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.

Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds.

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification. However, carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains. In this study, we utilized Arabidopsis as a model to establish carotenoid biofortification strategies in seeds. We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase (PSY) increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage, consistent with previous studies of provitamin A biofortified grains. In contrast, stacking with Orange (OR His ), a gene that initiates chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of ß-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing OR His and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with OR His and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of ß-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates ß-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00046-1.

Effects of Selenium Supplementation on Glucosinolate Biosynthesis in Broccoli.

Selenium (Se)-enriched broccoli has health-beneficial selenium-containing compounds, but it may contain reduced amounts of chemopreventive glucosinolates. To investigate the basis by which Se treatment influences glucosinolate levels, we treated two broccoli cultivars with 25 µM Na2SeO4. We found that Se supplementation suppressed the accumulation of total glucosinolates, particularly glucoraphanin, the direct precursor of a potent anticancer compound, in broccoli florets and leaves. We showed that the suppression was not associated with plant sulfur nutrition. The levels of the glucosinolate precursors methionine and phenylalanine as well as the expression of genes involved in glucosinolate biosynthesis were greatly decreased following Se supplementation. Comparative proteomic analysis identified proteins in multiple metabolic and cellular processes that were greatly affected and detected an enzyme affecting methionine biosynthesis that was reduced in the Se-biofortified broccoli. These results indicate that Se-conferred glucosinolate reduction is associated with negative effects on precursor amino acid biosynthesis and glucosinolate-biosynthetic-gene expression and provide information for a better understanding of glucosinolate accumulation in response to Se supplementation in broccoli.

Proteome Modification in Tomato Plants upon Long-Term Aluminum Treatment.

This study aimed to identify the aluminum (Al)-induced proteomes in tomato (Solanum lycopersicum, "Micro-Tom") after long-term exposure to the stress factor. Plants were treated in Magnavaca's solution (pH 4.5) supplemented with 7.5 µM Al(3+) ion activity over a 4 month period beginning at the emergence of flower buds and ending when the lower mature leaves started to turn yellow. Proteomes were identified using a 8-plex isobaric tags for relative and absolute quantification (iTRAQ) labeling strategy followed by a two-dimensional (high- and low-pH) chromatographic separation and final generation of tandem mass spectrometry (MS/MS) spectra of tryptic peptides on an LTQ-Orbitrap Elite mass spectrometer. Principal component analysis revealed that the Al-treatment had induced systemic alterations in the proteomes from roots and leaves but not seed tissues. The significantly changed root proteins were shown to have putative functions in Al(3+) ion uptake and transportation, root development, and a multitude of other cellular processes. Changes in the leaf proteome indicate that the light reaction centers of photosynthetic machinery are the primary targets of Al-induced stress. Embryo and seed-coat tissues derived from Al-treated plants were enriched with stress proteins. The biological processes involving these Al-induced proteins concur with the physiological and morphological changes, such as the disturbance of mineral homeostasis (higher contents of Al, P, and Fe and reduced contents of S, Zn, and Mn in Al-treated compared to nontreated plants) in roots and smaller sizes of roots and the whole plants. More importantly, the identified significant proteins might represent a molecular mechanism for plants to develop toward establishing the Al tolerance and adaptation mechanism over a long period of stress treatment.

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.