Laser capture of tomato pericarp tissues for microscale carotenoid analysis by supercritical fluid chromatography.

Plant organs and tissues are comprised of an array of cell types often superimposed on a gradient of developmental stages. As a result, the ability to analyze and understand the synthesis, metabolism, and accumulation of plant biomolecules requires improved methods for cell- and tissue-specific analysis. Tomato (Solanum lycopersicum) is the world's most valuable fruit crop and is an important source of health-promoting dietary compounds, including carotenoids. Furthermore, tomato possesses unique genetic activities at the cell and tissue levels, making it an ideal system for tissue- and cell-type analysis of important biochemicals. A sample preparation workflow was developed for cell-type-specific carotenoid analysis in tomato fruit samples. Protocols for hyperspectral imaging of tomato fruit samples, cryoembedding and sectioning of pericarp tissue, laser microdissection of specific cell types, metabolite extraction using cell wall digestion enzymes and pressure cycling, and carotenoid quantification by supercritical fluid chromatography were optimized and integrated into a working protocol. The workflow was applied to quantify carotenoids in the cuticle and noncuticle component of the tomato pericarp during fruit development from the initial ripening to full ripe stages. Carotenoids were extracted and quantified from cell volumes less than 10nL. This workflow for cell-type-specific metabolite extraction and quantification can be adapted for the analysis of diverse metabolites, cell types, and organisms.

Overexpression of native ORANGE (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress.

The carotenoid content of plants can be increased by overexpression of the regulatory protein ORANGE (OR) or a mutant variant known as the 'golden SNP'. In the present study, a strong light-inducible promoter was used to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine in the microalgae Chlamydomonas reinhardtii. Overexpression of CrORWT and CrORHis roughly doubled and tripled, respectively, the accumulation of several different carotenoids, including ß-carotene, α-carotene, lutein and violaxanthin in C. reinhardtii and upregulated the transcript abundance of nearly all relevant carotenoid biosynthetic genes. In addition, microscopic analysis revealed that the OR transgenic cells were larger than control cells and exhibited larger chloroplasts with a disrupted morphology. Moreover, both CrORWT and CrORHis cell lines showed increased tolerance to salt and paraquat stress. The levels of endogenous phytohormone abscisic acid (ABA) were also increased in CrORWT and CrORHis lines, not only in normal growth conditions but also in growth medium supplemented with salt and paraquat. Together these results offer new insights regarding the role of the native OR protein in regulating carotenoid biosynthesis and the accumulation of several carotenoids in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance potentially mediated by ABA.

The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor.

Modern tomatoes have narrow genetic diversity limiting their improvement potential. We present a tomato pan-genome constructed using genome sequences of 725 phylogenetically and geographically representative accessions, revealing 4,873 genes absent from the reference genome. Presence/absence variation analyses reveal substantial gene loss and intense negative selection of genes and promoters during tomato domestication and improvement. Lost or negatively selected genes are enriched for important traits, especially disease resistance. We identify a rare allele in the TomLoxC promoter selected against during domestication. Quantitative trait locus mapping and analysis of transgenic plants reveal a role for TomLoxC in apocarotenoid production, which contributes to desirable tomato flavor. In orange-stage fruit, accessions harboring both the rare and common TomLoxC alleles (heterozygotes) have higher TomLoxC expression than those homozygous for either and are resurgent in modern tomatoes. The tomato pan-genome adds depth and completeness to the reference genome, and is useful for future biological discovery and breeding.

Green tea ingestion by rats does not affect iron absorption but does alter the composition of the saliva proteome.

We tested the hypothesis that rats adapt to the iron absorption inhibitory effects of tea by modifying the expression of salivary proteins. Thirty-six weanling rats were allocated into 6 groups. Two control groups were fed a semipurified diet containing 20 mg Fe(2+)/kg diet. Two groups were fed spray dried green tea infusion mixed into the diet (28.6 g tea/kg diet) and 2 groups were fed the control diet with a twice daily gavage of a tea solution (0.25 g tea/mL). Saliva samples were collected in 3 groups (control, gavage, and oral) on day 8 (acute) and in the remaining groups on day 31 (chronic). Iron absorption was assessed using a (58)Fe(3+) tracer administered on day 1 (acute) and day 24 (chronic). 2D gel electrophoresis and mass spectrometry were used to assess the composition of the saliva proteome. There was no significant difference in iron absorption between the 3 groups on either day 1 or day 24. Salivary proline-rich proteins and submandibular gland secretory protein increased to a greater extent in the oral group than in the gavage group, when compared to control, within the same exposure time period. Amylase, chitinase, deoxyribonuclease, cysteine-rich secretory protein 1, and parotid secretory protein all decreased to a greater extent in the oral tea group, compared to the control, within the same exposure time period. Our results show that green tea did not decrease iron absorption in rats but it did have a marked effect on the saliva proteome when given orally.