Violaxanthin

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.[Pubmed:38666490]

J Agric Food Chem. 2024 Apr 26.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing Violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance Violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of Violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of Violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for Violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

Improvement of the Quality of Wild Rocket (Diplotaxis tenuifolia) with Respect to Health-Related Compounds by Enhanced Growth Irradiance.[Pubmed:38648561]

J Agric Food Chem. 2024 Apr 22.

For healthier human nutrition, it is desirable to provide food with a high content of nutraceuticals such as polyphenolics, vitamins, and carotenoids. We investigated to what extent high growth irradiance influences the content of phenolics, alpha-tocopherol and carotenoids, in wild rocket (Diplotaxis tenuifolia), which is increasingly used as a salad green. Potted plants were grown in a climate chamber with a 16 h day length at photosynthetic photon flux densities varying from 20 to 1250 mumol m(-2) s(-1). Measurements of the maximal quantum yield of photosystem II, F(V)/F(M), and of the epoxidation state of the Violaxanthin cycle (V-cycle) showed that the plants did not suffer from excessive light for photosynthesis. Contents of carotenoids belonging to the V-cycle, alpha-tocopherol and several quercetin derivatives, increased nearly linearly with irradiance. Nonintrusive measurements of chlorophyll fluorescence induced by UV-A and blue light relative to that induced by red light, indicating flavonoid and carotenoid content, allowed not only a semiquantitative measurement of both compounds but also allowed to follow their dynamic changes during reciprocal transfers between low and high growth irradiance. The results show that growth irradiance has a strong influence on the content of three different types of compounds with antioxidative properties and that it is possible to determine the contents of flavonoids and specific carotenoids in intact leaves using chlorophyll fluorescence. The results may be used for breeding to enhance healthy compounds in wild rocket leaves and to monitor their content for selection of appropriate genotypes.

Distinct growth patterns in seedling and tillering wheat plants suggests a developmentally restricted role of HYD2 in salt-stress response.[Pubmed:38632145]

Plant Cell Rep. 2024 Apr 17;43(5):119.

Mutants lacking functional HYD2 homoeologs showed improved seedling growth, but comparable or increased susceptibility to salt stress in tillering plants, suggesting a developmentally restricted role of HYD2 in salt response. Salinity stress threatens global food security by reducing the yield of staple crops such as wheat (Triticum ssp.). Understanding how wheat responds to salinity stress is crucial for developing climate resilient varieties. In this study, we examined the interplay between carotenoid metabolism and the response to salt (NaCl) stress, a specific form of salinity stress, in tetraploid wheat plants with mutations in carotenoid beta-hydroxylase 1 (HYD1) and HYD2. Our investigation encompassed both the vulnerable seedling stage and the more developed tillering stage of wheat plant growth. Mutant combinations lacking functional HYD2 homoeologs, including hyd-A2 hyd-B2, hyd-A1 hyd-A2 hyd-B2, hyd-B1 hyd-A2 hyd-B2, and hyd-A1 hyd-B1 hyd-A2 hyd-B2, had longer first true leaves and slightly enhanced root growth during germination under salt stress compared to the segregate wild-type (control) plants. Interestingly, these mutant seedlings also showed decreased levels of neoxanthin and Violaxanthin (xanthophylls derived from beta-carotene) and an increase in beta-carotene in roots. However, tillering hyd mutant and segregate wild-type plants generally did not differ in their height, tiller count, and biomass production under acute or prolonged salt stress, except for decreases in these parameters observed in the hyd-A1 hyd-B1 hyd-A2 hyd-B2 mutant that indicate its heightened susceptibility to salt stress. Taken together, these findings suggest a significant, yet developmentally restricted role of HYD2 homoeologs in salt-stress response in tetraploid wheat. They also show that hyd-A2 hyd-B2 mutant plants, previously demonstrated for possessing enriched nutritional (beta-carotene) content, maintain an unimpaired ability to withstand salt stress.

BnaABF3 and BnaMYB44 regulate the transcription of zeaxanthin epoxidase genes in carotenoid and abscisic acid biosynthesis.[Pubmed:38620011]

Plant Physiol. 2024 Apr 15:kiae184.

Zeaxanthin epoxidase (ZEP) is a key enzyme that catalyzes the conversion of zeaxanthin to Violaxanthin in the carotenoid and abscisic acid (ABA) biosynthesis pathways. The rapeseed (Brassica napus) genome has 4 ZEP (BnaZEP) copies that are suspected to have undergone subfunctionalization, yet the 4 genes' underlying regulatory mechanisms remain unknown. Here, we genetically confirmed the functional divergence of the gene pairs BnaA09.ZEP/BnaC09.ZEP and BnaA07.ZEP/BnaC07.ZEP, which encode enzymes with tissue-specific roles in carotenoid and ABA biosynthesis in flowers and leaves, respectively. Molecular and transgenic experiments demonstrated that each BnaZEP pair is transcriptionally regulated via ABA-responsive element-binding factor 3 s (BnaABF3s) and BnaMYB44s as common and specific regulators, respectively. BnaABF3s directly bound to the promoters of all 4 BnaZEPs and activated their transcription, with overexpression of individual BnaABF3s inducing BnaZEP expression and ABA accumulation under drought stress. Conversely, loss of BnaABF3s function resulted in lower expression of several genes functioning in carotenoid and ABA metabolism and compromised drought tolerance. BnaMYB44s specifically targeted and repressed the expression of BnaA09.ZEP/BnaC09.ZEP but not BnaA07.ZEP/BnaC07.ZEP. Overexpression of BnaA07.MYB44 resulted in increased carotenoid content and an altered carotenoid profile in petals. Additionally, RNA-seq analysis indicated that BnaMYB44s functions as a repressor in phenylpropanoid and flavonoid biosynthesis. These findings provide clear evidence for the subfunctionalization of duplicated genes and contribute to our understanding of the complex regulatory network involved in carotenoid and ABA biosynthesis in B. napus.

Tetraselmis chuii Edible Microalga as a New Source of Neuroprotective Compounds Obtained Using Fast Biosolvent Extraction.[Pubmed:38612712]

Int J Mol Sci. 2024 Mar 31;25(7):3897.

Tetraselmis chuii is an EFSA-approved novel food and dietary supplement with increasing use in nutraceutical production worldwide. This study investigated the neuroprotective potential of bioactive compounds extracted from T. chuii using green biobased solvents (ethyl acetate, AcOEt, and cyclopentyl methyl ether, CPME) under pressurized liquid extraction (PLE) conditions and supercritical fluid extraction (SFE). Response surface optimization was used to study the effect of temperature and solvent composition on the neuroprotective properties of the PLE extracts, including anticholinergic activity, reactive oxygen/nitrogen species (ROS/RNS) scavenging capacity, and anti-inflammatory activity. Optimized extraction conditions of 40 degrees C and 34.9% AcOEt in CPME resulted in extracts with high anticholinergic and ROS/RNS scavenging capacity, while operation at 180 degrees C and 54.1% AcOEt in CPME yielded extracts with potent anti-inflammatory properties using only 20 min. Chemical characterization revealed the presence of carotenoids (neoxanthin, Violaxanthin, zeaxanthin, alpha- and beta-carotene) known for their anti-cholinesterase, antioxidant, and anti-inflammatory potential. The extracts also exhibited high levels of omega-3 polyunsaturated fatty acids (PUFAs) with a favorable omega-3/omega-6 ratio (>7), contributing to their neuroprotective and anti-inflammatory effects. Furthermore, the extracts were found to be safe to use, as cytotoxicity assays showed no observed toxicity in HK-2 and THP-1 cell lines at or below a concentration of 40 mug mL(-1). These results highlight the neuroprotective potential of Tetraselmis chuii extracts, making them valuable in the field of nutraceutical production and emphasize the interest of studying new green solvents as alternatives to conventional toxic solvents.

A key gene, violaxanthin de-epoxidase-like 1, enhances fucoxanthin accumulation in Phaeodactylum tricornutum.[Pubmed:38566219]

Biotechnol Biofuels Bioprod. 2024 Apr 2;17(1):49.

BACKGROUND: Fucoxanthin has been widely investigated owing to its beneficial biological properties, and the model diatom Phaeodactylum tricornutum, possessing fucoxanthin (Fux) chlorophyll proteins as light-harvesting systems, is considered to have the potential to become a commercial cell factory for the pigment production. RESULTS: Here, we compared the pigment contents in 10 different P. tricornutum strains from the globe, and found that strain CCMP631 (Pt6) exhibited the highest Fux content but with a low biomass. Comparison of mRNA levels revealed that higher Fux content in Pt6 was related with the higher expression of gene Violaxanthin de-epoxidase-like (VDL) protein 1 (VDL1), which encodes the enzyme catalyzing the tautomerization of Violaxanthin to neoxanthin in Fux biosynthesis pathway. Single nucleotide variants of VDL1 gene and allele-specific expression in strains Pt1 (the whole genome sequenced strain CCMP632) and Pt6 were analyzed, and overexpressing of each of the 4 VDL1 alleles, two from Pt1 and two from Pt6, in strain Pt1 leads to an increase in downstream product diadinoxanthin and channels the pigments towards Fux biosynthesis. All the 8 VDL1 overexpression (OE) lines showed significant increases by 8.2 to 41.7% in Fux content without compromising growth, and VDL1 Allele 2 OE lines even exhibited the higher cell density on day 8, with an increase by 24.2-28.7% in two Pt1VDL1-allele 2 OE lines and 7.1-11.1% in two Pt6VDL1-allele 2 OE lines, respectively. CONCLUSIONS: The results reveal VDL1, localized in the plastid stroma, plays a key role in Fux over-accumulation in P. tricornutum. Overexpressing VDL1, especially allele 2, improved both the Fux content and growth rate, which provides a new strategy for the manipulation of Fux production in the future.

Developing carotenoids-enhanced tomato fruit with multi-transgene stacking strategies.[Pubmed:38554536]

Plant Physiol Biochem. 2024 Mar 28;210:108575.

As natural dominant pigments, carotenoids and their derivatives not only contribute to fruit color and flavor quality but are regarded as phytochemicals beneficial to human health because of various bioactivities. Tomato is one of the most important vegetables as well as a main dietary source of carotenoids. So, it's of great importance to generate carotenoid-biofortified tomatoes. The carotenoid biosynthesis pathway is a network co-regulated by multiple enzymes and regulatory genes. Here, we assembled four binary constructs containing different combinations of four endogenous carotenoids metabolic-related genes, including SlOR(His), SlDXS, SlPSY, and SlBHY by using a high efficiency multi-transgene stacking system and a series of fruit-specific promotors. Transgenic lines overexpression SlOR(His) alone, three genes (SlOR(His)/SlDXS/SlPSY), two genes (SlOR(His)/SlBHY), and all these four genes (SlOR(His)/SlDXS/SlPSY/SlBHY) were enriched with carotenoids to varying degrees. Notably, overexpressing SlOR(His) alone showed comparable effects with simultaneous overexpression of the key regulatory enzyme coding genes SlDXS, SlPSY, and SlOR(His) in promoting carotenoid accumulation. Downstream carotenoid derivatives zeaxanthin and Violaxanthin were detected only in lines containing SlBHY. In addition, the sugar content and total antioxidant capacity of these carotenoids-enhanced tomatoes was also increased. These data provided useful information for the future developing of biofortified tomatoes with different carotenoid profiles, and confirmed a promising system for generation of nutrients biofortified tomatoes by multiple engineering genes stacking strategy.

Valorization of Nannochloropsis oceanica for integrated co-production of violaxanthin cycle carotenoids.[Pubmed:38493940]

Bioresour Technol. 2024 May;399:130597.

The development of integrated co-production of multiple high-purity carotenoids from microalgal cells holds considerable significance for the valorization of microalgae. In this study, the economical microalga Nannochloropsis oceanica was identified as an accumulator of Violaxanthin cycle carotenoids, including Violaxanthin, antheraxanthin, and zeaxanthin. Notably, a novel and competent approach for the integrated co-production of Violaxanthin cycle carotenoids was explored, encompassing four steps: microalgal cultivation, solvent extraction, octadecylsilyl open-column chromatography, and ethanol precipitation. Under optimal co-production conditions, the purities of the obtained Violaxanthin, antheraxanthin, and zeaxanthin all exceeded 92%, with total recovery rates of approximately 51%, 40%, and 60%, respectively. Utilizing nuclear magnetic resonance techniques, the purified Violaxanthin, antheraxanthin, and zeaxanthin were identified as all-trans-Violaxanthin, all-trans-antheraxanthin, and all-trans-zeaxanthin, respectively. This method held significance for the multiproduct biorefinery of the microalga N. oceanica and carried potential future implications for the Violaxanthin cycle carotenoids.

Dynamics and interplay of photosynthetic regulatory processes depend on the amplitudes of oscillating light.[Pubmed:38482712]

Plant Cell Environ. 2024 Jun;47(6):2240-2257.

Plants have evolved multiple regulatory mechanisms to cope with natural light fluctuations. The interplay between these mechanisms leads presumably to the resilience of plants in diverse light patterns. We investigated the energy-dependent nonphotochemical quenching (qE) and cyclic electron transports (CET) in light that oscillated with a 60-s period with three different amplitudes. The photosystem I (PSI) and photosystem II (PSII) function-related quantum yields and redox changes of plastocyanin and ferredoxin were measured in Arabidopsis thaliana wild types and mutants with partial defects in qE or CET. The decrease in quantum yield of qE due to the lack of either PsbS- or Violaxanthin de-epoxidase was compensated by an increase in the quantum yield of the constitutive nonphotochemical quenching. The mutant lacking NAD(P)H dehydrogenase (NDH)-like-dependent CET had a transient significant PSI acceptor side limitation during the light rising phase under high amplitude of light oscillations. The mutant lacking PGR5/PGRL1-CET restricted electron flows and failed to induce effective photosynthesis control, regardless of oscillation amplitudes. This suggests that PGR5/PGRL1-CET is important for the regulation of PSI function in various amplitudes of light oscillation, while NDH-like-CET acts' as a safety valve under fluctuating light with high amplitude. The results also bespeak interplays among multiple photosynthetic regulatory mechanisms.

Integrated Metabolomic and Transcriptomic Profiles Provide Insights into the Mechanisms of Anthocyanin and Carotenoid Biosynthesis in Petals of Medicago sativa ssp. sativa and Medicago sativa ssp. falcata.[Pubmed:38475545]

Plants (Basel). 2024 Feb 29;13(5):700.

The petals of Medicago sativa ssp. sativa and M. sativa ssp. falcata are purple and yellow, respectively. Free hybridization between M. sativa ssp. sativa and M. sativa ssp. falcata has created hybrids with various flower colors in nature. Moreover, the flower colors of alfalfa are closely correlated with yield, nutritional quality, stress tolerance and other agronomic characteristics. To elucidate the underlying mechanisms of flower color formation in M. sativa ssp. sativa and M. sativa ssp. falcata, we conducted an integrative analysis of the transcriptome and metabolome of alfalfa with three different petal colors (purple, yellow and cream). The metabolic profiles suggested that anthocyanins and carotenoids are the crucial pigments in purple and yellow flowers, respectively. A quantitative exploration of the anthocyanin and carotenoid components indicated that the accumulations of cyanidin, delphinidin, peonidin, malvidin, pelargonidin and petunidin derivatives are significantly higher in purple flowers than in cream flowers. In addition, the content of carotenes (phytoene, alpha-carotene and beta-carotene) and xanthophylls (alpha-cryptoxanthin, lutein, beta-cryptoxanthin, zeaxanthin, antheraxanthin and Violaxanthin derivatives) was markedly higher in yellow flowers than in cream flowers. Furthermore, we found that delphinidin-3,5-O-diglucoside and lutein were the predominant pigments accumulated in purple and yellow flowers, respectively. The transcriptomic results revealed that twenty-five upregulated structural genes (one C4H, three 4CL, twelve CHS, two CHI, one F3H, one F3'H, one F3'5'H and four DFR) are involved in the accumulation of anthocyanins in purple flowers, and nine structural genes (two PSY, one ZDS, two CRTISO, two BCH, one ZEP and one ECH) exert an effect on the carotenoid biosynthesis pathway in yellow flowers. The findings of this study reveal the underlying mechanisms of anthocyanin and carotenoid biosynthesis in alfalfa with three classic flower colors.

Glyphosate hormesis stimulates tomato (Solanum lycopersicum L.) plant growth and enhances tolerance against environmental abiotic stress by triggering nonphotochemical quenching.[Pubmed:38456569]

Pest Manag Sci. 2024 Mar 8.

BACKGROUND: Glyphosate is the most widely applied herbicide in the world. Hormesis caused by low glyphosate doses has been widely documented in many plant species. However, the specific adaptative mechanism of plants responding to glyphosate hormesis stimulation remains unclear. This study focused on the biphasic relationship between glyphosate dose and tomato plant growth, and how glyphosate hormesis stimulates plant growth and enhances tolerance to environmental stress. RESULTS: We constructed a hormesis model to describe the biphasic relationship with a maximal stimulation (MAX) of 162% above control by glyphosate at 0.063 g ha(-1) . Low-dose glyphosate increased photosynthetic pigment contents and improve photosynthetic efficiency, leading to plant growth stimulation. We also found that glyphosate hormesis enhanced plant tolerance to diuron (DCMU; a representative photosynthesis inhibitor) by triggering the nonphotochemical chlorophyll fluorescence quenching (NPQ) reaction to dissipate excess energy stress from photosystem II (PSII). Transcriptomic analysis and quantitative real-time polymerase chain reaction results revealed that the photosynthesis-antenna proteins pathway was the most sensitive to glyphosate hormesis, and PsbS (encoding photosystem II subunit S), ZEP (encoding zeaxanthin epoxidase) and VDE (encoding Violaxanthin de-epoxidase) involved in NPQ played crucial roles in the plant response to glyphosate hormesis. CONCLUSION: These results provide novel insights into the mechanisms of plant hormesis and is meaningful to the application of glyphosate hormesis in agriculture. (c) 2024 Society of Chemical Industry.

Combined high light and salt stress enhances accumulation of PsbS and zeaxanthin in Chlamydomonas reinhardtii.[Pubmed:38433102]

Physiol Plant. 2024 Mar-Apr;176(2):e14233.

The performance and acclimation strategies of Chlamydomonas reinhardtii under stress conditions are typically studied in response to single stress factors. Under natural conditions, however, organisms rarely face only one stressor at a time. Here, we investigated the impact of combined salt and high light stress on the photoprotective response of C. reinhardtii. Compared to the single stress factors, the combination of both stressors decreased the photosynthetic performance, while the activation of energy dissipation remained unaffected. However, the PsbS protein was strongly accumulated and the conversion of Violaxanthin to zeaxanthin was enhanced. These results support an important photoprotective function of PsbS and zeaxanthin independently of energy dissipation under combined salt and high light stress in C. reinhardtii.

Tropical bloom-forming mesoalgae Cladophoropsis sp. and Laurencia sp.-responses to ammonium enrichment and a simulated heatwave.[Pubmed:38402562]

J Phycol. 2024 Apr;60(2):554-573.

Algal blooms are increasing worldwide, driven by elevated nutrient inputs. However, it is still unknown how tropical benthic algae will respond to heatwaves, which are expected to be more frequent under global warming. In the present study, a multifactorial experiment was carried out to investigate the potential synergistic effects of increased ammonium inputs (25 muM, control at 2.5 muM) and a heatwave (31 degrees C, control at 25 degrees C) on the growth and physiology (e.g., ammonium uptake, nutrient assimilation, photosynthetic performance, and pigment concentrations) of two bloom-forming algal species, Cladophoropsis sp. and Laurencia sp. Both algae positively responded to elevated ammonium concentrations with higher growth and chlorophyll a and lutein concentrations. Increased temperature was generally a less important driver, interacting with elevated ammonium by decreasing the algaes' %N content and N:P ratios. Interestingly, this stress response was not captured by the photosynthetic yield (Fv/Fm) nor by the carbon assimilation (%C), which increased for both algae at higher temperatures. The negative effects of higher temperature were, however, buffered by nutrient inputs, showing an antagonistic response in the combined treatment for the concentration of VAZ (Violaxanthin, antheraxanthin, zeaxanthin) and thalli growth. Ammonium uptake was initially higher for Cladophoropsis sp. and increased for Laurencia sp. over experimental time, showing an acclimation capacity even in a short time interval. This experiment shows that both algae benefited from increased ammonium pulses and were able to overcome the otherwise detrimental stress of increasingly emerging temperature anomalies, which provide them a strong competitive advantage and might support their further expansions in tropical marine systems.

Impact of sulfite use and acidification on chemical quality components in thermally processed watermelon juices.[Pubmed:38395581]

Food Res Int. 2024 Mar;180:114088.

The present study compared pasteurized and reconstituted (from vacuum-concentrated) watermelon juices with sulfite use ( approximately 40 mg/L) and acidification (pH = 4.2) to fresh watermelon juices. The products were evaluated for phenolics, free amino acids, carotenoids, sugars, organic acids, and alcohols by high-performance liquid chromatography-HPLC and the volatile profile by headspace-gas chromatography/mass spectrometry(HS-GC/MS). Pasteurization had no significant impact on most of the chemical components. Furthermore, it potentiated typical watermelon aromas (E,E)-2,6-nonadienal, (Z)-3-nonen-1-ol, 4-hexen-1-ol, (E,Z)-3,6-nonadien-1-ol, 6-amino-2-methyl-2-heptanol, (E)-6-nonenal, (E)-2-nonenal, pentanal, nonanal and 1-nonanol), without off-flavor compounds formation. On the other hand, the reconstituted juice showed reduced amino acids (serine, glutamine, and tryptophan), phenolics (epicatechin gallate, myricetin, and cis-resveratrol), carotenoids (lycopene, beta-carotene, and Violaxanthin) and most volatile compounds. Our results showed that sulfite and acidification could maintain watermelon juice's nutritional and quality parameters after pasteurization. The vacuum concentration and reconstitution processes negatively impacted the evaluated compounds. Our findings contribute to improving thermal processes in watermelon juices for better preservation of nutrients, flavor, and bioactive compounds.

Evaluation of the Content of Minerals, B-Group Vitamins, Tocols, and Carotenoids in Raw and In-House Cooked Wild Edible Plants.[Pubmed:38338607]

Foods. 2024 Feb 2;13(3):472.

Notwithstanding the increased interest in wild edible plants, little is known on how some domestic thermal processes can affect their content. The aim of this study was to investigate the amounts of minerals, B1 and B2 vitamins, tocols, and carotenoids in raw, boiled, and steamed wild edible plants, namely, Sonchus asper (L.) Hill s.l., Sonchus oleraceus L., Cichorium intybus L., and Beta vulgaris L. var cicla. All vegetables were confirmed as high sources of lutein (from 6 to 9 mg/100 g) and beta-carotene (from 2 to 5 mg/100 g). Quite high amounts of Violaxanthin and neoxanthin were found. Alfa-tocopherol and gamma-tocopherol were the main tocols, with same contents in raw and processed vegetables (about 2.5 mg/100 g). The most abundant macro element and trace element were, respectively, potassium and iron. B1 and B2 vitamins were found in low amounts in almost all plants, with the exception of thiamine in Beta vulgaris (about 1.6 mg/100 g). Boiling led to a significant loss of minerals (up to 60%) and B-group vitamins (up to 100%), while, among carotenoids, it only affected Violaxanthin levels (up to 90%). Steamed vegetables showed only a slight reduction, about 20%, in beta-carotene and lutein, with a marked decrease in Violaxanthin and neoxanthin. One hundred grams of all fresh and cooked plants can be claimed as a source of vitamin A and E.