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Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato.

Azari R, Reuveni M, Evenor D, Nahon S, Shlomo H, Chen L, Levin I - J. Exp. Bot. (2010)

Bottom Line: However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful.This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion.Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO Box 6, Bet Dagan 50250, Israel.

ABSTRACT
Fruits of tomato plants carrying the high pigment-1 mutations hp-1 and hp-1(w) are characterized by an increased number of plastids coupled with enhanced levels of functional metabolites. Unfortunately, hp-1 mutant plants are also typified by light-dependent retardation in seedling and whole-plant growth and development, which limits their cultivation. These mutations were mapped to the gene encoding UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1) and, recently, fruit-specific RNA interference studies have demonstrated an increased number of plastids and enhanced carotenoid accumulation in the transgenic tomato fruits. However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful. In this study, five transgenic lines constitutively overexpressing normal DDB1 in hp-1 mutant plants were analysed. Eleven-day-old seedlings, representing these lines, displayed up to approximately 73- and approximately 221-fold overexpression of the gene in hypocotyls and cotyledons, respectively. This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion. This reversion of phenotypes was generally accompanied by correlated responses in chlorophyll accumulation and altered expression of selected light signalling genes: PHYTOCHROME A, CRYPTOCHROME 1, ELONGATED HYPOCOTYL 5, and the gene encoding CHLOROPHYLL A/B-BINDING PROTEIN 4. Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

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Transcript level analysis of DDB1, PHYA, CRY1, HY5, and CAB4 in hypocotyls (A, B, C, D, and E, respectively) and cotyledons (F, G, H, I, and J, respectively) of 11-d-old transgenic hp-1 tomato seedlings overexpressing normal DDB1 in comparison with their normal and mutant control lines under different light treatments (black bars, pooled hp-1 mutant and azygous control lines; white bars, non-mutant line; grey bars, pooled T3 hp-1 mutant seedlings overexpressing normal DDB1 under the CaMV 35S promoter); different letters below the bars indicate statistically significant differences between genotypes within each gene and light condition.
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fig3: Transcript level analysis of DDB1, PHYA, CRY1, HY5, and CAB4 in hypocotyls (A, B, C, D, and E, respectively) and cotyledons (F, G, H, I, and J, respectively) of 11-d-old transgenic hp-1 tomato seedlings overexpressing normal DDB1 in comparison with their normal and mutant control lines under different light treatments (black bars, pooled hp-1 mutant and azygous control lines; white bars, non-mutant line; grey bars, pooled T3 hp-1 mutant seedlings overexpressing normal DDB1 under the CaMV 35S promoter); different letters below the bars indicate statistically significant differences between genotypes within each gene and light condition.

Mentions: Based on the results to this point, it could be concluded that DDB1 significantly affects developmental as well as metabolomic processes in developing seedlings and young plants, and that these effects are light and organ dependent. Therefore, transcript levels of selected light-regulated genes were analysed separately in the hypocotyls and cotyledons of seedlings representing the transgenic lines and compared with their normal controls under the three illumination conditions. In addition to DDB1, four light signalling genes (Quail, 2002) were examined: two photoreceptor genes, PHYA and CRY1, a downstream effector gene encoding a bZIP transcription factor, HY5, and a light-regulated structural gene, CAB4. To simplify the presentation, all five transgenic lines and the two hp-1 controls were separately pooled prior to the final analysis. The analysis of the data, generally displayed in Fig. 3, indicated the following. (i) With the exception of PHYA in hypocotyls and generally as expected, the light treatment had a strong and highly significant effect on the transcript levels of the analysed genes, substantiating the choice of genes [P(F) values for light treatment in the hypocotyls were 4.9×10−24, 5.0×10−1, 1.2×10−9, 1.9×10−4, and 1.6×10−24, while in the cotyledons they were 1.6×10−8, 9.0×10−38, 3.3×10−7, 7.9×10−7, and 9.0×10−7, for DDB1, PHYA, CRY1, HY5, and CAB4, respectively]. (ii) Similarly to DDB1, no significant transcriptional differences were generally found between the two hp-1 lines and their normal counterpart in either hypocotyls or cotyledons within each light treatment. The exceptions in this respect were the transcriptional up-regulation of HY5 in cotyledons under yellow light (Fig. 3I) and of CAB4 in hypocotyls under complete darkness (Fig. 3E) in the hp-1 genotypes. Interestingly, the latter gene was significantly down-regulated in hypocotyls of the hp-1 genotypes grown under the yellow screen (Fig. 3E). (iii) Contrary to the results obtained in the comparison between hp-1 and normal genotypes, wider transcriptional effects were observed in the five transgenic lines (Fig. 3). These effects were light dependent; in some cases they were organ specific and in a few others they were opposite to the DDB1 transcript levels.


Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato.

Azari R, Reuveni M, Evenor D, Nahon S, Shlomo H, Chen L, Levin I - J. Exp. Bot. (2010)

Transcript level analysis of DDB1, PHYA, CRY1, HY5, and CAB4 in hypocotyls (A, B, C, D, and E, respectively) and cotyledons (F, G, H, I, and J, respectively) of 11-d-old transgenic hp-1 tomato seedlings overexpressing normal DDB1 in comparison with their normal and mutant control lines under different light treatments (black bars, pooled hp-1 mutant and azygous control lines; white bars, non-mutant line; grey bars, pooled T3 hp-1 mutant seedlings overexpressing normal DDB1 under the CaMV 35S promoter); different letters below the bars indicate statistically significant differences between genotypes within each gene and light condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921201&req=5

fig3: Transcript level analysis of DDB1, PHYA, CRY1, HY5, and CAB4 in hypocotyls (A, B, C, D, and E, respectively) and cotyledons (F, G, H, I, and J, respectively) of 11-d-old transgenic hp-1 tomato seedlings overexpressing normal DDB1 in comparison with their normal and mutant control lines under different light treatments (black bars, pooled hp-1 mutant and azygous control lines; white bars, non-mutant line; grey bars, pooled T3 hp-1 mutant seedlings overexpressing normal DDB1 under the CaMV 35S promoter); different letters below the bars indicate statistically significant differences between genotypes within each gene and light condition.
Mentions: Based on the results to this point, it could be concluded that DDB1 significantly affects developmental as well as metabolomic processes in developing seedlings and young plants, and that these effects are light and organ dependent. Therefore, transcript levels of selected light-regulated genes were analysed separately in the hypocotyls and cotyledons of seedlings representing the transgenic lines and compared with their normal controls under the three illumination conditions. In addition to DDB1, four light signalling genes (Quail, 2002) were examined: two photoreceptor genes, PHYA and CRY1, a downstream effector gene encoding a bZIP transcription factor, HY5, and a light-regulated structural gene, CAB4. To simplify the presentation, all five transgenic lines and the two hp-1 controls were separately pooled prior to the final analysis. The analysis of the data, generally displayed in Fig. 3, indicated the following. (i) With the exception of PHYA in hypocotyls and generally as expected, the light treatment had a strong and highly significant effect on the transcript levels of the analysed genes, substantiating the choice of genes [P(F) values for light treatment in the hypocotyls were 4.9×10−24, 5.0×10−1, 1.2×10−9, 1.9×10−4, and 1.6×10−24, while in the cotyledons they were 1.6×10−8, 9.0×10−38, 3.3×10−7, 7.9×10−7, and 9.0×10−7, for DDB1, PHYA, CRY1, HY5, and CAB4, respectively]. (ii) Similarly to DDB1, no significant transcriptional differences were generally found between the two hp-1 lines and their normal counterpart in either hypocotyls or cotyledons within each light treatment. The exceptions in this respect were the transcriptional up-regulation of HY5 in cotyledons under yellow light (Fig. 3I) and of CAB4 in hypocotyls under complete darkness (Fig. 3E) in the hp-1 genotypes. Interestingly, the latter gene was significantly down-regulated in hypocotyls of the hp-1 genotypes grown under the yellow screen (Fig. 3E). (iii) Contrary to the results obtained in the comparison between hp-1 and normal genotypes, wider transcriptional effects were observed in the five transgenic lines (Fig. 3). These effects were light dependent; in some cases they were organ specific and in a few others they were opposite to the DDB1 transcript levels.

Bottom Line: However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful.This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion.Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, PO Box 6, Bet Dagan 50250, Israel.

ABSTRACT
Fruits of tomato plants carrying the high pigment-1 mutations hp-1 and hp-1(w) are characterized by an increased number of plastids coupled with enhanced levels of functional metabolites. Unfortunately, hp-1 mutant plants are also typified by light-dependent retardation in seedling and whole-plant growth and development, which limits their cultivation. These mutations were mapped to the gene encoding UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1) and, recently, fruit-specific RNA interference studies have demonstrated an increased number of plastids and enhanced carotenoid accumulation in the transgenic tomato fruits. However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful. In this study, five transgenic lines constitutively overexpressing normal DDB1 in hp-1 mutant plants were analysed. Eleven-day-old seedlings, representing these lines, displayed up to approximately 73- and approximately 221-fold overexpression of the gene in hypocotyls and cotyledons, respectively. This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion. This reversion of phenotypes was generally accompanied by correlated responses in chlorophyll accumulation and altered expression of selected light signalling genes: PHYTOCHROME A, CRYPTOCHROME 1, ELONGATED HYPOCOTYL 5, and the gene encoding CHLOROPHYLL A/B-BINDING PROTEIN 4. Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

Show MeSH