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Tomato (Solanum lycopersicum) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine (t) appear orange due to accumulation of 7,9,7′,9′-tetra- cis -lycopene (prolycopene) as a result of a mutation in the carotenoid cis – trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh . This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r ²⁹⁹⁷ of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine , transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis -carotenoid metabolites in a feedback regulation of PSY1 gene expression.

The tomato (Lycopersicon esculentum) chloroplast small heat shock protein (sHSP), HSP21, is induced by heat treatment in leaves, but also under normal growth conditions in developing fruits during the transition of chloroplasts to chromoplasts. We used transgenic tomato plants constitutively expressing HSP21 to study the role of the protein under stress conditions and during fruit maturation. Although we did not find any effect for the transgene on photosystem II (PSII) thermotolerance, our results show that the protein protects PSII from temperature-dependent oxidative stress. In addition, we found direct evidence of the protein's role in fruit reddening and the conversion of chloroplasts to chromoplasts. When plants were grown under normal growth temperature, transgenic fruits accumulated carotenoids earlier than controls. Furthermore, when detached mature green fruits were stored for 2 weeks at 2°C and then transferred to room temperature, the natural accumulation of carotenoids was blocked. In a previous study, we showed that preheat treatment, which induces HSP21, allowed fruit color change at room temperature, after a cold treatment. Here, we show that mature green transgenic fruits constitutively expressing HSP21 do not require the heat treatment to maintain the ability to accumulate carotenoids after cold storage. This study demonstrates that a sHSP plays a role in plant development under normal growth conditions, in addition to its protective effect under stress conditions.

Fleshy tomato fruit typically lacks stomata; therefore, a proper cuticle is particularly vital for fruit development and interaction with the surroundings. Here, we characterized the tomato SlSHINE3 (SlSHN3) transcription factor to extend our limited knowledge regarding the regulation of cuticle formation in fleshy fruits. We created SlSHN3 overexpressing and silenced plants, and used them for detailed analysis of cuticular lipid compositions, phenotypic characterization, and the study on the mode of SlSHN3 action. Heterologous expression of SlSHN3 in Arabidopsis phenocopied overexpression of the Arabidopsis SHNs. Silencing of SlSHN3 results in profound morphological alterations of the fruit epidermis and significant reduction in cuticular lipids. We demonstrated that SlSHN3 activity is mediated by control of genes associated with cutin metabolism and epidermal cell patterning. As with SlSHN3 RNAi lines, mutation in the SlSHN3 target gene, SlCYP86A69, resulted in severe cutin deficiency and altered fruit surface architecture. In vitro activity assays demonstrated that SlCYP86A69 possesses NADPH-dependent ω-hydroxylation activity, particularly of C18:1 fatty acid to the 18-hydroxyoleic acid cutin monomer. This study provided insights into transcriptional mechanisms mediating fleshy fruit cuticle formation and highlighted the link between cutin metabolism and the process of fruit epidermal cell patterning.

Mapping of a mutation in a tomato deficient in the plant cuticle component cutin yields the first cutin synthase, as shown via accumulation of polymer precursors and in vitro oligomerization of synthetic substrates. A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol. CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo , indicating that it is a cutin synthase.

Most enzymes in the central pathway of carotenoid biosynthesis in plants have been identified and studied at the molecular level. However, the specificity and role of cis-trans-isomerization of carotenoids, which occurs in vivo during carotene biosynthesis, remained unresolved. We have previously cloned from tomato (Solanum lycopersicum) the CrtISO gene, which encodes a carotene cis-trans-isomerase. To study the biochemical properties of the enzyme, we developed an enzymatic in vitro assay in which a purified tomato CRTISO polypeptide overexpressed in Escherichia coli cells is active in the presence of an E. coli lysate that includes membranes. We show that CRTISO is an authentic carotene isomerase. Its catalytic activity of cis-to-trans isomerization requires redox-active components, suggesting that isomerization is achieved by a reversible redox reaction acting at specific double bonds. Our data demonstrate that CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9'. We conclude that CRTISO functions in the carotenoid biosynthesis pathway in parallel with zeta-carotene desaturation, by converting 7,9,9'-tri-cis-neurosporene to 9'-cis-neurosporene and 7'9'-di-cis-lycopene into all-trans-lycopene. These results establish that in plants carotene desaturation to lycopene proceeds via cis-carotene intermediates.

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.

Ornithogalum dubium is a popular ornamental monocot native to South Africa with flower colors ranging from pure white to deep orange. Gene editing based on the CRISPR/Cas9 system has recently been shown to hold potential for color improvement in ornamental flower crops. To apply this approach to Ornithogalum color manipulation, genomic or transcriptomic data must first be collected. Here, cDNA libraries of O. dubium leaves and flowers were constructed and sequenced using the Illumina HiSeq 2500. Over 155 million 100-bp paired-end reads were assembled into a transcriptome database of 360,689 contigs, of which 18,660 contigs were differentially expressed between leaves and flowers. Carotenoids are the main pigment imparting spectrum of orange hues to O. dubium flowers. By querying our database, we identified a total of 16 unique transcripts (unigenes) predicted to be involved in the carotenoid biosynthesis pathway of Ornithogalum. Combining carotenoid profiles, we further inferred several key unigenes responsible for floral coloration and accumulation in O. dubium, of which the gene LCYB/comp146645_c0 was found as a suitable target to generate potentially red flower varieties of O. dubium. Our research thus provides a framework for the application of CRISPR/Cas9 technology to improve this ornamental crop.

Background The concept of metabolite profiling has been around for decades and technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. However, studies are generally confined to polar compounds alone. Here we describe a simple method for lipophilic compounds analysis in various plant tissues. Results We choose the same preparative and instrumental platform for lipophilic profiling as that we routinely use for polar metabolites measurements. The method was validated in terms of linearity, carryover, reproducibility and recovery rates, as well as using various plant tissues. As a first case study we present metabolic profiling of Arabidopsis root and shoot tissue of wild type (C24) and mutant ( rsr4-1 ) plants deficient on vitamin B6. We found significant alterations in lipid constituent contents, especially in the roots, which were characterised by dramatic increases in several fatty acids, thus providing further hint for the role of pyridoxine in oxidative stress and lipid peroxidation. The second example is the lipophilic profiling of red and green tomato fruit cuticles of wild type (Alisa Craig) and the DFD (delayed fruit deterioration) mutant, which we compared and contrasted with the more focused wax analysis of these plants reported before. Conclusion We can rapidly and reliably detect and quantify over 40 lipophilic metabolites including fatty acids, fatty alcohols, alkanes, sterols and tocopherols. The method presented here affords a simple and rapid, yet robust complement to previously validated methods of polar metabolite profiling by gas-chromatography mass-spectrometry.

Key message Through meta-genetic analysis of Cucumis melo sweetness, we expand the description of the complex genetic architecture of this trait. Integration of extensive new results with published QTL data provides an outline towards construction of a melon sweetness pan-QTLome. An ultimate objective in crop genetics is describing the complete repertoire of genes and alleles that shape the phenotypic variation of a quantitative trait within a species. Flesh sweetness is a primary determinant of fruit quality and consumer acceptance of melons . Cucumis melo is a diverse species that, among other traits, displays extensive variation in total soluble solids (TSS) content in fruit flesh, ranging from 2 0 Brix in non-sweet to 18 0 Brix in sweet accessions. We present here meta-genetic analysis of TSS and sugar variation in melon, using six different populations and fruit measurements collected from more than 30,000 open-field and greenhouse-grown plants, integrated with 15 published melon sweetness-related quantitative trait loci (QTL) studies. Starting with characterization of sugar composition variation across 180 diverse accessions that represent 3 subspecies and 12 of their cultivar-groups, we mapped TSS and sugar QTLs, and confirmed that sucrose accumulation is the key variable explaining TSS variation. All modes-of-inheritance for TSS were displayed by multi-season analysis of a broad half-diallel population derived from 20 diverse founders, with significant prevalence of the additive component. Through parallel genetic mapping in four advanced bi-parental populations, we identified common as well as unique TSS QTLs in 12 chromosomal regions. We demonstrate the cumulative less-than-additive nature of favorable TSS QTL alleles and the potential of a QTL-stacking approach. Using our broad dataset, we were additionally able to show that TSS variation displays weak genetic correlations with melon fruit size and ripening behavior, supporting effective breeding for sweetness per se. Our integrated analysis, combined with additional layers of published QTL data, broadens the perspective on the complex genetic landscape of melon sweetness and proposes a scheme towards future construction of a crop community-driven melon sweetness pan-QTLome.