical pathway of 2-acetyl-1-pyrroline accumulation, additional gene loci involved in aroma metabolism need to be identified. to harness the positive effects of osbadh gene suppression in aromatic rice production without the collateral negative effects on crop performance, we could inhibit accumulation of the functional osbadh mrna either by rna interference specifically in rice grain using osbadh2-derived inverted-repeat constructs driven by endosperms-specific promoters, or by incorporating the loss-of-function spontaneous mutant aromatic allele into one of the parental lines in hybrid rice. due to the recessive nature of spontaneous mutant aromatic allele, the vegetative growth of the heterozygous f plants of the hybrid rice varieties will not be adversely affected, while the endosperm homozygous for the mutant allele derived from the heterozygous f hybrids will produce and accumulate fragrant compound.  this study indicates that down-regulated expression level of osbadh gene favorably enhances the accumulation of aromatic compound, 2-acetyl-1-pyrroline. in addition, the sequencing data suggests the prevalent aromatic allele of spontaneous mutation has a single evolutionary origin. 
genetic studies and physical fine mapping reveal that a candidate gene homologous to betaine aldehyde dehydrogenase is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of osbadh is lacking. the results showed that multiple mutations identical to fgr allele occur in the fragrant rice accessions across china; osbadh is expressed constitutively, with less expression abundance in mature roots; the disrupted osbadh by rna interference leads to significantly increased 2-acetyl-1-pyrroline production. semi-quantitative, real-time reverse transcription-polymerase chain reaction, as well as gas chromatography-mass spectrometry were conducted to determine the expression levels of osbadh and the fragrant compound in wild type and transgenic osbadh2-rnai repression lines, respectively. we have found that the altered expression levels of osbadh gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin. to elucidate the physiological roles of osbadh sequencing approach and rna interference technique were employed to analyze allelic variation and functions of osbadh gene in aroma production. aromatic rice is popular worldwide because of its characteristic fragrance.
 aromatic rice is popular worldwide because of its characteristic fragrance. genetic studies and physical fine mapping reveal that a candidate gene homologous to betaine aldehyde dehydrogenase is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of osbadh is lacking. to elucidate the physiological roles of osbadh sequencing approach and rna interference technique were employed to analyze allelic variation and functions of osbadh gene in aroma production. semi-quantitative, real-time reverse transcription-polymerase chain reaction, as well as gas chromatography-mass spectrometry were conducted to determine the expression levels of osbadh and the fragrant compound in wild type and transgenic osbadh2-rnai repression lines, respectively.  the results showed that multiple mutations identical to fgr allele occur in the fragrant rice accessions across china; osbadh is expressed constitutively, with less expression abundance in mature roots; the disrupted osbadh by rna interference leads to significantly increased 2-acetyl-1-pyrroline production.  we have found that the altered expression levels of osbadh gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin. 