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Specific Upregulation of a Cotton Phytoene Synthase Gene Produces Golden Cottonseeds with Enhanced Provitamin A.

ABSTRACT: Provitamin A (PVA) bio-fortification of crops offers a sustainable strategy to prevent the prevalence of vitamin A deficiency (VAD), one of the world's major public health problems. The present work aimed to enhance PVA accumulation in cottonseed, the main by-product in the production of cotton fibers and the third largest source of edible plant oil in the world. On the basis of comprehensive identification of carotenoid synthase genes and their expression levels in various cotton tissues, we selected phytoene synthase as the target for manipulating carotenoid biosynthesis in the developing cottonseeds. After functional verification in transgenic tobacco, a cotton phytoene synthase gene (GhPSY2D) driven by a seed-specific promoter was transformed into cotton. The transgenic cottonseeds showed golden appearance and contained over 6-fold higher carotenoid contents in the extracted oil than the non-transgenic control. Thin layer chromatograph analysis indicated that the main PVA carotenoid ?-carotene was predominant in the transgenic cottonseeds, but undetectable in the wild-type control. By simultaneously providing economically valuable fibers and edible oils, the transgenic cottons bio-fortified with ?-carotene in seeds may be a new powerful tool against VAD in low-income regions.

SUBMITTER: Yao D

PROVIDER: S-EPMC5778082 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Specific Upregulation of a Cotton Phytoene Synthase Gene Produces Golden Cottonseeds with Enhanced Provitamin A.

Yao Dan D Wang Yi Y Li Qian Q Ouyang Xufen X Li Yaohua Y Wang Chuannan C Ding Lingli L Hou Lei L Luo Ming M Xiao Yuehua Y

Scientific reports 20180122 1

Provitamin A (PVA) bio-fortification of crops offers a sustainable strategy to prevent the prevalence of vitamin A deficiency (VAD), one of the world's major public health problems. The present work aimed to enhance PVA accumulation in cottonseed, the main by-product in the production of cotton fibers and the third largest source of edible plant oil in the world. On the basis of comprehensive identification of carotenoid synthase genes and their expression levels in various cotton tissues, we se ...[more]

PMID: 29358599

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Project description:BackgroundCarotenoids play important roles in photosynthesis, hormone signaling, and secondary metabolism. Phytoene synthase (PSY) catalyzes the first step of the carotenoid biosynthetic pathway. In this study, we aimed to characterize the PSY genes in tobacco and analyze their function.ResultsIn this study, we identified three groups of PSY genes, namely PSY1, PSY2, and PSY3, in four Nicotiana species; phylogenetic analysis indicated that these genes shared a high similarity with those in tomato but not with those in monocots such as rice and maize. The expression levels of PSY1 and PSY2 were observed to be highest in leaves compared to other tissues, and they could be elevated by treatment with certain phytohormones and exposure to strong light. No PSY3 expression was detected under these conditions. We constructed virus-induced PSY1 and PSY2 silencing in tobacco and found that the newly emerged leaves in these plants were characterized by severe bleaching and markedly decreased carotenoid and chlorophyll content. Thylakoid membrane protein complex levels in the gene-silenced plants were also less than those in the control plants. The chlorophyll fluorescence parameters such as Fv/Fm, ΦPSII, qP, and NPQ, which reflect photosynthetic system activities, of the gene-silenced plants were also significantly decreased. We further performed RNA-Seq and metabonomics analysis between gene-silenced tobacco and control plants. RNA-Seq results showed that abiotic stress, isoprenoid compounds, and amino acid catabolic processes were upregulated, whereas the biosynthesis of cell wall components was downregulated. Metabolic analysis results were consistent with the RNA-Seq. We also found the downstream genes in carotenoid biosynthesis pathways were upregulated, and putative transcription factors that regulate carotenoid biosynthesis were identified.ConclusionsOur results suggest that PSY can regulate carotenoid contents not only by controlling the first biosynthesis step but also by exerting effects on the expression of downstream genes, which would thereby affect photosynthetic activity. Meanwhile, PSY may affect other processes such as amino acid catabolism and cell wall organization. The information we report here may aid further research on PSY genes and carotenoid biosynthesis.

Project description:Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.

Project description:BACKGROUND:Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains ?-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. RESULTS:To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 ?mol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 ?mol m(-2) s(-1)). When grown under continuous light at 920 ?mol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. CONCLUSIONS:We identified genes encoding CrtE and CrtB in E. gracilis and found that their protein products catalyze the early steps of carotenoid biosynthesis. Further, we found that the response of the carotenoid biosynthetic pathway to light stress in E. gracilis is controlled, at least in part, by the level of crtB transcription. This is the first functional analysis of crtE and crtB in Euglena.

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Specific Upregulation of a Cotton Phytoene Synthase Gene Produces Golden Cottonseeds with Enhanced Provitamin A.

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Specific Upregulation of a Cotton Phytoene Synthase Gene Produces Golden Cottonseeds with Enhanced Provitamin A.

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