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Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers.

Diretto G, Welsch R, Tavazza R, Mourgues F, Pizzichini D, Beyer P, Giuliano G - BMC Plant Biol. (2007)

Bottom Line: Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes .These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin.Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

View Article: PubMed Central - HTML - PubMed

Affiliation: ENEA, Casaccia Research Center, Rome, Italy. gianfranco.diretto@casaccia.enea.it

ABSTRACT

Background: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.

Results: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species.

Conclusion: Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

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Schematic representation of metabolite and gene expression changes in engineered tubers. Boxes represent the metabolic intermediates, arrows represent the genes catalyzing the various reactions. Fold induction or repression with respect to the wild-type – averaged over three transgenic lines- is represented by different hues of red or green, respectively (see legend). White means that no data are available. Asterisks indicate significance of the fold variation with respect to the Wt in an ANOVA test (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). (A) AS-e lines 1,2,3 [3]. (B) AS-h lines 1,2,3 (this paper).
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Figure 2: Schematic representation of metabolite and gene expression changes in engineered tubers. Boxes represent the metabolic intermediates, arrows represent the genes catalyzing the various reactions. Fold induction or repression with respect to the wild-type – averaged over three transgenic lines- is represented by different hues of red or green, respectively (see legend). White means that no data are available. Asterisks indicate significance of the fold variation with respect to the Wt in an ANOVA test (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). (A) AS-e lines 1,2,3 [3]. (B) AS-h lines 1,2,3 (this paper).

Mentions: Expression of endogenous carotenoid genes is often altered as a consequence of manipulations modifying the levels of biosynthetic intermediates in the pathway. This phenomenon has been observed both in tomato leaves [13,14] and in potato tubers [2,3]. We measured the expression of carotenoid gene transcripts (PSY1, PSY2, PDS, ZDS, CrtISO, LCY-b, LCY-e, CHY1, CHY2, LUT1, LUT5, ZEP, NXS) in tubers, using Real Time RT-PCR. The tuber transcript levels, normalized first for the β-tubulin transcript and then for the Wt transcript levels, are shown in Figure 1. The biosynthetic steps catalyzed by these genes are shown in Figure 2. The GUS line, as well as "non expressor" lines AS-h 5 and 6, showed only minor variations in gene expression with respect to the Wt line. This indicates that 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 CHY2 gene is silenced in the four lines showing alterations in carotenoid content (AS-h 1 to 4). Line AS-h 3, which is the one showing the highest increase in total carotenoids (Table 2), also shows the most efficient silencing of endogenous CHY2. Alongside CHY2, also the CHY1 transcript is silenced, albeit to different extent, in transgenic tubers of lines AS-h1 to 4. This result indicates that the homology between the two transcripts in the region chosen for silencing is sufficiently high to warrant cross-silencing.


Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers.

Diretto G, Welsch R, Tavazza R, Mourgues F, Pizzichini D, Beyer P, Giuliano G - BMC Plant Biol. (2007)

Schematic representation of metabolite and gene expression changes in engineered tubers. Boxes represent the metabolic intermediates, arrows represent the genes catalyzing the various reactions. Fold induction or repression with respect to the wild-type – averaged over three transgenic lines- is represented by different hues of red or green, respectively (see legend). White means that no data are available. Asterisks indicate significance of the fold variation with respect to the Wt in an ANOVA test (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). (A) AS-e lines 1,2,3 [3]. (B) AS-h lines 1,2,3 (this paper).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828156&req=5

Figure 2: Schematic representation of metabolite and gene expression changes in engineered tubers. Boxes represent the metabolic intermediates, arrows represent the genes catalyzing the various reactions. Fold induction or repression with respect to the wild-type – averaged over three transgenic lines- is represented by different hues of red or green, respectively (see legend). White means that no data are available. Asterisks indicate significance of the fold variation with respect to the Wt in an ANOVA test (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). (A) AS-e lines 1,2,3 [3]. (B) AS-h lines 1,2,3 (this paper).
Mentions: Expression of endogenous carotenoid genes is often altered as a consequence of manipulations modifying the levels of biosynthetic intermediates in the pathway. This phenomenon has been observed both in tomato leaves [13,14] and in potato tubers [2,3]. We measured the expression of carotenoid gene transcripts (PSY1, PSY2, PDS, ZDS, CrtISO, LCY-b, LCY-e, CHY1, CHY2, LUT1, LUT5, ZEP, NXS) in tubers, using Real Time RT-PCR. The tuber transcript levels, normalized first for the β-tubulin transcript and then for the Wt transcript levels, are shown in Figure 1. The biosynthetic steps catalyzed by these genes are shown in Figure 2. The GUS line, as well as "non expressor" lines AS-h 5 and 6, showed only minor variations in gene expression with respect to the Wt line. This indicates that 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 CHY2 gene is silenced in the four lines showing alterations in carotenoid content (AS-h 1 to 4). Line AS-h 3, which is the one showing the highest increase in total carotenoids (Table 2), also shows the most efficient silencing of endogenous CHY2. Alongside CHY2, also the CHY1 transcript is silenced, albeit to different extent, in transgenic tubers of lines AS-h1 to 4. This result indicates that the homology between the two transcripts in the region chosen for silencing is sufficiently high to warrant cross-silencing.

Bottom Line: Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes .These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin.Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

View Article: PubMed Central - HTML - PubMed

Affiliation: ENEA, Casaccia Research Center, Rome, Italy. gianfranco.diretto@casaccia.enea.it

ABSTRACT

Background: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.

Results: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species.

Conclusion: Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

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