CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Evolution and origin of bread wheat.

Bread wheat (Triticum aestivum, genome BBAADD) is a young hexaploid species formed only 8,500-9,000 years ago through hybridization between a domesticated free-threshing tetraploid progenitor, genome BBAA, and Aegilops tauschii, the diploid donor of the D subgenome. Very soon after its formation, it spread globally from its cradle in the fertile crescent into new habitats and climates, to become a staple food of humanity. This extraordinary global expansion was probably enabled by allopolyploidy that accelerated genetic novelty through the acquisition of new traits, new intergenomic interactions, and buffering of mutations, and by the attractiveness of bread wheat's large, tasty, and nutritious grain with high baking quality. New genome sequences suggest that the elusive donor of the B subgenome is a distinct (unknown or extinct) species rather than a mosaic genome. We discuss the origin of the diploid and tetraploid progenitors of bread wheat and the conflicting genetic and archaeological evidence on where it was formed and which species was its free-threshing tetraploid progenitor. Wheat experienced many environmental changes throughout its evolution, therefore, while it might adapt to current climatic changes, efforts are needed to better use and conserve the vast gene pool of wheat biodiversity on which our food security depends.

Independent evolution of transcript abundance and gene regulatory dynamics.

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

Homoeologous exchanges occur through intragenic recombination generating novel transcripts and proteins in wheat and other polyploids.

Recombination between homeologous chromosomes, also known as homeologous exchange (HE), plays a significant role in shaping genome structure and gene expression in interspecific hybrids and allopolyploids of several plant species. However, the molecular mechanisms that govern HEs are not well understood. Here, we studied HE events in the progeny of a nascent allotetraploid (genome AADD) derived from two diploid progenitors of hexaploid bread wheat using cytological and whole-genome sequence analyses. In total, 37 HEs were identified and HE junctions were mapped precisely. HEs exhibit typical patterns of homologous recombination hotspots, being biased toward low-copy, subtelomeric regions of chromosome arms and showing association with known recombination hotspot motifs. But, strikingly, while homologous recombination preferentially takes place upstream and downstream of coding regions, HEs are highly enriched within gene bodies, giving rise to novel recombinant transcripts, which in turn are predicted to generate new protein fusion variants. To test whether this is a widespread phenomenon, a dataset of high-resolution HE junctions was analyzed for allopolyploid Brassica, rice, Arabidopsis suecica, banana, and peanut. Intragenic recombination and formation of chimeric genes was detected in HEs of all species and was prominent in most of them. HE thus provides a mechanism for evolutionary novelty in transcript and protein sequences in nascent allopolyploids.

Targeted Inter-Homologs Recombination in Arabidopsis Euchromatin and Heterochromatin.

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both "hot and cold" regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.

T-DNA-genome junctions form early after infection and are influenced by the chromatin state of the host genome.

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

The Israeli-Palestinian wheat landraces collection: restoration and characterization of lost genetic diversity.

BACKGROUND: For over a century, genetic diversity of wheat worldwide was eroded by continual selection for high yields and industrial demands. Wheat landraces cultivated in Israel and Palestine demonstrate high genetic diversity and a potentially wide repertoire of adaptive alleles. While most Israeli-Palestinian wheat landraces were lost in the transition to 'Green Revolution' semi-dwarf varieties, some germplasm collections made at the beginning of the 20th century survived in gene banks and private collections worldwide. However, fragmentation and poor conservation place this unique genetic resource at a high risk of genetic erosion. Herein, we describe a long-term initiative to restore, conserve, and characterize a collection of Israeli and Palestinian wheat landraces (IPLR). RESULTS: We report on (i) the IPLR construction (n = 932), (ii) the historical and agronomic context to this collection, (iii) the characterization and assessment of the IPLR's genetic diversity, and (iv) a data comparison from two distinct subcollections within IPLR: a collection made by N. Vavilov in 1926 (IPLR-VIR) and a later one (1979-1981) made by Y. Mattatia (IPLR-M). Though conducted in the same eco-geographic space, these two collections were subjected to considerably different conservation pathways. IPLR-M, which underwent only one propagation cycle, demonstrated marked genetic and phenotypic variability (within and between accessions) in comparison with IPLR-VIR, which had been regularly regenerated over ∼90 years. CONCLUSION: We postulate that long-term ex situ conservation involving human and genotype × environment selection may significantly reduce accession heterogeneity and allelic diversity. Results are further discussed in a broader context of pre-breeding and conservation. © 2019 Society of Chemical Industry.

The effects of AtRad52 over-expression on homologous recombination in Arabidopsis.

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.

Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.

Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana.

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

DNA Crossover Motifs Associated with Epigenetic Modifications Delineate Open Chromatin Regions in Arabidopsis.

The rate of crossover, the reciprocal exchanges of homologous chromosomal segments, is not uniform along chromosomes differing between male and female meiocytes. To better understand the factors regulating this variable landscape, we performed a detailed genetic and epigenetic analysis of 737 crossover events in Arabidopsis thaliana. Crossovers were more frequent than expected in promoters. Three DNA motifs enriched in crossover regions and less abundant in crossover-poor pericentric regions were identified. One of these motifs, the CCN repeat, was previously unknown in plants. The A-rich motif was preferentially associated with promoters, while the CCN repeat and the CTT repeat motifs were preferentially associated with genes. Analysis of epigenetic modifications around the motifs showed, in most cases, a specific epigenetic architecture. For example, we show that there is a peak of nucleosome occupancy and of H3K4me3 around the CCN and CTT repeat motifs while nucleosome occupancy was lowest around the A-rich motif. Cytosine methylation levels showed a gradual decrease within ∼2 kb of the three motifs, being lowest at sites where crossover occurred. This landscape was conserved in the decreased DNA methylation1 mutant. In summary, the crossover motifs are associated with epigenetic landscapes corresponding to open chromatin and contributing to the nonuniformity of crossovers in Arabidopsis.

Heterosis as a consequence of regulatory incompatibility.

BACKGROUND: The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. Heterosis is typically viewed as the opposite of hybrid incompatibility. In this view, the superior performance of the hybrid is attributed to heterozygote combinations that compensate for deleterious mutations accumulating in each individual genome, or lead to new, over-dominating interactions with improved performance. Still, only fragmented knowledge is available on genes and processes contributing to heterosis. RESULTS: We describe a budding yeast hybrid that grows faster than both its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increased signs of DNA damage. A systematic genetic screen identified hundreds of S. cerevisiae alleles whose deletion reduced growth of the hybrid. These growth-affecting alleles were condition-dependent, and differed greatly from alleles that reduced the growth of the S. cerevisiae parent. CONCLUSIONS: Our results define a budding yeast hybrid that is perturbed in multiple regulatory processes but still shows a clear growth heterosis. We propose that heterosis results from incompatibilities that perturb regulatory mechanisms, which evolved to protect cells against damage or prepare them for future challenges by limiting cell growth.

Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

Use of multicopy transposons bearing unfitness genes in weed control: four example scenarios.

We speculate that multicopy transposons, carrying both fitness and unfitness genes, can provide new positive and negative selection options to intractable weed problems. Multicopy transposons rapidly disseminate through populations, appearing in approximately 100% of progeny, unlike nuclear transgenes, which appear in a proportion of segregating populations. Different unfitness transgenes and modes of propagation will be appropriate for different cases: (1) outcrossing Amaranthus spp. (that evolved resistances to major herbicides); (2) Lolium spp., important pasture grasses, yet herbicide-resistant weeds in crops; (3) rice (Oryza sativa), often infested with feral weedy rice, which interbreeds with the crop; and (4) self-compatible sorghum (Sorghum bicolor), which readily crosses with conspecific shattercane and with allotetraploid johnsongrass (Sorghum halepense). The speculated outcome of these scenarios is to generate weed populations that contain the unfitness gene and thus are easily controllable. Unfitness genes can be under chemically or environmentally inducible promoters, activated after gene dissemination, or under constitutive promoters where the gene function is utilized only at special times (e.g. sensitivity to an herbicide). The transposons can be vectored to the weeds by introgression from the crop (in rice, sorghum, and Lolium spp.) or from planted engineered weed (Amaranthus spp.) using a gene conferring the degradation of a no longer widely used herbicide, especially in tandem with an herbicide-resistant gene that kills all nonhybrids, facilitating the rapid dissemination of the multicopy transposons in a weedy population.

Deficiency in DNA methylation increases meiotic crossover rates in euchromatic but not in heterochromatic regions in Arabidopsis.

Meiotic recombination is tightly regulated by cis- and trans-acting factors. Although DNA methylation and chromatin remodeling affect chromosome structure, their impact on meiotic recombination is not well understood. To study the effect of DNA methylation on the landscape of chromosomal recombination, we analyzed meiotic recombination in the decreased DNA methylation 1 (ddm1) mutant. DDM1 is a SWI2/SNF2-like chromatin-remodeling protein necessary for DNA methylation and heterochromatin maintenance in Arabidopsis thaliana. The rate of meiotic recombination between markers located in euchromatic regions was significantly higher in both heterozygous (DDM1/ddm1) and homozygous (ddm1/ddm1) backgrounds than in WT plants. The effect on recombination was similar for both male and female meiocytes. Contrary to expectations, ddm1 had no effect on the number of crossovers between markers in heterochromatic pericentric regions that underwent demethylation. These results are surprising, because the pericentromeric regions are hypermethylated and were expected to be the regions most affected by demethylation. Thus, DDM1 loss of function may trigger changes that enhance meiotic recombination in euchromatin regions but are not sufficient to induce the same events in heterochromatic segments. This work uncovers the repressive role of methylation on meiotic recombination in euchromatic regions and suggests that additional factors may have a role in controlling the suppression of recombination in heterochromatin.

Identification of plant RAD52 homologs and characterization of the Arabidopsis thaliana RAD52-like genes.

RADiation sensitive52 (RAD52) mediates RAD51 loading onto single-stranded DNA ends, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, including animals and fungi. This article reports that RAD52 homologs are present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications, during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins. Remarkably, the two identified Arabidopsis thaliana RAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria, or chloroplast. The A. thaliana RAD52-1A ORF provided partial complementation to the yeast rad52 mutant. A. thaliana mutants and RNA interference lines defective in the expression of RAD52-1 or RAD52-2 showed reduced fertility, sensitivity to mitomycin C, and decreased levels of intrachromosomal recombination compared with the wild type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.

Uncovering the dynamics of precise repair at CRISPR/Cas9-induced double-strand breaks.

CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of this process requires measuring the rates of cutting, error-prone, and precise repair, which have remained elusive so far. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterize the dynamics of DSB induction, processing and repair at endogenous loci along a 72 h time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is determined by the combined effect of the rates of DSB induction processing of broken ends, and precise versus error repair. In this study, 64-88% of the molecules were cleaved in the three targets analyzed, while indels ranged between 15-41%. Precise repair accounts for most of the gap between cleavage and error repair, representing up to 70% of all repair events. Altogether, this system exposes flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting the efficiency of CRISPR-mediated mutagenesis.

A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta.

Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase(®) reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases' cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established 'QQR' zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.

An orange ripening mutant links plastid NAD(P)H dehydrogenase complex activity to central and specialized metabolism during tomato fruit maturation.

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.