BACKGROUND
potato is the fourth most important source of calories for mankind after the cereals wheat, rice and maize.
potato production worldwide stands at  <dig> million tons, of which 36% in developing countries, and covers more than  <dig> million hectares  <cit> .
therefore, the nutritional improvement of potato is of paramount importance to help eradicate nutritional deficiencies.
vitamin a deficiency affects several hundred million people worldwide  <cit> .
vitamin a is ingested by humans in two forms: preformed vitamin a , found in foods of animal origin, and β-carotene , found in dark green vegetables like spinach, in palm oil, and in orange vegetables like yams or carrots.
the first dedicated step in the biosynthesis of carotenoids is the synthesis of phytoene, a colorless compound which is desaturated and isomerized into all-trans lycopene .
lycopene is the substrate for two competing cyclases: epsilon-cyclase, introducing a single epsilon-ring, and beta-cyclase introducing one or two beta-rings.
successive hydroxylation and epoxidation reactions result in the synthesis of lutein, on the branch starting at ε-cyclase, and β-β-ring xanthophylls on the branch starting at β-cyclase.
metabolic engineering of plant carotenoids has been achieved through different strategies .
these can be summarized in:
i) "push strategies", in which a gene encoding a rate-limiting step in the pathway is overexpressed.
examples of "push" strategies are the overexpression of the psy gene, encoding phytoene synthase, in rice  <cit> , canola  <cit> , tomato  <cit>  and potato  <cit> , of later genes in the pathway, such as those encoding lycopene β-cyclases  <cit>  or of "mini-pathways" composed of more than one gene, directing the formation of β-carotene in rice endosperm  <cit>  or of zeaxanthin in tomato fruits  <cit> .
a variant of "push" strategies consists in the introduction of non-plant genes to redirect metabolite flux and synthesize new compounds  <cit> .
ii) "block" strategies, relying on the silencing of a biosynthetic step situated immediately downstream of the compound whose levels are to be increased.
examples of "block" strategies are the increase in lycopene in tomato fruits obtained through the silencing of lycopene β-cyclases  <cit> , or the increase of zeaxanthin in potato tubers obtained through the silencing of zeaxanthin epoxidase  <cit> .
in this paper, we present a variant "block" strategy, aimed at redirecting the metabolic flux into the β-β branch of the bifurcated carotenoid pathway by silencing the first step of the competing ε-β branch.
we present the phenotypic analysis of transgenic potato plants in which the first biosynthetic step in the branch leading to lutein, lycopene ε-cyclase , has been inhibited through a tuber-specific antisense approach.
RESULTS
construction of transgenic plants
the first dedicated step in lutein biosynthesis is the ε-cyclization of lycopene by lcy-e .
in cv.
desirée, lutein constitutes about 12% of tuber and 48% of leaf carotenoids, respectively.
in arabidopsis, lut <dig> mutants, which are deficient in ε-cyclization, are viable  <cit>  but, under stress conditions, show reduced chlorophyll triplet quenching and, therefore, enhanced photooxidation  <cit> .
in order not to interfere with leaf lutein biosynthesis, we decided to silence the lycopene ε-cyclase transcript  specifically in tubers.
several examples of organ-specific silencing are known in plants, such as the fruit-specific silencing of lycopene β-cyclase   <cit>  or of the hp- <dig> gene  <cit>  in tomato.
in the present work, the patatin promoter  <cit>  was used to target the expression of the silencing transcript in tubers.
appropriate fragments of the patatin b <dig> promoter  <cit>  and of the lcy-e transcript were amplified by pcr, re-sequenced, and cloned in the pbi <dig> vector for agrobacterium-mediated transformation  <cit>  .
two plasmids were constructed: plasmid pb33:gus contains the gus reporter gene under the control of the b <dig> promoter, while plasmid pbi33:as-e contains an antisense lcy-e cdna fragment replacing gus .
the two constructs were introduced in potato  using published methods  <cit>  and transgenic plants were selected on kanamycin.
the presence of the transgene was confirmed via pcr .
two independent transgenic lines for gus and  <dig> for as-e were selected for further characterization.
for each clone, two plants were acclimated in the greenhouse for tuberization.
at the end of the life cycle, tubers were harvested and the tuber production was evaluated.
neither gus nor as-e transgenic plants showed major alterations in tuber weight or number .
tuber and leaf carotenoid composition
the carotenoid composition of tubers from the wt, one of the gus lines and all the as-e lines was analyzed through diode array hplc .
the gus line did not show significant changes in carotenoid content, with respect to wild-type desirée.
in contrast, four out of the six as-e lines  showed significant  increases in total tuber carotenoids, as well as changes in carotenoid composition.
this increase was reflected in a minor increase in yellow pigmentation of the tubers .
consistent with the hypothesized silencing of lcy-e, the levels of β-β-carotenoids  showed significant increases  in these lines.
β-carotene was the single carotenoid compound showing the highest relative increases, up to 14-fold in line as-e  <dig>  somewhat surprisingly, the levels of lutein remained fairly constant in most of the as-e lines.
the ratio of β-β- to β-ε-carotenoids increased from  <dig>  in the wild-type, up to  <dig>  in line as-e  <dig>
a slight increase of the colorless biosynthetic intermediate, phytofluene, was observed in lines as-e  <dig> to  <dig>  the other early intermediates  were below detection in all lines.
these changes in carotenoid content must be attributed to a specific effect of the introduced as-e transgene, and not to non-specific events occurring during plant regeneration, such as somaclonal variation.
these non-specific events account for a very limited variation in carotenoid levels, as shown by the data relative to the gus line  which shows only minor changes with respect to the wild-type.
consistently with the tuber-specific nature of the promoter used for the silencing construct, no significant variations in carotenoid or chlorophyll content were observed in leaves of gus or as-e lines .
transgene expression in tubers and leaves
we performed fluorimetric gus assays on the gus lines and real time rt-pcr assays on four as-e lines showing significant variations in tuber carotenoids  and one showing wild-type carotenoid composition .
the results are shown in figure  <dig>  gus activity was found in tissues of gus transgenic plants, but not wt plants, and was preferentially expressed in tubers.
the as-e silencing transcript was detected in the tubers of all the as-e lines, except for as-e  <dig>  lack of expression of the as-e transcript in this line is not due to improper transgene integration, since the transgene is present and is not rearranged .
the silencing transcript was below the levels of detection of the real time assay in the leaves of all the as-e lines .
the levels of expression of the transgene in the tubers were rather low, ranging from  <dig> -fold to  <dig> -fold β-tubulin.
other than the lack of transgene expression in line as-e  <dig>  no evident correlation between the levels of transgene expression and carotenoid composition of tubers  were observed.
perturbations in endogenous carotenoid gene expression
expression of some genes in the carotenoid pathway can be forward- or feedback-regulated by changes of the levels of pathway intermediates: for instance, the pds transcript, encoding phytoene desaturase, is induced in leaves accumulating phytoene as a result of the ghost mutation or after treatment with the herbicide norflurazon  <cit> , suggesting that expression of this gene may be driven by the concentration of the substrate of the reaction.
in agreement with this hypothesis, tubers overexpressing a bacterial phytoene synthase show increased levels of the pds transcript  <cit> .
we measured the expression of carotenoid gene transcripts in the tubers, using real time quantitative rt-pcr.
alongside the wt, we analyzed lines gus <dig> and as-e <dig>  showing wild-type carotenoid levels, and lines as-e  <dig>   <dig> and  <dig>  showing significant changes in carotenoid composition.
we analyzed transcripts for most genes in the carotenoid pathway .
the biosynthetic steps catalyzed by these genes are shown in figure  <dig>  the identity of the transcripts used for designing the oligonucleotides used for rt-pcr, is indicated in methods.
the tuber transcript levels, normalized first for the β-tubulin transcript and then for the wt, are shown in figure  <dig>
the gus <dig> line, as well as line as-e <dig>  showed only minor variations in gene expression with respect to the wt line.
this indicates that in vitro culture conditions, somaclonal effects due to regeneration procedures, or the presence of the silencing transgene by itself, do not cause any major variability in endogenous carotenoid gene expression.
the endogenous lcy-e transcript shows consistent silencing in as-e lines  <dig>  and  <dig>  but not in the other lines.
line as-e  <dig>  which is the one showing the highest increase in β-β-xanthophylls , also shows the most efficient silencing of endogenous lcy-e.
some clear trends in gene expression can be identified: crtiso, lcy-b and zep show an increase in all the as-e lines, with the exception of line as-e  <dig>  which has a wild-type carotenoid content.
other transcripts show more variable trends, being induced or repressed in only one or two of the as-e lines.
line as-e <dig>  which is the one showing the highest increase in total carotenoid levels , stands out from the other lines analyzed, in that it shows a simultaneous induction of several transcripts: psy <dig>  pds, crtiso, lcy-b, lut <dig>  and chy <dig>
the transcript levels of the three genes showing significant changes in tubers  were measured also in leaves .
in this tissue, they showed only minor oscillations.
this confirms that, similarly to the alterations in carotenoid content, also the perturbations in endogenous carotenoid transcript levels were limited to tubers, the target tissue of as-e transgene overexpression.
discussion
like for any other metabolic pathway, flux through the carotenoid pathway is thought to be controlled by rate-limiting enzymatic steps, whose modification through metabolic engineering should be able to change carotenoid composition.
metabolic flux analysis  <cit>  should allow the identification of these rate-limiting steps, and predict the effects of their engineering.
however, the experimental verification of these predictions often contradicts theory, mainly because the levels of expression of some of the genes, and thus of the corresponding enzymes, are sensitive to the levels of the intermediates and of the final products of the pathway.
in potato, overexpression of early genes of the carotenoid pathway is a promising strategy for increasing total carotenoids and β-carotene in the tuber.
overexpression of the bacterial crtb gene, encoding phytoene synthase, leads to up to 7-fold increase in total carotenoids, mainly β-carotene and lutein  <cit> .
this indicates that psy is a rate-limiting step in wild-type tubers.
the role of lcy-e and lcy-b in directing the flux towards the synthesis of β-β and β-ε carotenoids can hardly be underestimated.
modulating the levels of these two competing cyclases is a strategy for obtaining tubers with a defined carotenoid composition.
the results presented here indicate that the lcy-e transcript can be silenced in a tuber-specific fashion, resulting in a tuber-specific increase of β-β-carotenoids , and, more specifically, of β- carotene .
real time rt-pcr measurements showed that the silencing transgene, introduced under the control of the patatin promoter, was expressed in a tuber-specific fashion in all but one of the transgenic lines.
the transgene was expressed at rather low levels, which is not surprising, since homology-driven post-transcriptional gene silencing is known to be a bidirectional phenomenon, affecting both the target gene and the silencing construct  <cit> .
the lack of expression of the transgene in line as-e  <dig> awaits an explanation, since the construct is stably integrated in the genome and it is not rearranged .
whatever the case, this line, together with the gus <dig> line, shows wild-type carotenoid composition, reinforcing the hypothesis that the alterations in carotenoid content observed in lines as-e  <dig> to  <dig> are indeed the effect of expression of the as-e transgene, and thus of the silencing of endogenous lcy-e. in fact, in all the lines expressing the as-e transgene, the endogenous lcy-e transcript was silenced, to various extents.
the silencing was confined to tubers.
this result suggests that, similarly to tomato fruits  <cit> , a physical barrier must exist, preventing the spreading of the silencing signal outside of the tuber tissue.
the tuber-specific changes in lcy-e gene expression were accompanied by tuber-specific changes in carotenoid composition: first, total carotenoid levels increased significantly, albeit to variable extents; second, this increase involved, as expected, mostly carotenoids of the β-β- branch; third, β-carotene showed the highest relative increases  among all carotenoid compounds.
surprisingly, lutein showed little, if any, reduction in the silenced tubers.
this observation suggests that lcy-e activity is not rate-limiting for tuber lutein levels.
it is possible that, to decrease lutein, much higher levels of silencing of lcy-e are required, similar to the ones obtained with rnai constructs  <cit> .
several additional enzymes, such as carotenoid hydroxylases  <cit>  are involved in lutein synthesis.
furthermore, it is also possible that lutein levels are also controlled by hitherto undescribed carotenoid cleavage activities  <cit> .
endogenous carotenoid gene expression shows two distinct patterns of regulation in silenced tubers: three transcripts  are significantly induced in all the silenced lines.
this indicates that these transcripts are sensitive either to the levels of lcy-e expression, or to the levels of one or more carotenoid pathway intermediates.
in one of the lines, as-e  <dig>  showing the highest total carotenoid and β-carotene levels, a generalized induction of carotenoid transcripts is observed .
this result can be interpreted in two different ways:
a) the generalized induction of carotenoid gene transcripts could be an effect of the more extreme carotenoid level perturbation observed in this line with respect to the other ones
b) instead, the generalized induction could be the effect of a mutation, or of another somaclonal event, occurred during transformation or regeneration of this line, and having a generalized inductive effect on the carotenoid pathway.
in this case, the induction of carotenoid gene expression, added to the silencing of lcy-e, would be the cause of the extreme variation in carotenoid content observed in this line.
no major changes in tuber productivity or leaf carotenoid composition were observed in any of the lines, suggesting that silencing of lcy-e is a viable strategy for changing tuber carotenoid composition without affecting agronomic performance.
field trials are however needed to verify the agronomic performance of these lines.
the presence of gene paralogs showing preferential expression in tubers constitutes an interesting parallel with tomato, a close relative of potato, where it has been suggested that duplication of the psy and chy genes has created two parallel pathways, one active in leaves and the second active in chromoplast-containing organs   <cit> .
in agreement with this hypothesis, mutation or silencing of psy <dig> in tomato affects fruit and flower, but not leaf carotenoids  <cit> .
it remains to be demonstrated what is the relative contribution of the different members of the psy and chy gene families to potato tuber carotenogenesis.
CONCLUSIONS
using an antisense construct under the control of the tuber-specific patatin promoter, we obtained the tuber-specific silencing of the potato lcy-e transcript.
the amount of β-β-carotenoids increased accordingly in a tuber-specific fashion, without a parallel decrease of the major tuber β-ε- carotenoid, lutein.
this modification in carotenoid content is paralleled by modifications in endogenous carotenoid gene expression.
the strategy described here, in combination with previously described ones, such as the overexpression of phytoene synthase and the antisense silencing of zeaxanthin epoxidase, constitutes a toolbox for the metabolic engineering of the carotenoid content of potato tubers.

 potato is the fourth most important source of calories for mankind after the cereals wheat, rice and maize. potato production worldwide stands at million tons, of which 36% in developing countries, and covers more than million hectares. therefore, the nutritional improvement of potato is of paramount importance to help eradicate nutritional deficiencies. vitamin a deficiency affects several hundred million people worldwide. vitamin a is ingested by humans in two forms: preformed vitamin a, found in foods of animal origin, and β-carotene, found in dark green vegetables like spinach, in palm oil, and in orange vegetables like yams or carrots. the first dedicated step in the biosynthesis of carotenoids is the synthesis of phytoene, a colorless compound which is desaturated and isomerized into all-trans lycopene. lycopene is the substrate for two competing cyclases: epsilon-cyclase, introducing a single epsilon-ring, and beta-cyclase introducing one or two beta-rings. successive hydroxylation and epoxidation reactions result in the synthesis of lutein, on the branch starting at ε-cyclase, and β-β-ring xanthophylls on the branch starting at β-cyclase. metabolic engineering of plant carotenoids has been achieved through different strategies. these can be summarized in: i) "push strategies", in which a gene encoding a rate-limiting step in the pathway is overexpressed. examples of "push" strategies are the overexpression of the psy gene, encoding phytoene synthase, in rice, canola, tomato and potato, of later genes in the pathway, such as those encoding lycopene β-cyclases or of "mini-pathways" composed of more than one gene, directing the formation of β-carotene in rice endosperm or of zeaxanthin in tomato fruits. a variant of "push" strategies consists in the introduction of non-plant genes to redirect metabolite flux and synthesize new compounds. ii) "block" strategies, relying on the silencing of a biosynthetic step situated immediately downstream of the compound whose levels are to be increased. examples of "block" strategies are the increase in lycopene in tomato fruits obtained through the silencing of lycopene β-cyclases, or the increase of zeaxanthin in potato tubers obtained through the silencing of zeaxanthin epoxidase. in this paper, we present a variant "block" strategy, aimed at redirecting the metabolic flux into the β-β branch of the bifurcated carotenoid pathway by silencing the first step of the competing ε-β branch. we present the phenotypic analysis of transgenic potato plants in which the first biosynthetic step in the branch leading to lutein, lycopene ε-cyclase, has been inhibited through a tuber-specific antisense approach.  construction of transgenic plants the first dedicated step in lutein biosynthesis is the ε-cyclization of lycopene by lcy-e. in cv. desirée, lutein constitutes about 12% of tuber and 48% of leaf carotenoids, respectively. in arabidopsis, lut mutants, which are deficient in ε-cyclization, are viable but, under stress conditions, show reduced chlorophyll triplet quenching and, therefore, enhanced photooxidation. in order not to interfere with leaf lutein biosynthesis, we decided to silence the lycopene ε-cyclase transcript specifically in tubers. several examples of organ-specific silencing are known in plants, such as the fruit-specific silencing of lycopene β-cyclase or of the hp- gene in tomato. in the present work, the patatin promoter was used to target the expression of the silencing transcript in tubers. appropriate fragments of the patatin b promoter and of the lcy-e transcript were amplified by pcr, re-sequenced, and cloned in the pbi vector for agrobacterium-mediated transformation. two plasmids were constructed: plasmid pb33:gus contains the gus reporter gene under the control of the b promoter, while plasmid pbi33:as-e contains an antisense lcy-e cdna fragment replacing gus. the two constructs were introduced in potato using published methods and transgenic plants were selected on kanamycin. the presence of the transgene was confirmed via pcr. two independent transgenic lines for gus and for as-e were selected for further characterization. for each clone, two plants were acclimated in the greenhouse for tuberization. at the end of the life cycle, tubers were harvested and the tuber production was evaluated. neither gus nor as-e transgenic plants showed major alterations in tuber weight or number. tuber and leaf carotenoid composition the carotenoid composition of tubers from the wt, one of the gus lines and all the as-e lines was analyzed through diode array hplc. the gus line did not show significant changes in carotenoid content, with respect to wild-type desirée. in contrast, four out of the six as-e lines showed significant increases in total tuber carotenoids, as well as changes in carotenoid composition. this increase was reflected in a minor increase in yellow pigmentation of the tubers. consistent with the hypothesized silencing of lcy-e, the levels of β-β-carotenoids showed significant increases in these lines. β-carotene was the single carotenoid compound showing the highest relative increases, up to 14-fold in line as-e somewhat surprisingly, the levels of lutein remained fairly constant in most of the as-e lines. the ratio of β-β- to β-ε-carotenoids increased from in the wild-type, up to in line as-e a slight increase of the colorless biosynthetic intermediate, phytofluene, was observed in lines as-e to the other early intermediates were below detection in all lines. these changes in carotenoid content must be attributed to a specific effect of the introduced as-e transgene, and not to non-specific events occurring during plant regeneration, such as somaclonal variation. these non-specific events account for a very limited variation in carotenoid levels, as shown by the data relative to the gus line which shows only minor changes with respect to the wild-type. consistently with the tuber-specific nature of the promoter used for the silencing construct, no significant variations in carotenoid or chlorophyll content were observed in leaves of gus or as-e lines. transgene expression in tubers and leaves we performed fluorimetric gus assays on the gus lines and real time rt-pcr assays on four as-e lines showing significant variations in tuber carotenoids and one showing wild-type carotenoid composition. the results are shown in figure gus activity was found in tissues of gus transgenic plants, but not wt plants, and was preferentially expressed in tubers. the as-e silencing transcript was detected in the tubers of all the as-e lines, except for as-e lack of expression of the as-e transcript in this line is not due to improper transgene integration, since the transgene is present and is not rearranged. the silencing transcript was below the levels of detection of the real time assay in the leaves of all the as-e lines. the levels of expression of the transgene in the tubers were rather low, ranging from -fold to -fold β-tubulin. other than the lack of transgene expression in line as-e no evident correlation between the levels of transgene expression and carotenoid composition of tubers were observed. perturbations in endogenous carotenoid gene expression expression of some genes in the carotenoid pathway can be forward- or feedback-regulated by changes of the levels of pathway intermediates: for instance, the pds transcript, encoding phytoene desaturase, is induced in leaves accumulating phytoene as a result of the ghost mutation or after treatment with the herbicide norflurazon, suggesting that expression of this gene may be driven by the concentration of the substrate of the reaction. in agreement with this hypothesis, tubers overexpressing a bacterial phytoene synthase show increased levels of the pds transcript. we measured the expression of carotenoid gene transcripts in the tubers, using real time quantitative rt-pcr. alongside the wt, we analyzed lines gus and as-e showing wild-type carotenoid levels, and lines as-e and showing significant changes in carotenoid composition. we analyzed transcripts for most genes in the carotenoid pathway. the biosynthetic steps catalyzed by these genes are shown in figure the identity of the transcripts used for designing the oligonucleotides used for rt-pcr, is indicated in methods. the tuber transcript levels, normalized first for the β-tubulin transcript and then for the wt, are shown in figure the gus line, as well as line as-e showed only minor variations in gene expression with respect to the wt line. this indicates that in vitro culture conditions, somaclonal effects due to regeneration procedures, or the presence of the silencing transgene by itself, do not cause any major variability in endogenous carotenoid gene expression. the endogenous lcy-e transcript shows consistent silencing in as-e lines and but not in the other lines. line as-e which is the one showing the highest increase in β-β-xanthophylls, also shows the most efficient silencing of endogenous lcy-e. some clear trends in gene expression can be identified: crtiso, lcy-b and zep show an increase in all the as-e lines, with the exception of line as-e which has a wild-type carotenoid content. other transcripts show more variable trends, being induced or repressed in only one or two of the as-e lines. line as-e which is the one showing the highest increase in total carotenoid levels, stands out from the other lines analyzed, in that it shows a simultaneous induction of several transcripts: psy pds, crtiso, lcy-b, lut and chy the transcript levels of the three genes showing significant changes in tubers were measured also in leaves. in this tissue, they showed only minor oscillations. this confirms that, similarly to the alterations in carotenoid content, also the perturbations in endogenous carotenoid transcript levels were limited to tubers, the target tissue of as-e transgene overexpression. discussion like for any other metabolic pathway, flux through the carotenoid pathway is thought to be controlled by rate-limiting enzymatic steps, whose modification through metabolic engineering should be able to change carotenoid composition. metabolic flux analysis should allow the identification of these rate-limiting steps, and predict the effects of their engineering. however, the experimental verification of these predictions often contradicts theory, mainly because the levels of expression of some of the genes, and thus of the corresponding enzymes, are sensitive to the levels of the intermediates and of the final products of the pathway. in potato, overexpression of early genes of the carotenoid pathway is a promising strategy for increasing total carotenoids and β-carotene in the tuber. overexpression of the bacterial crtb gene, encoding phytoene synthase, leads to up to 7-fold increase in total carotenoids, mainly β-carotene and lutein. this indicates that psy is a rate-limiting step in wild-type tubers. the role of lcy-e and lcy-b in directing the flux towards the synthesis of β-β and β-ε carotenoids can hardly be underestimated. modulating the levels of these two competing cyclases is a strategy for obtaining tubers with a defined carotenoid composition. the results presented here indicate that the lcy-e transcript can be silenced in a tuber-specific fashion, resulting in a tuber-specific increase of β-β-carotenoids, and, more specifically, of β- carotene. real time rt-pcr measurements showed that the silencing transgene, introduced under the control of the patatin promoter, was expressed in a tuber-specific fashion in all but one of the transgenic lines. the transgene was expressed at rather low levels, which is not surprising, since homology-driven post-transcriptional gene silencing is known to be a bidirectional phenomenon, affecting both the target gene and the silencing construct. the lack of expression of the transgene in line as-e awaits an explanation, since the construct is stably integrated in the genome and it is not rearranged. whatever the case, this line, together with the gus line, shows wild-type carotenoid composition, reinforcing the hypothesis that the alterations in carotenoid content observed in lines as-e to are indeed the effect of expression of the as-e transgene, and thus of the silencing of endogenous lcy-e. in fact, in all the lines expressing the as-e transgene, the endogenous lcy-e transcript was silenced, to various extents. the silencing was confined to tubers. this result suggests that, similarly to tomato fruits, a physical barrier must exist, preventing the spreading of the silencing signal outside of the tuber tissue. the tuber-specific changes in lcy-e gene expression were accompanied by tuber-specific changes in carotenoid composition: first, total carotenoid levels increased significantly, albeit to variable extents; second, this increase involved, as expected, mostly carotenoids of the β-β- branch; third, β-carotene showed the highest relative increases among all carotenoid compounds. surprisingly, lutein showed little, if any, reduction in the silenced tubers. this observation suggests that lcy-e activity is not rate-limiting for tuber lutein levels. it is possible that, to decrease lutein, much higher levels of silencing of lcy-e are required, similar to the ones obtained with rnai constructs. several additional enzymes, such as carotenoid hydroxylases are involved in lutein synthesis. furthermore, it is also possible that lutein levels are also controlled by hitherto undescribed carotenoid cleavage activities. endogenous carotenoid gene expression shows two distinct patterns of regulation in silenced tubers: three transcripts are significantly induced in all the silenced lines. this indicates that these transcripts are sensitive either to the levels of lcy-e expression, or to the levels of one or more carotenoid pathway intermediates. in one of the lines, as-e showing the highest total carotenoid and β-carotene levels, a generalized induction of carotenoid transcripts is observed. this result can be interpreted in two different ways: a) the generalized induction of carotenoid gene transcripts could be an effect of the more extreme carotenoid level perturbation observed in this line with respect to the other ones b) instead, the generalized induction could be the effect of a mutation, or of another somaclonal event, occurred during transformation or regeneration of this line, and having a generalized inductive effect on the carotenoid pathway. in this case, the induction of carotenoid gene expression, added to the silencing of lcy-e, would be the cause of the extreme variation in carotenoid content observed in this line. no major changes in tuber productivity or leaf carotenoid composition were observed in any of the lines, suggesting that silencing of lcy-e is a viable strategy for changing tuber carotenoid composition without affecting agronomic performance. field trials are however needed to verify the agronomic performance of these lines. the presence of gene paralogs showing preferential expression in tubers constitutes an interesting parallel with tomato, a close relative of potato, where it has been suggested that duplication of the psy and chy genes has created two parallel pathways, one active in leaves and the second active in chromoplast-containing organs. in agreement with this hypothesis, mutation or silencing of psy in tomato affects fruit and flower, but not leaf carotenoids. it remains to be demonstrated what is the relative contribution of the different members of the psy and chy gene families to potato tuber carotenogenesis.  using an antisense construct under the control of the tuber-specific patatin promoter, we obtained the tuber-specific silencing of the potato lcy-e transcript. the amount of β-β-carotenoids increased accordingly in a tuber-specific fashion, without a parallel decrease of the major tuber β-ε- carotenoid, lutein. this modification in carotenoid content is paralleled by modifications in endogenous carotenoid gene expression. the strategy described here, in combination with previously described ones, such as the overexpression of phytoene synthase and the antisense silencing of zeaxanthin epoxidase, constitutes a toolbox for the metabolic engineering of the carotenoid content of potato tubers. 
the data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. real time measurements confirmed the tuber-specific silencing of lcy-e. antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase. upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified. tuber-specific changes in expression of several genes in the pathway were observed. total carotenoids increased up to -fold. we silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase, by introducing, via agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter.
 potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin a. potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. none of these carotenoids have provitamin a activity.  we silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase, by introducing, via agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. real time measurements confirmed the tuber-specific silencing of lcy-e. antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase. total carotenoids increased up to -fold. these changes were not accompanied by a decrease in lutein, suggesting that lcy-e is not rate-limiting for lutein accumulation. tuber-specific changes in expression of several genes in the pathway were observed.  the data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified. 