Defective mutations in STAY-GREEN 1, PHYTOENE SYNTHASE 1, and MYB12 genes lead to formation of green ripe fruit in tomato.

Modern tomatoes produce colorful mature fruits, but many wild tomato ancestors form green or gray green ripe fruits. Here, tomato cultivar 'Lvbaoshi' (LBS) that produces green ripe fruits was found to contain three recessive loci responsible for fruit development. The colorless peel of LBS fruits was caused by a 603 bp deletion in the promoter of SlMYB12. The candidate genes of the remaining two loci were identified as STAY-GREEN 1 (SlSGR1) and PHYTOENE SYNTHASE 1 (SlPSY1). SGR1 and PSY1 co-suppression by RNAi converted the pink fruits into green ripe fruits in transgenic plants. An amino acid change in PSY1 and a deletion in the promoter of SGR1 were also identified in several wild tomatoes bearing green or gray ripe fruits. Overexpression of PSY1 from green ripe fruit wild tomatoes in LBS plants could only partially rescue the green ripe fruit phenotype of LBS, and transgenic lines expressing ProSGR1::SGR1 from Solanum pennellii also failed to convert purple-flesh into red-flesh fruits. This work uncovers a novel regulatory mechanism by which SlMYB12, SlPSY1, and SlSGR1 control fruit color in cultivated and some wild tomato species.

Potato Solanum tuberosum L. Phytoene Synthase Genes (StPSY1, StPSY2, and StPSY3) Are Involved in the Plant Response to Cold Stress.

The structure and phylogeny of the Solanum tuberosum L. phytoene synthase genes StPSY1, StPSY2, and StPSY3 were characterized. Their expression was studied in potato seedlings exposed to cold stress in the dark phase of the diurnal cycle to simulate night cooling. All of the three genes were activated as the temperature decreased, and the greatest response was observed for StPSY1. StPSY3 was for the first time shown to respond to cold stress and photoperiod. A search for cis-regulatory elements was carried out in the promoter regions and 5'-UTRs of the StPSY genes, and the regulation of all three genes proved associated with the response to light. A high level of cold-induced activation of StPSY1 was tentatively attributed to the presence of cis elements associated with sensitivity to cold and ABA.

Nudix hydrolase 23 post-translationally regulates carotenoid biosynthesis in plants.

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

Reducing PHYTOENE SYNTHASE activity fine-tunes the abundance of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis.

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

Overlapping and specialized roles of tomato phytoene synthases in carotenoid and abscisic acid production.

Carotenoids are plastidial isoprenoids required for photoprotection and phytohormone production in all plants. In tomato (Solanum lycopersicum), carotenoids also provide color to flowers and ripe fruit. Phytoene synthase (PSY) catalyzes the first and main flux-controlling step of the carotenoid pathway. Three genes encoding PSY isoforms are present in tomato, PSY1 to PSY3. Mutants have shown that PSY1 is the isoform providing carotenoids for fruit pigmentation, but it is dispensable in photosynthetic tissues. No mutants are available for PSY2 or PSY3, but their expression profiles suggest a main role for PSY2 in leaves and PSY3 in roots. To further investigate isoform specialization with genetic tools, we created gene-edited lines defective in PSY1 and PSY2 in the MicroTom background. The albino phenotype of lines lacking both PSY1 and PSY2 confirmed that PSY3 does not contribute to carotenoid biosynthesis in shoot tissues. Our work further showed that carotenoid production in tomato shoots relies on both PSY1 and PSY2 but with different contributions in different tissues. PSY2 is the main isoform for carotenoid biosynthesis in leaf chloroplasts, but PSY1 is also important in response to high light. PSY2 also contributes to carotenoid production in flower petals and, to a lesser extent, fruit chromoplasts. Most interestingly, our results demonstrate that fruit growth is controlled by abscisic acid (ABA) specifically produced in the pericarp from PSY1-derived carotenoid precursors, whereas PSY2 is the main isoform associated with ABA synthesis in seeds and salt-stressed roots.

Tomato geranylgeranyl diphosphate synthase isoform 1 is involved in the stress-triggered production of diterpenes in leaves and strigolactones in roots.

Carotenoids are photoprotectant pigments and precursors of hormones such as strigolactones (SL). Carotenoids are produced in plastids from geranylgeranyl diphosphate (GGPP), which is diverted to the carotenoid pathway by phytoene synthase (PSY). In tomato (Solanum lycopersicum), three genes encode plastid-targeted GGPP synthases (SlG1 to SlG3) and three genes encode PSY isoforms (PSY1 to PSY3). Here, we investigated the function of SlG1 by generating loss-of-function lines and combining their metabolic and physiological phenotyping with gene co-expression and co-immunoprecipitation analyses. Leaves and fruits of slg1 lines showed a wild-type phenotype in terms of carotenoid accumulation, photosynthesis, and development under normal growth conditions. In response to bacterial infection, however, slg1 leaves produced lower levels of defensive GGPP-derived diterpenoids. In roots, SlG1 was co-expressed with PSY3 and other genes involved in SL production, and slg1 lines grown under phosphate starvation exuded less SLs. However, slg1 plants did not display the branched shoot phenotype observed in other SL-defective mutants. At the protein level, SlG1 physically interacted with the root-specific PSY3 isoform but not with PSY1 and PSY2. Our results confirm specific roles for SlG1 in producing GGPP for defensive diterpenoids in leaves and carotenoid-derived SLs (in combination with PSY3) in roots.

Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants.

Chlorophylls and carotenoids are essential photosynthetic pigments. Plants spatiotemporally coordinate the needs of chlorophylls and carotenoids for optimal photosynthesis and fitness in response to diverse environmental and developmental cues. However, how the biosynthesis pathways of these two pigments are coordinated, particularly at posttranslational level to allow rapid control, remains largely unknown. Here, we report that the highly conserved ORANGE (OR) family proteins coordinate both pathways via posttranslationally mediating the first committed enzyme in each pathway. We demonstrate that OR family proteins physically interact with magnesium chelatase subunit I (CHLI) in chlorophyll biosynthesis pathway in addition to phytoene synthase (PSY) in carotenoid biosynthesis pathway and concurrently stabilize CHLI and PSY enzymes. We show that loss of OR genes hinders both chlorophyll and carotenoid biosynthesis, limits light-harvesting complex assembly, and impairs thylakoid grana stacking in chloroplasts. Overexpression of OR safeguards photosynthetic pigment biosynthesis and enhances thermotolerance in both Arabidopsis and tomato plants. Our findings establish a novel mechanism by which plants coordinate chlorophyll and carotenoid biosynthesis and provide a potential genetic target to generate climate-resilient crops.

Genome-wide identification of the geranylgeranyl pyrophosphate synthase (GGPS) gene family involved in chlorophyll synthesis in cotton.

BACKGROUND: Geranylgeranyl pyrophosphate synthase (GGPS) is a structural enzyme of the terpene biosynthesis pathway that is involved in regulating plant photosynthesis, growth and development, but this gene family has not been systematically studied in cotton. RESULTS: In the current research, genome-wide identification was performed, and a total of 75 GGPS family members were found in four cotton species, Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii. The GGPS genes were divided into three subgroups by evolutionary analysis. Subcellular localization prediction showed that they were mainly located in chloroplasts and plastids. The closely related GGPS contains a similar gene structure and conserved motif, but some genes are quite different, resulting in functional differentiation. Chromosome location analysis, collinearity and selection pressure analysis showed that many fragment duplication events occurred in GGPS genes. Three-dimensional structure analysis and conservative sequence analysis showed that the members of the GGPS family contained a large number of α-helices and random crimps, and all contained two aspartic acid-rich domains, DDxxxxD and DDxxD (x is an arbitrary amino acid), suggesting its key role in function. Cis-regulatory element analysis showed that cotton GGPS may be involved in light response, abiotic stress and other processes. A GGPS gene was silenced successfully by virus-induced gene silencing (VIGS), and it was found that the chlorophyll content in cotton leaves decreased significantly, suggesting that the gene plays an important role in plant photosynthesis. CONCLUSIONS: In total, 75 genes were identified in four Gossypium species by a series of bioinformatics analysis. Gene silencing from GGPS members of G. hirsutum revealed that GGPS plays an important regulatory role in photosynthesis. This study provides a theoretical basis for the biological function of GGPS in cotton growth and development.

Identification and Functional Analysis of Two Novel Genes-Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)-from Aurantiochytrium limacinum Significantly Enhance De Novo ß-Carotene Biosynthesis in Escherichia coli.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo ß-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of ß-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in ß-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, ß-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

A Small Subunit of Geranylgeranyl Diphosphate Synthase Functions as an Active Regulator of Carotenoid Synthesis in Nicotiana tabacum.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis and photoprotectors for plants to adapt to high light stress. Here, we report a small subunit (SSU) of geranylgeranyl diphosphate synthase (GGPPS) in Nicotiana tabacum, NtSSU II, which takes part in the regulation carotenoid biosynthesis by forming multiple enzymatic components with NtGGPPS1 and downstream phytoene synthase (NtPSY1). NtSSU II transcript is widely distributed in various tissues and stimulated by low light and high light treatments. The confocal image revealed that NtSSU II was localized in the chloroplast. Bimolecular fluorescence complementation (BiFC) indicated that NtSSU II and NtGGPPS1 formed heterodimers, which were able to interact with phytoene synthase (NtPSY1) to channel GGPP into the carotenoid production. CRISPR/Cas9-induced ntssu II mutant exhibited decreased leaf area and biomass, along with a decline in carotenoid and chlorophyll accumulation. Moreover, the genes involved in carotenoid biosynthesis were also downregulated in transgenic plants of ntssu II mutant. Taken together, the newly identified NtSSU II could form multiple enzymatic components with NtGGPPS1 and NtPSY1 to regulate carotenoid biosynthesis in N. tabacum, in addition to the co-expression of genes in carotenoids biosynthetic pathways.

NtDREB-1BL1 Enhances Carotenoid Biosynthesis by Regulating Phytoene Synthase in Nicotiana tabacum.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis as well as photoprotectors for plants to adapt to high light stress. Phytoene synthase (PSY) is the entry enzyme and also the major rate-limiting enzyme in the carotenoid pathway. Here, we report a dehydration-responsive element-binding protein (DREB) transcription factor member in Nicotiana tabacum K326, NtDREB-1BL1, which regulates carotenoids biosynthesis by binding to the NtPSY promoter. The NtDREB-1BL1 transcript was widely distributed in leaves by Real-time PCR. Confocal image revealed that NtDREB-1BL1 was localized in the nucleus. The chromatin immunoprecipitation (ChIP) with the qPCR technique indicated that NtDREB-1BL1 could anchor the promoter region of NtPSY. Overexpression (NtDREB-1BL1 OE) and RNA interference (NtDREB-1BL1 RNAi) of NtDREB-1BL1 were performed to evaluate its biological function in N. tabacum. Both carotenoid and chlorophyll contents increased in transgenic plants of NtDREB-1BL1 OE compared with wild-type (WT) plants, with the augment of the genes involved in carotenoid biosynthesis. In contrast, the contents of carotenoid and chlorophyll significantly decreased in transgenic plants of NtDREB-1BL1 RNAi compared to WT, along with the decline in the expression of genes related to carotenoid biosynthesis. Moreover, transgenic plants of NtDREB-1BL1 OE exhibited enhanced tolerance under drought stress, with the weakened tolerance of drought stress in transgenic plants of NtDREB-1BL1 RNAi. In conclusion, our results illustrated the new role of transcription factor NtDREB-1BL1 in improving carotenoid biosynthesis through regulating NtPSY expression.

Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation.

Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

Nucleotide variation in the phytoene synthase (ClPsy1) gene contributes to golden flesh in watermelon (Citrullus lanatus L.).

KEY MESSAGE: A gene controlling golden flesh trait in watermelon was discovered and fine mapped to a 39.08 Kb region on chromosome 1 through a forward genetic strategy, and Cla97C01G008760 (annotated as phytoene synthase protein, ClPsy1 ) was recognized as the most likely candidate gene. Vitamin A deficiency is a worldwide public nutrition problem, and ß-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, which occurs due to an accumulated abundance of ß-carotene) is an important germplasm resource. In this study, a genetic analysis of segregated gf gene populations indicated that gf was controlled by a single recessive gene. BSA-seq (Bulked segregation analysis) and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population including over 1000 F2 plants narrowed this region to 39.08 Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for the gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines co-segregated with the gf trait only among individuals in the genetic population and were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs exhibited expression trends similar to that of ClPsy1 in the parental lines and may regulate the ClPsy1 expression. Further research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in watermelon ß-carotene accumulation.

Functional diversity of isoprenoid lipids in Methylobacterium extorquens PA1.

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1.

Isolation, expression, and functional analysis of the geranylgeranyl pyrophosphate synthase (GGPPS) gene from Liriodendron tulipifera.

Terpenoids are important secondary metabolites in plants and are involved in stress responses and pollinator attraction. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key synthase in the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway of terpenoid synthesis, catalyzing the synthesis of diterpenoids. Liriodendron tulipifera is a nectar plant in North America. Little is known about the key genes involved in the biosynthetic pathways of terpenoids, the precursors of most compounds related to nectar, fragrance and coloring in flowers in L. tulipifera. In this study, the LtuGGPPS2 gene and its promoter (LtuGGPPS2-pro) were cloned from L. tulipifera. The results of sequence alignment showed that the LtuGGPPS2 gene is highly homologous to GGPPS genes of other plants. Subcellular localization analysis showed that the LtuGGPPS2 protein localizes to chloroplasts, suggesting that the LtuGGPPS2 gene is probably related to carotenoid and chlorophyll synthesis. Based on tissue expression profiles revealed by RT-qPCR, the expression level of the LtuGGPPS2 gene was highest in petals. These results were consistent with the changes in volatile and nonvolatile terpenoids in the flowers of L. tulipifera. GUS staining to examine the LtuGGPPS2 promoter indicated that it is responsive to hormones. Overexpression of the LtuGGPPS2 gene increased the carotenoid content and GGPPS enzyme activity in Arabidopsis thaliana, indicating that LtuGGPPS2 is the key terpenoid synthase in the flowers of L. tulipifera. Our findings lay a foundation for further functional analysis of the LtuGGPPS2 gene and deeper investigation of the terpenoid biosynthetic pathway in L. tulipifera.

Comparative transcriptome analysis of unripe and ripe banana (cv. Nendran) unraveling genes involved in ripening and other related processes.

Banana is one of the most important fruit crops consumed globally owing to its high nutritional value. Previously, we demonstrated that the ripe pulp of the banana cultivar (cv.) Nendran (AAB) contained a high amount of pro-vitamin A carotenoids. However, the molecular factors involved in the ripening process in Nendran fruit are unexplored. Hence, we commenced a transcriptome study by using the Illumina HiSeq 2500 at two stages i.e. unripe and ripe fruit-pulp of Nendran. Overall, 3474 up and 4727 down-regulated genes were obtained. A large number of identified transcripts were related to genes involved in ripening, cell wall degradation and aroma formation. Gene ontology analysis highlighted differentially expressed genes that play a key role in various pathways. These pathways were mainly linked to cellular, molecular and biological processes. The present transcriptome study also reveals a crucial role of up-regulated carotenoid biosynthesis pathway genes namely, lycopene beta cyclase and geranylgeranyl pyrophosphate synthase at the ripening stage. Genes related to the ripening and other processes like aroma and flavor were highly expressed in the ripe pulp. Expression of numerous transcription factor family genes was also identified. This study lays a path towards understanding the ripening, carotenoid accumulation and other related processes in banana.

Chloroplast-to-chromoplast transition envisions provitamin A biofortification in green vegetables.

The carotenoids available in food are vital dietary micronutrients for human health. Plants synthesize and accumulate different carotenoids in plastids in a tissue-specific manner. The level of ß-carotene (provitamin A) and other nutritionally important carotenoids is substantially low in the green tissues such as leaves compared to the fruits and roots. In photosynthetic tissues, chloroplasts can accumulate a moderate level of carotenoids, mainly to facilitate photosynthesis and environmental stress tolerance. However, chromoplasts from the storage tissues such as tomato fruit and carrot root can synthesize and accumulate carotenoids to a substantially higher level. A synthetic biology approach that utilizes a transient expression of bacterial phytoene synthase (crtB) gene in the photosynthetic leaves can induce the transition of chloroplasts into chromoplasts. The plastid-localized heterologous expression of crtB in leaves can induce the overaccumulation of phytoene, triggering the chloroplast-to-chromoplast transition; therefore, enhancing the biosynthesis and accumulation of carotenoids, including provitamin A. The transition of chloroplasts into chromoplasts, however, altered the photosynthetic thylakoids, consequently reducing the photosynthetic efficiency and plant growth. An efficient metabolic engineering strategy is desirable to enhance the production of targeted carotenoids in leaves without perturbing the photosynthetic efficiency and plant growth. Collectively, a synthetic biology strategy that triggers the transformation of chloroplasts into chromoplasts in photosynthetic tissues unfolds new avenues for carotenoid biofortification in the leafy food and vegetable crops, which can increase the dietary intake of carotenoids, therefore, combating the crisis of vitamin A deficiency.

Integrated metabolic profiling and transcriptome analysis of pigment accumulation in Lonicera japonica flower petals during colour-transition.

BACKGROUND: Plants have remarkable diversity in petal colour through the biosynthesis and accumulation of various pigments. To better understand the mechanisms regulating petal pigmentation in Lonicera japonica, we used multiple approaches to investigate the changes in carotenoids, anthocyanins, endogenous hormones and gene expression dynamics during petal colour transitions, i.e., green bud petals (GB_Pe), white flower petals (WF_Pe) and yellow flower petals (YF_Pe). RESULTS: Metabolome analysis showed that YF_Pe contained a much higher content of carotenoids than GB_Pe and WF_Pe, with α-carotene, zeaxanthin, violaxanthin and γ-carotene identified as the major carotenoid compounds in YF_Pe. Comparative transcriptome analysis revealed that the key differentially expressed genes (DEGs) involved in carotenoid biosynthesis, such as phytoene synthase, phytoene desaturase and ζ-carotene desaturase, were significantly upregulated in YF_Pe. The results indicated that upregulated carotenoid concentrations and carotenoid biosynthesis-related genes predominantly promote colour transition. Meanwhile, two anthocyanins (pelargonidin and cyanidin) were significantly increased in YF_Pe, and the expression level of an anthocyanidin synthase gene was significantly upregulated, suggesting that anthocyanins may contribute to vivid yellow colour in YF_Pe. Furthermore, analyses of changes in indoleacetic acid, zeatin riboside, gibberellic acid, brassinosteroid (BR), methyl jasmonate and abscisic acid (ABA) levels indicated that colour transitions are regulated by endogenous hormones. The DEGs involved in the auxin, cytokinin, gibberellin, BR, jasmonic acid and ABA signalling pathways were enriched and associated with petal colour transitions. CONCLUSION: Our results provide global insight into the pigment accumulation and the regulatory mechanisms underlying petal colour transitions during the flower development process in L. japonica.

Characterization and functional analysis of phytoene synthase gene family in tobacco.

BACKGROUND: Carotenoids 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. RESULTS: In 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. CONCLUSIONS: Our 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.

Variability and Phylogeny of the Pepper Phytoene Synthase Paralogs PSY1 and PSY2 in Species of Various Capsicum Complexes.

Genes homologous to PSY1 and PSY2 that encode phytoene synthase isoforms in Capsicum species C. baccatum, C. chinense, C. frutescens, C. tovarii, C. eximium, and C. chacoense were identified. High conservatism of functionally significant sites of phytoene synthases of the analyzed accessions was revealed. It was found that only PSY1-based clustering of pepper species corresponds to the traditional Capsicum phylogeny; C. eximium was a part of the Purple corolla complex, and C. chacoense was equidistant from Annuum and Baccatum clades. The absence of significant differences between PSY1 and PSY2 of yellow-fruited C. chinense and red-fruited pepper accessions was shown. The yellow color of C. chinense fruit may be the result of both decreased PSY1 expression and increased PSY2 transcription. Thus, it was demonstrated that the acquired fruit pigmentation retains strict phylogenetic limitations, which, however, can be overcome using artificial selection for the activity of phytoene synthase PSY1.