BACKGROUND
carotenoids constitute a group of naturally occurring pigments that play diverse roles in plants.
structurally carotenoids are composed of eight isoprene units joined to form a c <dig> hydrocarbon skeleton containing conjugated double bonds and linear or cyclic end groups.
in chloroplasts, they are part of light-harvesting complexes and also function as antioxidants.
carotenoids accumulate in chromoplasts as secondary metabolites, and impart attractive colors to flowers and fruits.
they are important in human diet as they provide β-carotene, the vitamin a precursor.
owing to their antioxidant activity, they are also commercially used by cosmetic and pharmaceutical industries  <cit> .
carotenoid biosynthesis has been extensively studied in plants such as tomato, arabidopsis and pepper  <cit> .
genes coding for enzymes catalyzing main steps of the carotenoid biosynthesis pathway have been cloned and their expression profiles have also been studied in different species  <cit> .
tomato fruit is a model system for studying the carotenogenesis in plants.
ripening in tomato fruit is associated with vivid changes in color.
the change in fruit color from green to orange, pink and then red is accompanied by shift in carotenoid profile from β-carotene at breaker stage to lycopene at red ripe stage.
these changes are brought about by transcriptional upregulation of phytoene synthase  and phytoene desaturase  genes  <cit>  and down regulation of lycopene β-cyclase  and lycopene ε-cyclase  genes  <cit> .
significant increase in carotenoid content was achieved by genetic engineering of carotenoid biosynthesis pathway in canola, rice, potato and maize  <cit> .
in contrast to these transgenic crops, only limited success has been achieved in increasing the carotenoid levels in tomato  <cit> .
the carotenoid biosynthetic pathway is controlled by a complex regulatory mechanism that includes transcriptional, post transcriptional and feed-back inhibition by end-products  <cit> .
moreover, isoprene precursors required for the carotenoid pathway also serve as precursors for phytohormones such as abscisic acid , gibberellins and secondary metabolites  <cit> .
constitutive over-expression of chromoplast-specific psy <dig> in tomato has been shown to result in dwarf phenotype, probably by interfering with the gibberellin biosynthesis pathway  <cit> .
contrary to the expectation, the psy <dig> over-expressing transgenic tomato fruit also had reduced lycopene content as compared to untransformed plants  <cit> .
tomato high-pigment  <dig>  mutant showed 30% increase in carotenoid content in the mature fruit, but exhibited aba deficiency  <cit> .
therefore, understanding the regulatory network and metabolic cross-talk between pathways is necessary for metabolic engineering of carotenoids in plants.
the success of desired modification in transgenics depends upon the source of transgene; organ to which it is targeted, choice of promoter used, and the key nodes in pathway targeted for modification.
the key steps in carotenoid biosynthetic pathway predominantly targeted for transgenic modifications are catalyzed by enzymes such as psy, pds and lcy-b.
although cdnas of genes encoding carotenoid biosynthetic pathway enzymes have been well characterized in tomato, their promoters have received limited attention.
only pds promoter has been characterized in tomato  <cit> .
isolation and characterization of promoters of carotenoid biosynthesis pathway genes will help understand the regulation of carotenoid biosynthesis pathway in tomato.
in this study, we describe the isolation and characterization of cyc-b  promoter from a green fruited solanum habrochaites genotype ec <dig>  the cyc-b full-length promoter and its 5' truncated promoter regions were analyzed by transient and stable expression systems using gus as reporter gene in tomato.
a short promoter region with higher expression level and developmental expression similar to that of full-length promoter was identified.
RESULTS
tomato genome contains two types of lycopene β-cyclase genes, lcy-b and cyc-b, encoding chloroplast- and chromoplast-specific lycopene β-cyclase enzymes, respectively.
lcy-b is expressed in leaves, flowers and in fruits until breaker-stage of fruit ripening  <cit> .
the cyc-b encodes a chromoplast-specific lycopene β-cyclase and is expressed exclusively in flowers and at breaker-stage of fruit  <cit> .
lycopene β-cyclase is one of the crucial enzymes for carotenoid biosynthesis.
lycopene β-cyclase along with lycopene ε-cyclase  bring about the cyclization of lycopene.
activities of both of these enzymes together make α-carotene, while activity of lycopene β-cyclase alone leads to formation of β-carotene  <cit> .
in pepper also, lyc-b and capsanthin-capsorubin synthase  genes have been proposed to bring about major changes in carotenoid profiles during ripening in pepper  <cit> .
ccs is a protein with high homology to cyc-b of tomato, and is highly induced during fruit development  <cit> .
in watermelon, co-dominant caps markers based on a snp in lycopene β-cyclase gene has been developed for allelic selection between canary yellow and red watermelon  <cit> .
thus, lycopene β-cyclase plays a major role in carotenogenesis of different colored flowers and fruits.
isolation and characterization of cyc-b promoter will help understand the regulation of cyc-b expression in chromoplast-rich organs, and the promoter may be useful in genetic engineering of carotenoid content in plants.
expression of cyc-b gene in s. lycopersicum and s. habrochaites
the mrna levels of cyc-b gene in leaf and flower, and at different stages of fruit development of s. lycopersicum cv.
pusa ruby and s. habrochaites genotype ec <dig> were determined by semi-quantitative rt-pcr analysis.
pusa ruby fruits show typical color change from green to red during ripening, while s. habrochaites  fruits remain green even at fully ripe stage.
cyc-b expression was high in the chromoplast-rich tissues such as flowers and fruits, while a low basal level expression was detected in fully developed leaves in both the genotypes .
in pusa ruby fruits, cyc-b expression was highest in breaker stage, and thereafter reduced to a very low level at red-ripe stage.
on the contrary, in s. habrochaites  fruits, cyc-b expression levels were similar in different stages of fruit ripening, and remained high even at fully ripe stage.
expression pattern of cyc-b in s. habrochaites observed in this study was similar to that of cyc-b gene expression in beta mutant reported by ronen et al  <cit> .
although, ronen et al  <cit>  could not detect cyc-b expression in wild-type s. lycopersicum cv.
m <dig>  in this study we could detect expression of cyc-b in leaves, and in fruits at all the stages of ripening in both s. lycopersicum cv.
pusa ruby and s. habrochaites genotype ec <dig>  expression pattern of cyc-b gene, a low level in leaves and high level in chromoplast-rich flowers and fruits, observed in our study is consistent with the expression patterns previously reported for pds and psy <dig>  genes  <cit> .
similarly, crtr-b <dig> , a chromoplast-specific gene is highly expressed in petals and anthers, and expresses albeit at low level in carpels and sepals, while crtr-b <dig>  a chloroplast-specific gene shows high level of expression in leaves and sepals but show a low level of expression in flower tissues  <cit> .
moreover, it was shown that transgenic plants expressing antisense b  did not show any biochemical or developmental alterations in leaves and stems  <cit> .
thereby implicating that the basal expression level of cyc-b found in leaves may not have critical role in vegetative tissues.
isolation of cyc-b promoter
the promoter region of cyc-b gene from s. habrochaites genotype ec <dig> and s. lycopersicum genotype ec <dig> was isolated by directional genome walking pcr using a set of walker primers and gene-specific primers.
comparison of the sequence of the dna fragment cloned from s. habrochaites with the coding sequence of cyc-b coding sequence  revealed the presence of  <dig> bp fragment upstream to the start codon of cyc-b gene.
the  <dig> bp fragment upstream to the atg codon was designated as shcyc-b full-length promoter .
the nucleotide sequence of shcyc-b promoter cloned in this study was deposited at ncbi .
the dna fragment cloned from s. lycopersicum contained  <dig> bp promoter region .
the shcyc-b promoter sequence was analyzed for transcription start site  and potential cis-acting transcription factor binding sites.
neural network promoter prediction  <cit>  identified a tss at  <dig> bp 5' to atg.
place  <cit>  and plantcare  <cit>  analysis revealed a potential tata box at  <dig> bp 5' to atg and  <dig> bp upstream to the potential tss.
several caat boxes, a rap <dig>  transcription factor binding site, an ethylene responsive element , circadian elements and light responsive elements were found in the shcyc-b promoter .
a list of some of the relevant cis-elements detected and their relative positions from the translational start site atg is given in table  <dig>  a comparison of the cyc-b promoters from s. lycopersicum and s. habrochaites revealed that ere  cis-element was conserved between these two promoters .
this ere cis-element was also reported in cyc-b promoter of beta gene  <cit> .
this indicates ethylene responsive regulation of cyc-b promoter during fruit ripening.
the rap <dig>  transcription factor binding site atcta  <cit>  was also found in both the cyc-b promoters cloned in this study.
the cis-element atcta has been found to be conserved in promoters of genes involved in carotenoid and tocopherol biosynthesis, and certain photosynthesis-related genes in arabidopsis  <cit> .
however, there were many elements that were exclusively present in shcyc-b promoter but not in slcyc-b promoter.
these include rbcs general consensus sequence, carg consensus sequence found in the promoter of flowering-time gene , l-box  and gt- <dig> element, which are known to play important role in gene expression .
transient and stable expression of shcyc-b promoter in tomato
to characterize the putative shcyc-b promoter, promoter::β-glucuronidase  reporter gene fusion constructs were prepared for full-length and its 5' deletion fragments, and transformed into agrobacterium .
binary vector pbi <dig> having constitutive camv35s promoter::gus reporter gene was used as control.
functional analysis was carried out by transient in fruto expression in tomato fruits, and stable expression in transgenic tomato.
in transient expression analysis, gus activity was evident in columella and placental tissues in both green stage and ripe stage of tomato fruit .
this qualitative gus assay revealed that the full-length shcyc-b promoter as well as its deletion fragments were able to drive the expression of reporter gene in different developmental stages of tomato fruit.
to identify putative cis-elements required for developmental and tissue-specific expression of shcyc-b promoter, tomato transgenic plants were generated.
six to nine independent transgenic lines for each construct were screened in t <dig> generation on the basis of pcr and histochemical gus staining.
there was no difference in the localization of gus activity in fruits among full-length and truncated promoter constructs at various stages of fruit development.
gus staining was visible in vascular bundles, columella, placental tissue and seeds.
in case of d0- <dig>  and d3- <dig>  lines, locular tissue was also highly stained.
however, there was no gus activity in epidermis.
the intensity of gus staining was relatively similar among independent events for each construct, though one or two events of d1- <dig> and d2- <dig> transgenics showed variation in the intensity of gus stain.
among the  <dig> independent events examined for d2-  <dig> transgenic plants,  <dig> plants showed consistently lower gus intensity as compared to that of full-length and other deletion constructs .
about 4- <dig> individual events for each construct were carried forward to t <dig> for further analyses.
t <dig> seeds were selected on kanamycin and screened by pcr.
for each construct, 2- <dig> events were subjected to southern analysis to determine transgene copy number .
subsequently, plants having single copy insertions were analyzed for promoter activity by northern analysis, and histochemical as well as quantitative gus assays.
since there was no visible difference in gus staining of d0- <dig> and d1- <dig> transgenic fruits, and no single copy t <dig> plants were available in d1- <dig>  plants harboring this construct were not included in further analysis.
to examine the tissue-specific expression, leaf, root, flower and fruits at different developmental stages from single copy transgenic plants for each construct were subjected to histochemical gus staining.
gus activity was apparently not detectable by visual observations in transgenic roots  and leaves  transformed with the shcyc-b full-length or truncated promoter constructs.
the transgenic flowers harboring either full-length shcyc-b promoter or its 5' deletion fragments showed gus staining mainly in stamens, while there was little or no gus staining in petals .
similar kind of gus staining was observed in the flowers of transgenic tomato expressing pds promoter-driven gus reporter gene  <cit> .
the camv35s::gus transgenic plants showed gus staining both in leaves and flowers .
in t <dig> fruits, localization of gus activity was similar to that observed in t <dig> fruits for all shcyc-b promoter constructs.
in fruits, d2- <dig> consistently showed lower gus intensity, while the gus staining was highest in d3- <dig> .
the activity of full-length and truncated promoter driven gus was low at green fruit stage, and showed an upregulation at breaker and orange stages of fruit development .
since ere  cis-element was found at - <dig> bp 5' to atg of shcyc-b promoter, we examined whether ethylene could induce the expression of cyc-b promoter in vegetative tissues.
foliar spray of ethephon  did not induce cyc-b promoter in the seedlings of full-length and deletion transgenic lines .
this suggests that developmental  cues are required to induce cyc-b promoter.
quantitative gus assay
the visual observations made by histochemical gus staining in leaf, flower and different developmental stages of fruit, were quantified by fluorometric mug  assay.
tomato transgenic plants harboring pbi <dig>  were also included in the analysis.
camv35s promoter driven gus activity was lowest in flowers, and increased about 4- and > 8-fold in leaves and fruits, respectively .
the expression pattern of full-length shcyc-b promoter as determined by gus activity  was similar to that of the expression pattern observed by rt-pcr analysis in s. habrochaites .
in full-length shcyc-b promoter transgenic plants, gus activity was about 5- and > 12-fold higher in flower and fruits, respectively, as compared with leaves.
similarly, shcyc-b d2- <dig> and d3- <dig> promoter transgenic plants also showed lower gus activity in leaves as compared to flower and fruits .
it appears that shcyc-b promoter activity in leaves is too low to be detected by histochemical gus staining.
the d2- <dig> promoter transgenic plants consistently showed lowest gus activity as compared to full-length and d3- <dig> truncated-promoter transgenic plants in all the tissues.
although the promoter strength of d3- <dig> was similar to that of full-length promoter in green fruits, d3- <dig> promoter showed higher activity than full-length promoter in leaves, flowers, and breaker-, orange- and red-stages of fruits .
most noticeably, the shortest promoter fragment d3- <dig> showed  <dig>  and  <dig> -fold higher gus activity in flowers and leaves, respectively, as compared to that of d0- <dig> full-length promoter .
northern-blot analysis
northern blot was performed to analyze the relative levels of cyc-b full-length or truncated promoter driven gus expression in different tissues namely leaf, flower and fruits from transgenic tomato plants.
blots used for detecting gus mrna levels were reprobed with ef1α that served as a loading control for amount of total rna.
the concentration of total rna loaded per lane for a particular stage of development or tissue was essentially same for transgenic lines of each construct, but was varying in the range of 20- <dig> μg across different stages or tissues used.
shcyc-b full-length promoter and its deletion fragments showed low expression in chloroplast-rich green leaves and green fruits, while their expression was high in chromoplast-rich flowers, and fruits at breaker and orange stage of ripening .
consistent with gus assay, in northern analysis also, d3- <dig> promoter fragment showed highest promoter activity in flowers .
however, in contrast to the higher gus-activity observed in fruits of transgenic plants expressing d3- <dig> promoter driven gus reporter, the transcript levels of d3- <dig> promoter driven gus were not so apparently higher than that of d0- <dig> promoter driven gus reporter.
the deletion fragment d2- <dig> showed lowest promoter strength as compared to the full-length promoter and d3- <dig>  the reduction in gus expression in d2- <dig> transgenic plant does not appear to be due to transgene position and/or silencing effect, as seven out of nine independent events showed reduced gus staining, and the transgenic plants with single insertion for d2- <dig> fragment were selected for analysis.
the present study clearly showed that the shcyc-b promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits at different stages of ripening.
the promoter strength was drastically decreased by a deletion of - <dig> to - <dig> bp 5' to atg as compared to full-length promoter, while the shortest d3- <dig> promoter fragment showed highest activity.
this suggests that nucleotide sequence - <dig> to - <dig> bp upstream to initiation codon may contain cis-elements involved in down regulation of cyc-b expression, while nucleotide sequences - <dig> to - <dig> bp upstream to initiation codon might be involved in negating the repressive nature of regulatory sequences in - <dig> to - <dig> bp 5' to atg.
the rap <dig>  binding cis-element atcta has been shown to confer strong basal activity in psy promoter from arabidopsis  <cit> .
this cis-element was found in shcyc-b full-length promoter at - <dig> to - <dig> bp upstream to atg, and might contribute to the basal activity of full-length promoter, as deletion of this cis-element in d2- <dig> resulted in considerable decrease in promoter activity.
the presence of three gt- <dig> cis-element , one at - <dig> to - <dig> and two at - <dig> to - <dig> and - <dig> to - <dig> bp upstream of atg might be playing a role in the gene expression.
gt- <dig> element was initially identified as cis-element regulating cell-type specific expression specifically in light regulated genes.
gt- <dig> protein binds to tfiia and tata-binding proteins.
the gt- <dig> cis-element is conserved in many plant promoters, and may have positive or negative regulatory effect on transcription depending upon cell type  <cit> .
CONCLUSIONS
in this study, lycopene β-cyclase  promoter from solanum habrochaites, a green fruited tomato genotype, was isolated and functionally characterized in transient in fruto and stable transformation systems.
conservation of cis-elements such as rap <dig>  binding cis-element and ere cis-element between cyc-b and psy promoter suggests a common regulatory mechanism for carotenoid accumulation in fruits.
a short promoter region with promoter activity and developmental expression pattern comparable to that of full-length shcyc-b promoter was identified.
as signal transduction events and transcription factors that developmentally regulate the cyc-b expression are not known, the short promoter region identified in this study can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in cyc-b expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato.

 carotenoids constitute a group of naturally occurring pigments that play diverse roles in plants. structurally carotenoids are composed of eight isoprene units joined to form a c hydrocarbon skeleton containing conjugated double bonds and linear or cyclic end groups. in chloroplasts, they are part of light-harvesting complexes and also function as antioxidants. carotenoids accumulate in chromoplasts as secondary metabolites, and impart attractive colors to flowers and fruits. they are important in human diet as they provide β-carotene, the vitamin a precursor. owing to their antioxidant activity, they are also commercially used by cosmetic and pharmaceutical industries. carotenoid biosynthesis has been extensively studied in plants such as tomato, arabidopsis and pepper. genes coding for enzymes catalyzing main steps of the carotenoid biosynthesis pathway have been cloned and their expression profiles have also been studied in different species. tomato fruit is a model system for studying the carotenogenesis in plants. ripening in tomato fruit is associated with vivid changes in color. the change in fruit color from green to orange, pink and then red is accompanied by shift in carotenoid profile from β-carotene at breaker stage to lycopene at red ripe stage. these changes are brought about by transcriptional upregulation of phytoene synthase and phytoene desaturase genes and down regulation of lycopene β-cyclase and lycopene ε-cyclase genes. significant increase in carotenoid content was achieved by genetic engineering of carotenoid biosynthesis pathway in canola, rice, potato and maize. in contrast to these transgenic crops, only limited success has been achieved in increasing the carotenoid levels in tomato. the carotenoid biosynthetic pathway is controlled by a complex regulatory mechanism that includes transcriptional, post transcriptional and feed-back inhibition by end-products. moreover, isoprene precursors required for the carotenoid pathway also serve as precursors for phytohormones such as abscisic acid, gibberellins and secondary metabolites. constitutive over-expression of chromoplast-specific psy in tomato has been shown to result in dwarf phenotype, probably by interfering with the gibberellin biosynthesis pathway. contrary to the expectation, the psy over-expressing transgenic tomato fruit also had reduced lycopene content as compared to untransformed plants. tomato high-pigment mutant showed 30% increase in carotenoid content in the mature fruit, but exhibited aba deficiency. therefore, understanding the regulatory network and metabolic cross-talk between pathways is necessary for metabolic engineering of carotenoids in plants. the success of desired modification in transgenics depends upon the source of transgene; organ to which it is targeted, choice of promoter used, and the key nodes in pathway targeted for modification. the key steps in carotenoid biosynthetic pathway predominantly targeted for transgenic modifications are catalyzed by enzymes such as psy, pds and lcy-b. although cdnas of genes encoding carotenoid biosynthetic pathway enzymes have been well characterized in tomato, their promoters have received limited attention. only pds promoter has been characterized in tomato. isolation and characterization of promoters of carotenoid biosynthesis pathway genes will help understand the regulation of carotenoid biosynthesis pathway in tomato. in this study, we describe the isolation and characterization of cyc-b promoter from a green fruited solanum habrochaites genotype ec the cyc-b full-length promoter and its 5' truncated promoter regions were analyzed by transient and stable expression systems using gus as reporter gene in tomato. a short promoter region with higher expression level and developmental expression similar to that of full-length promoter was identified.  tomato genome contains two types of lycopene β-cyclase genes, lcy-b and cyc-b, encoding chloroplast- and chromoplast-specific lycopene β-cyclase enzymes, respectively. lcy-b is expressed in leaves, flowers and in fruits until breaker-stage of fruit ripening. the cyc-b encodes a chromoplast-specific lycopene β-cyclase and is expressed exclusively in flowers and at breaker-stage of fruit. lycopene β-cyclase is one of the crucial enzymes for carotenoid biosynthesis. lycopene β-cyclase along with lycopene ε-cyclase bring about the cyclization of lycopene. activities of both of these enzymes together make α-carotene, while activity of lycopene β-cyclase alone leads to formation of β-carotene. in pepper also, lyc-b and capsanthin-capsorubin synthase genes have been proposed to bring about major changes in carotenoid profiles during ripening in pepper. ccs is a protein with high homology to cyc-b of tomato, and is highly induced during fruit development. in watermelon, co-dominant caps markers based on a snp in lycopene β-cyclase gene has been developed for allelic selection between canary yellow and red watermelon. thus, lycopene β-cyclase plays a major role in carotenogenesis of different colored flowers and fruits. isolation and characterization of cyc-b promoter will help understand the regulation of cyc-b expression in chromoplast-rich organs, and the promoter may be useful in genetic engineering of carotenoid content in plants. expression of cyc-b gene in s. lycopersicum and s. habrochaites the mrna levels of cyc-b gene in leaf and flower, and at different stages of fruit development of s. lycopersicum cv. pusa ruby and s. habrochaites genotype ec were determined by semi-quantitative rt-pcr analysis. pusa ruby fruits show typical color change from green to red during ripening, while s. habrochaites fruits remain green even at fully ripe stage. cyc-b expression was high in the chromoplast-rich tissues such as flowers and fruits, while a low basal level expression was detected in fully developed leaves in both the genotypes. in pusa ruby fruits, cyc-b expression was highest in breaker stage, and thereafter reduced to a very low level at red-ripe stage. on the contrary, in s. habrochaites fruits, cyc-b expression levels were similar in different stages of fruit ripening, and remained high even at fully ripe stage. expression pattern of cyc-b in s. habrochaites observed in this study was similar to that of cyc-b gene expression in beta mutant reported by ronen et al. although, ronen et al could not detect cyc-b expression in wild-type s. lycopersicum cv. m in this study we could detect expression of cyc-b in leaves, and in fruits at all the stages of ripening in both s. lycopersicum cv. pusa ruby and s. habrochaites genotype ec expression pattern of cyc-b gene, a low level in leaves and high level in chromoplast-rich flowers and fruits, observed in our study is consistent with the expression patterns previously reported for pds and psy genes. similarly, crtr-b, a chromoplast-specific gene is highly expressed in petals and anthers, and expresses albeit at low level in carpels and sepals, while crtr-b a chloroplast-specific gene shows high level of expression in leaves and sepals but show a low level of expression in flower tissues. moreover, it was shown that transgenic plants expressing antisense b did not show any biochemical or developmental alterations in leaves and stems. thereby implicating that the basal expression level of cyc-b found in leaves may not have critical role in vegetative tissues. isolation of cyc-b promoter the promoter region of cyc-b gene from s. habrochaites genotype ec and s. lycopersicum genotype ec was isolated by directional genome walking pcr using a set of walker primers and gene-specific primers. comparison of the sequence of the dna fragment cloned from s. habrochaites with the coding sequence of cyc-b coding sequence revealed the presence of bp fragment upstream to the start codon of cyc-b gene. the bp fragment upstream to the atg codon was designated as shcyc-b full-length promoter. the nucleotide sequence of shcyc-b promoter cloned in this study was deposited at ncbi. the dna fragment cloned from s. lycopersicum contained bp promoter region. the shcyc-b promoter sequence was analyzed for transcription start site and potential cis-acting transcription factor binding sites. neural network promoter prediction identified a tss at bp 5' to atg. place and plantcare analysis revealed a potential tata box at bp 5' to atg and bp upstream to the potential tss. several caat boxes, a rap transcription factor binding site, an ethylene responsive element, circadian elements and light responsive elements were found in the shcyc-b promoter. a list of some of the relevant cis-elements detected and their relative positions from the translational start site atg is given in table a comparison of the cyc-b promoters from s. lycopersicum and s. habrochaites revealed that ere cis-element was conserved between these two promoters. this ere cis-element was also reported in cyc-b promoter of beta gene. this indicates ethylene responsive regulation of cyc-b promoter during fruit ripening. the rap transcription factor binding site atcta was also found in both the cyc-b promoters cloned in this study. the cis-element atcta has been found to be conserved in promoters of genes involved in carotenoid and tocopherol biosynthesis, and certain photosynthesis-related genes in arabidopsis. however, there were many elements that were exclusively present in shcyc-b promoter but not in slcyc-b promoter. these include rbcs general consensus sequence, carg consensus sequence found in the promoter of flowering-time gene, l-box and gt- element, which are known to play important role in gene expression. transient and stable expression of shcyc-b promoter in tomato to characterize the putative shcyc-b promoter, promoter::β-glucuronidase reporter gene fusion constructs were prepared for full-length and its 5' deletion fragments, and transformed into agrobacterium. binary vector pbi having constitutive camv35s promoter::gus reporter gene was used as control. functional analysis was carried out by transient in fruto expression in tomato fruits, and stable expression in transgenic tomato. in transient expression analysis, gus activity was evident in columella and placental tissues in both green stage and ripe stage of tomato fruit. this qualitative gus assay revealed that the full-length shcyc-b promoter as well as its deletion fragments were able to drive the expression of reporter gene in different developmental stages of tomato fruit. to identify putative cis-elements required for developmental and tissue-specific expression of shcyc-b promoter, tomato transgenic plants were generated. six to nine independent transgenic lines for each construct were screened in t generation on the basis of pcr and histochemical gus staining. there was no difference in the localization of gus activity in fruits among full-length and truncated promoter constructs at various stages of fruit development. gus staining was visible in vascular bundles, columella, placental tissue and seeds. in case of d0- and d3- lines, locular tissue was also highly stained. however, there was no gus activity in epidermis. the intensity of gus staining was relatively similar among independent events for each construct, though one or two events of d1- and d2- transgenics showed variation in the intensity of gus stain. among the independent events examined for d2- transgenic plants, plants showed consistently lower gus intensity as compared to that of full-length and other deletion constructs. about 4- individual events for each construct were carried forward to t for further analyses. t seeds were selected on kanamycin and screened by pcr. for each construct, 2- events were subjected to southern analysis to determine transgene copy number. subsequently, plants having single copy insertions were analyzed for promoter activity by northern analysis, and histochemical as well as quantitative gus assays. since there was no visible difference in gus staining of d0- and d1- transgenic fruits, and no single copy t plants were available in d1- plants harboring this construct were not included in further analysis. to examine the tissue-specific expression, leaf, root, flower and fruits at different developmental stages from single copy transgenic plants for each construct were subjected to histochemical gus staining. gus activity was apparently not detectable by visual observations in transgenic roots and leaves transformed with the shcyc-b full-length or truncated promoter constructs. the transgenic flowers harboring either full-length shcyc-b promoter or its 5' deletion fragments showed gus staining mainly in stamens, while there was little or no gus staining in petals. similar kind of gus staining was observed in the flowers of transgenic tomato expressing pds promoter-driven gus reporter gene. the camv35s::gus transgenic plants showed gus staining both in leaves and flowers. in t fruits, localization of gus activity was similar to that observed in t fruits for all shcyc-b promoter constructs. in fruits, d2- consistently showed lower gus intensity, while the gus staining was highest in d3-. the activity of full-length and truncated promoter driven gus was low at green fruit stage, and showed an upregulation at breaker and orange stages of fruit development. since ere cis-element was found at - bp 5' to atg of shcyc-b promoter, we examined whether ethylene could induce the expression of cyc-b promoter in vegetative tissues. foliar spray of ethephon did not induce cyc-b promoter in the seedlings of full-length and deletion transgenic lines. this suggests that developmental cues are required to induce cyc-b promoter. quantitative gus assay the visual observations made by histochemical gus staining in leaf, flower and different developmental stages of fruit, were quantified by fluorometric mug assay. tomato transgenic plants harboring pbi were also included in the analysis. camv35s promoter driven gus activity was lowest in flowers, and increased about 4- and > 8-fold in leaves and fruits, respectively. the expression pattern of full-length shcyc-b promoter as determined by gus activity was similar to that of the expression pattern observed by rt-pcr analysis in s. habrochaites. in full-length shcyc-b promoter transgenic plants, gus activity was about 5- and > 12-fold higher in flower and fruits, respectively, as compared with leaves. similarly, shcyc-b d2- and d3- promoter transgenic plants also showed lower gus activity in leaves as compared to flower and fruits. it appears that shcyc-b promoter activity in leaves is too low to be detected by histochemical gus staining. the d2- promoter transgenic plants consistently showed lowest gus activity as compared to full-length and d3- truncated-promoter transgenic plants in all the tissues. although the promoter strength of d3- was similar to that of full-length promoter in green fruits, d3- promoter showed higher activity than full-length promoter in leaves, flowers, and breaker-, orange- and red-stages of fruits. most noticeably, the shortest promoter fragment d3- showed and -fold higher gus activity in flowers and leaves, respectively, as compared to that of d0- full-length promoter. northern-blot analysis northern blot was performed to analyze the relative levels of cyc-b full-length or truncated promoter driven gus expression in different tissues namely leaf, flower and fruits from transgenic tomato plants. blots used for detecting gus mrna levels were reprobed with ef1α that served as a loading control for amount of total rna. the concentration of total rna loaded per lane for a particular stage of development or tissue was essentially same for transgenic lines of each construct, but was varying in the range of 20- μg across different stages or tissues used. shcyc-b full-length promoter and its deletion fragments showed low expression in chloroplast-rich green leaves and green fruits, while their expression was high in chromoplast-rich flowers, and fruits at breaker and orange stage of ripening. consistent with gus assay, in northern analysis also, d3- promoter fragment showed highest promoter activity in flowers. however, in contrast to the higher gus-activity observed in fruits of transgenic plants expressing d3- promoter driven gus reporter, the transcript levels of d3- promoter driven gus were not so apparently higher than that of d0- promoter driven gus reporter. the deletion fragment d2- showed lowest promoter strength as compared to the full-length promoter and d3- the reduction in gus expression in d2- transgenic plant does not appear to be due to transgene position and/or silencing effect, as seven out of nine independent events showed reduced gus staining, and the transgenic plants with single insertion for d2- fragment were selected for analysis. the present study clearly showed that the shcyc-b promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits at different stages of ripening. the promoter strength was drastically decreased by a deletion of - to - bp 5' to atg as compared to full-length promoter, while the shortest d3- promoter fragment showed highest activity. this suggests that nucleotide sequence - to - bp upstream to initiation codon may contain cis-elements involved in down regulation of cyc-b expression, while nucleotide sequences - to - bp upstream to initiation codon might be involved in negating the repressive nature of regulatory sequences in - to - bp 5' to atg. the rap binding cis-element atcta has been shown to confer strong basal activity in psy promoter from arabidopsis. this cis-element was found in shcyc-b full-length promoter at - to - bp upstream to atg, and might contribute to the basal activity of full-length promoter, as deletion of this cis-element in d2- resulted in considerable decrease in promoter activity. the presence of three gt- cis-element, one at - to - and two at - to - and - to - bp upstream of atg might be playing a role in the gene expression. gt- element was initially identified as cis-element regulating cell-type specific expression specifically in light regulated genes. gt- protein binds to tfiia and tata-binding proteins. the gt- cis-element is conserved in many plant promoters, and may have positive or negative regulatory effect on transcription depending upon cell type.  in this study, lycopene β-cyclase promoter from solanum habrochaites, a green fruited tomato genotype, was isolated and functionally characterized in transient in fruto and stable transformation systems. conservation of cis-elements such as rap binding cis-element and ere cis-element between cyc-b and psy promoter suggests a common regulatory mechanism for carotenoid accumulation in fruits. a short promoter region with promoter activity and developmental expression pattern comparable to that of full-length shcyc-b promoter was identified. as signal transduction events and transcription factors that developmentally regulate the cyc-b expression are not known, the short promoter region identified in this study can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in cyc-b expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. 
moreover, this short promoter region of shcyc-b may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits. genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. however, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. a bp region upstream to the initiation codon of the lycopene β-cyclase gene was cloned and identified as full-length promoter. to identify promoter region necessary for regulating developmental expression of the shcyc-b gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of gus reporter cdna in binary vectors. agrobacterium-mediated transient and stable expression systems were used to study the gus expression driven by the full-length promoter and its 5' deletion fragments in tomato. the full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. functional characterization of the full-length shcyc-b promoter and its deletion fragments in transient expression system in fruto as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. promoter deletion analysis led to the identification of a short promoter region that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length shcyc-b promoter. our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length shcyc-b promoter. tomato is an important vegetable in human diet and provides the vitamin a precursor β-carotene. lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. this bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in cyc-b expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. deletion of - to - bp 5' to atg decreases the shcyc-b promoter strength, while deletion of - to - bp 5' to atg led to significant increase in the activity of gus in the transgenic plants. one of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. this study describes the isolation and characterization of chromoplast-specific lycopene β-cyclase promoter from a green fruited s. habrochaites genotype ec 
 carotenoids are a group of c isoprenoid molecules that play diverse biological and ecological roles in plants. tomato is an important vegetable in human diet and provides the vitamin a precursor β-carotene. genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. however, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. one of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. this study describes the isolation and characterization of chromoplast-specific lycopene β-cyclase promoter from a green fruited s. habrochaites genotype ec  a bp region upstream to the initiation codon of the lycopene β-cyclase gene was cloned and identified as full-length promoter. to identify promoter region necessary for regulating developmental expression of the shcyc-b gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of gus reporter cdna in binary vectors. these four plant transformation vectors were separately transformed in to agrobacterium. agrobacterium-mediated transient and stable expression systems were used to study the gus expression driven by the full-length promoter and its 5' deletion fragments in tomato. the full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. deletion of - to - bp 5' to atg decreases the shcyc-b promoter strength, while deletion of - to - bp 5' to atg led to significant increase in the activity of gus in the transgenic plants. promoter deletion analysis led to the identification of a short promoter region that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length shcyc-b promoter.  functional characterization of the full-length shcyc-b promoter and its deletion fragments in transient expression system in fruto as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length shcyc-b promoter. this bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in cyc-b expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. moreover, this short promoter region of shcyc-b may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits. 