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, 22 (6), 1977-97

An Orange Ripening Mutant Links Plastid NAD(P)H Dehydrogenase Complex Activity to Central and Specialized Metabolism During Tomato Fruit Maturation

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An Orange Ripening Mutant Links Plastid NAD(P)H Dehydrogenase Complex Activity to Central and Specialized Metabolism During Tomato Fruit Maturation

Shai Nashilevitz et al. Plant Cell.

Abstract

In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of Orr(Ds), a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in Orr(Ds), as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly beta-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, Orr(Ds) leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism.

Figures

Figure 1.
Figure 1.
Phenotypes of the OrrDs Mutation and Complementation of the Phenotype by the ex-35S-Orr Allele and ORR Gene Overexpression. (A) Somatic activity (sectors) in fruit of a plant (OrrDs) harboring both the Ds and Ac elements. (B) Fruit phenotypes in progeny of an OrrDs/ORR plant. Orange-colored fruit (homozygous, left), yellow-colored fruit (heterozygous, middle), and red (wild type, right). (C) The phenotype of peel isolated from mature fruit of the plants shown in (B). (D) Whole ripe fruit and isolated peel derived from the ex-35S-Orr plant compared with the homozygous mutant fruit (OrrDs/OrrDs). (E) Whole ripe fruit and isolated peel derived from a plant in which the full-length ORR cDNA (starting from AUG-I) was overexpressed in the OrrDs/OrrDs plant. (F) Whole ripe fruit and isolated peel derived from an F1 line obtained by a cross between a plant in which the ORR cDNA (starting from AUG-II) was overexpressed in the OrrDs/OrrDs plant. (G) Whole ripe fruit and isolated peel derived from a plant in which the ORR cDNA (starting from AUG-II) was overexpressed in the OrrDs/OrrDs plant. In (E) to (G), the plus and minus symbols represent the presence or absence of the transgene conferring ORR gene overexpression, respectively.
Figure 2.
Figure 2.
Structure of the OrrDs Allele and the 5′ Regions Obtained by RACE Analysis in the Different OrrDs Genotypes and the Wild Type. (A) The diagram displays the structure of the OrrDs allele. The ORR gene has two exons (black closed boxes) flanking one intron (black line between the exons). The position of composite Ds element (CDE) in the ORR 5′ end is marked. The CDE is composed of the CaMV 35S promoter flanked (downstream) by a complete Ds element and another element (possibly partial) upstream. ATG-I to ATG-IV are marked. At the bottom part, results of 5′ RACE analysis of the various OrrDs genotypes and the wild type (WT) are depicted. Long and short 5′ RACE products were obtained in the ex-35S-Orr genotype (ex-35S-Orr_L and ex-35S-Orr_S, respectively). The location of the corresponding Met along the amino acid (aa) sequence is indicated in parentheses next to each AUG codon. NRP, nested RACE primer. Black areas mark the putative protein formed in each transcript. (B) The exact position of the CDE in the ORR gene sequence between ATG-I and ATG-II. The CDE and the three ATGs (I, II, and III) are marked by a bigger and different font.
Figure 3.
Figure 3.
ORR Gene Expression and Protein Subcellular Localization. (A) Expression of ORR in various plant tissues (cv MicroTom) detected by qRT-PCR. Peel (P), flesh (F), and seeds (S) were dissected from IG (immature green), MG (mature green), Br (breaker), Or (orange), and R (red) fruit. Expression data were normalized to the expression of the ASR1 gene, and values are means ± se (n = 3). (B) The subcellular localization of ORR (starting from AUG-I) was investigated using confocal microscopy after transient expression of ORR-GFP fusions in tobacco leaves. The natural chlorophyll red fluorescence signal was used to visualize chloroplasts and was merged with the GFP signal. The pattern of ORR-GFP starting from AUG-I coincided with that of chlorophyll confirming its chloroplast localization. Bars = 30.25 μm.
Figure 4.
Figure 4.
ORR Gene Expression and Activity and Levels of the Ndh Complex in the OrrDs Mutants. (A) Expression of ORR in immature green fruit (30 d after anthesis) of the wild type (WT) and OrrDs mutants. qRT-PCR expression data were normalized to the expression of the ASR1 gene and presented as percentage of the wild type, which was set at 100% (values are means ± se; n = 3). Asterisk indicates values that are significantly different from the wild type (P < 0.01). The amplified part is located in the coding region downstream of the insertion. (B) to (D) Protein gel blot analysis. Thylakoids were extracted from wild-type, homozygous (Hom; OrrDs/OrrDs), heterozygous (Het; OrrDs/ORR), and the ex-35S-Orr mutants leaves and immature green fruit. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and analyzed using anti-NDH-H antibodies. The lanes were loaded with samples corresponding to 15 μg of chlorophyll (100%) and a series of dilutions as indicated. Immunodetection of OEC33 was used as a loading control. (E) In vivo detection of Ndh complex activity by chlorophyll fluorescence measurements. Leaf and immature green fruits chlorophyll fluorescence of wild-type and mutant tomato plants was monitored with a PAM fluorometer. The top curve is a typical trace of chlorophyll fluorescence in the wild type. After a 5-min actinic illumination (AL; 250 μmol photons m−2 s−1), Fs levels reached similar levels in wild-type and mutant leaves. After switching the light off (AL off), transient increases in chlorophyll fluorescence were recorded under low nonactinic light. Insets are magnified traces from the boxed area. SF, saturating flash of white light. [See online article for color version of this figure.]
Figure 5.
Figure 5.
The Effect of the OrrDs Mutation on Metabolite Levels in the Isoprenoid and Flavonoid Pathways. (A) HPLC analysis of various flavonoids in extracts (ripe fruit, 14 d after breaker stage) of the wild type (WT; red bars) and OrrDs mutants (OrrDs/ORR, yellow bars; OrrDs/OrrDs, orange bars). Numbers correspond to the peak area representing each metabolite; data are shown as means ± se (n = 4). Asterisk indicates values that are significantly different from the wild type (P < 0.01). (B) Composition of carotenoids in mature fruit derived from the different OrrDs genotypes and wild-type plants detected by HPLC. Total carotenoids are the sum of all the peaks area of each genotype. Data are shown as means ± se (n = 4). Asterisk indicates values that are significantly different from the wild type (P < 0.01). (C) Chlorophylls and tocopherols levels in mature fruit derived from the various OrrDs genotypes and wild-type plants detected by HPLC. Data are shown as means ± se (n = 4). Asterisk indicates values that are significantly different from the wild type (P < 0.01). [See online article for color version of this figure.]
Figure 6.
Figure 6.
Gene Expression Changes in the OrrDs Mutants. Venn diagrams of genes changed in expression in fruit (breaker stage) of the three OrrDs genotypes compared with the wild type (WT). The Venn diagrams show significantly up- and downregulated genes in fruit of the OrrDs/ORR (red), OrrDs/OrrDs (green) and the ex-35S-Orr (blue) lines when compared with the wild type. Genes being significantly up- or downregulated in several of the genotypes are shown in the respective overlap regions. [See online article for color version of this figure.]
Figure 7.
Figure 7.
The Effect of the OrrDs Mutation on Gene Expression in the Isoprenoid and Phenylpropanoid/Flavonoid Biosynthesis Pathways. (A) Representation of gene expression and metabolite levels changes in fruit derived from the OrrDs/ORR genotype compared with that of wild-type plants. Microarray probe sets reporting the gene expression of likely pathway genes are depicted by color-coded boxes indicating the log2 fold change of the respective transcripts according to a false-color scale reproduced in the figure, where white represents no change, red an upregulation, and green a downregulation. Each individual box represents a unique probe set present on the chip hybridizing to isoforms of the respective gene indicated next to it. Nonsignificantly changed probe sets are depicted in gray. Metabolites where quantitative data were available are represented as black text on colored boxes, where the box color indicates log2 fold changes following the same color scheme as transcripts. Metabolites not detectable in the mutant are painted on a blue box, whereas the ones not detectable in the wild type are painted on a yellow box. The electron transport pathway involving the Ndh complex and PTOX (adapted from Carol and Kuntz, 2001) is represented in a box on the left. GGPP, geranylgeranylpyrophosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; DXS, 1-deoxy-d-xylulose 5-phosphate synthase; PSY, phytoene synthase; CrtISO, carotene isomerase; CRTL-E, LCY-E, lycopene e-cyclase; LCY-B, lycopene β-cyclase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; CAO, chlorophyll a oxidase. (B) Expression of genes in the isoprenoid pathway (including carotenoid-related) in the hetrozygous OrrDs mutant and wild-type breaker stage fruit as detected by qRT-PCR. Full names of genes are given in (C). Expression data were normalized to the expression of the CLATHRIN ADAPTOR COMPLEXES SUBUNIT (CAC) gene; values are means ± se (n = 3). Asterisks indicate values that are significantly different from the wild type at *P < 0.05 and **P < 0.01. (C) Representation of gene expression and metabolite levels changes in fruit derived from the OrrDs/ORR genotype compared with wild-type plants. The array and metabolite analysis data are presented in the same way as in Figure 7A. PAL, Phe-ammonia lyase; 4CL, 4-coumaroyl-CoA synthase 2; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; 3GT, anthocyanidin 3-O-glucosyltransferase; RT, rhamnosyltransferase; CAD, cinnamyl-alcohol dehydrogenase; F3′5'H, flavanone 3′, 5′hydroxylase. (D) Expression of genes related with phenylpropanoids/flavonoids pathway in the hetrozygous OrrDs mutant and wild-type breaker stage fruit as detected by qRT-PCR. Full names of genes are given in (B). Expression data were normalized to the expression of the CAC gene; values are means ± se (n = 3).
Figure 8.
Figure 8.
Ethylene Emission from the OrrDs Mutants and Wild-Type Fruit. The chart shows ethylene emission from fruit of the OrrDs genotypes and the wild type (WT) measured 11 times during 19 d after the mature green fruit stage. Emission of each line is represented by the patterned strip between two graphs or between the graph and x axis (in the case of the OrrDs/OrrDs genotype). The letters a to c represent significance groups of ethylene emission per day (n = 3; P < 0.05; Student's t test).

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