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Physalis pubescens and Physalis alkekengi, members of the Physalis genus, are valued for their delicious and medicinal fruits as well as their different ripened fruit colors—golden for P. pubescens and scarlet for P. alkekengi. This study aimed to elucidate the pigment composition and genetic mechanisms during fruit maturation in these species. Fruit samples were collected at four development stages, analyzed using spectrophotometry and high-performance liquid chromatography (HPLC), and complemented with transcriptome sequencing to assess gene expression related to pigment biosynthesis. β-carotene was identified as the dominant pigment in P. pubescens, contrasting with P. alkekengi, which contained both lycopene and β-carotene. The carotenoid biosynthesis pathway was central to fruit pigmentation in both species. Key genes pf02G043370 and pf06G178980 in P. pubescens, and TRINITY_DN20150_c1_g3, TRINITY_DN10183_c0_g1, and TRINITY_DN23805_c0_g3 in P. alkekengi were associated with carotenoid production. Notably, the MYB-related and bHLH transcription factors (TFs) regulated zeta-carotene isomerase and β-hydroxylase activities in P. pubescens with the MYB-related TF showing dual regulatory roles. In P. alkekengi, six TF families—bHLH, HSF, WRKY, M-type MADS, AP2, and NAC—were implicated in controlling carotenoid synthesis enzymes. Our findings highlight the intricate regulatory network governing pigmentation and provide insights into Physalis germplasm's genetic improvement and conservation. [ABSTRACT FROM AUTHOR]

Key message: Approximately 119 MADS-box genes have been identified in durian. Moreover, DzAGL6-1 primarily expressed during fruit development, activates the DzPSY promoter. Transient expression of DzAGL6-1 in tomatoes influences carotenoid production. MADS-box transcription factors play a crucial role in regulating plant biological processes, including fruit ripening and associated events. This study aimed to comprehend the mechanisms involved in durian fruit development and ripening and carotenoid production by conducting a genome-wide analysis of MADS-box proteins in durian (Durio zibethinus L.), an economically important fruit in Southeast Asia. A total of 119 durian MADS-box proteins were identified from the genome of the ‘Musang King’ cultivar. Based on the phylogenetic analysis, the proteins were classified into types I and II, which exhibited similar conserved motif compositions. Notably, only 16 durian MADS-box genes exhibited fruit-specific expression patterns. Among these genes, DzAGL6-1 was predominantly expressed during fruit development, a stage at which carotenoid biosynthesis is activated. Transient expression of DzAGL6-1 in tomato fruit increased the transcript level of the carotenoid biosynthetic gene phytoene synthase (PSY) and the β-carotene content. Furthermore, DzAGL6-1 activated the promoter activity of DzPSY, as demonstrated by a dual-luciferase assay. These findings provide insights into the role of MADS-box transcription factors in regulating carotenoid biosynthesis during durian fruit development. [ABSTRACT FROM AUTHOR]

Key message: We identified seven OfNCEDs and analyzed the potential function of OfNCED4, which is involved in the biosynthesis of carotenoids and abscisic acid in Osmanthus fragrans. In plants, 9-cis-epoxycarotenoid dioxygenases (NCEDs) can cleave carotenoids and are rate-limiting enzymes for the biosynthesis of abscisic acid (ABA). Sweet osmanthus (Osmanthus fragrans Lour.), an important ornamental and fragrant tree, contains abundant carotenoids in its flowers. To examine the function of the 9-cis-epoxycarotenoid dioxygenases (NCEDs) involved in ABA biosynthesis and floral coloration in O. fragrans, the sequences of seven OfNCEDs were isolated from the genomic DNA and cDNA of O. fragrans. A bioinformatics analysis showed that these OfNCEDs encode 422 ~ 591 amino acids, and they all contain the RPE65 domain. These OfNCEDs showed different patterns of expression, and that of OfNCED4 was most consistent to the patterns of ABA accumulation during the flowering process. Moreover, the levels of expression of seven OfNCEDs in the O. fragrans flowers were significantly induced by exogenous ABA. ABA-responsive elements were found in the promoters of OfNCEDs. The overexpression of OfNCED4 resulted in increased contents of chlorophylls, carotenoids and ABA, and up-regulation of NtCRTISO, NtLCYE, NtLCYB, and NtNXS, and down-regulation of NtCCD1 and NtCCD4 in tobacco leaves. It suggests that OfNCED4 probably mediates the biosynthesis of ABA. Taken together, this study systematically identified the bioinformatics, pattern of expression of OfNCED genes and the potential function of OfNCED4, which could provide molecular evidence for further research on the regulation of carotenoid biosynthesis and floral color of O. fragrans. [ABSTRACT FROM AUTHOR]

Carotenoids are widely distributed and structurally diverse, which have significant roles in the photosynthesis of plants. As a precursor of vitamin A, carotenoids are also antioxidants that reduce various chronic diseases, which are beneficial for human health. Currently, the existing studies concerned the biological roles of APETALA2 (AP2)/ethylene-responsive factor (ERF) genes originated from higher plants. The AP2 superfamily of the transcriptional regulator was identified in higher plants, which was related to growth, development, carotenoid metabolism, and responses to various stresses. However, the regulatory mechanisms of the AP2-modulating carotenoid metabolism have not been reported in microalgae, which remain to be elucidated. Dunaliella parva AP2 (i.e., DpAP2), an important transcription factor, promotes carotenoid accumulation by binding to the promoter of target gene. Here, we identified an important AP2/ERF transcription factor, DpAP2, which could promote carotenoid accumulation by binding to the promoter of target gene. To demonstrate the function of DpAP2, the interacting proteins were identified by the yeast two-hybrid system. The results showed that DpAP2 could interact with three proteins with different activities (DNAbinding transcription factor activity, protein kinase activity, and alpha-Dphosphohexomutase activity); these proteins may be associated with multiple biological processes. This paper laid a good foundation for a deep understanding of the regulatory mechanisms of DpAP2 and genetic engineering breeding in D. parva. [ABSTRACT FROM AUTHOR]

SUMMARY: Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)‐mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene‐responsive element‐binding factor‐associated amphiphilic repression (EAR)‐containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44‐MdTPR1‐mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44‐mediated carotenoid biosynthesis. MdMYB44 enhanced β‐branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co‐repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1‐MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation‐mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR‐harboring TF/TPL repressive complex plays a universal role in histone deacetylation‐mediated inhibition of gene expression in various plants. Significance Statement: MdMYB44 regulated carotenoid biosynthesis partially by interacting with co‐repressor TPR1 via EAR motif to inhibit MdCCD4 and MdCYP97A3 expression through histone deacetylation, providing evidence that the EAR‐harboring TF/TPL repressive complex has a universal role in histone deacetylation‐mediated gene expression inhibition in various plants. [ABSTRACT FROM AUTHOR]

Carotenoids are important nutrients for human health that must be obtained from plants since they cannot be biosynthesized by the human body. Dissecting the regulatory mechanism of carotenoid metabolism in plants represents the first step toward manipulating carotenoid contents in plants by molecular design breeding. In this study, we determined that SlAP2c, an APETALA2 (AP2) family member, acts as a transcriptional repressor to regulate carotenoid biosynthesis in tomato (Solanum lycopersicum). Knockout of SlAP2c in both the "MicroTom" and "Ailsa Craig" backgrounds resulted in greater lycopene accumulation, whereas overexpression of this gene led to orange‐ripe fruit with significantly lower lycopene contents than the wild type. We established that SlAP2c represses the expression of genes involved in lycopene biosynthesis by directly binding to the cis‐elements in their promoters. Moreover, SlAP2c relies on its EAR motif to recruit the co‐repressors TOPLESS (TPL)2/4 and forms a complex with histone deacetylase (had)1/3, thereby reducing the histone acetylation levels of lycopene biosynthesis genes. Furthermore, SlAP2a, a homolog of SlAP2c, acts upstream of SlAP2c and alleviates the SlAP2c‐induced repression of lycopene biosynthesis genes by inhibiting SlAP2c transcription during fruit ripening. Therefore, we identified a transcriptional cascade mediated by AP2 family members that regulates lycopene biosynthesis during fruit ripening in tomato, laying the foundation for the manipulation of carotenoid metabolism in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis—a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis. [ABSTRACT FROM AUTHOR]

Ilex × attenuata 'Sunny Foster' represents a yellow leaf mutant originating from I. × attenuata 'Foster#2', a popular ornamental woody cultivar. However, the molecular mechanisms underlying this leaf color mutation remain unclear. Using a comprehensive approach encompassing cytological, physiological, and transcriptomic methodologies, notable distinctions were discerned between the mutant specimen and its wild type. The mutant phenotype displayed aberrant chloroplast morphology, diminished chlorophyll content, heightened carotenoid/chlorophyll ratios, and a decelerated rate of plant development. Transcriptome analysis identified differentially expressed genes (DEGs) related to chlorophyll metabolism, carotenoid biosynthesis and photosynthesis. The up-regulation of CHLD and CHLI subunits leads to decreased magnesium chelatase activity, while the up-regulation of COX10 increases heme biosynthesis—both impair chlorophyll synthesis. Conversely, the down-regulation of HEMD hindered chlorophyll synthesis, and the up-regulation of SGR enhanced chlorophyll degradation, resulting in reduced chlorophyll content. Additionally, genes linked to carotenoid biosynthesis, flavonoid metabolism, and photosynthesis were significantly down-regulated. We also identified 311 putative differentially expressed transcription factors, including bHLHs and GLKs. These findings shed light on the molecular mechanisms underlying leaf color mutation in I. × attenuata 'Sunny Foster' and provide a substantial gene reservoir for enhancing leaf color through breeding techniques. [ABSTRACT FROM AUTHOR]

(1) Background: Oxygen has exerted a great effect in shaping the environment and driving biological diversity in Earth's history. Green lineage has evolved primary and secondary carotenoid biosynthetic systems to adapt to Earth's oxygenation, e.g., Haematococcus lacustris, which accumulates the highest amount of secondary astaxanthin under stresses. The two systems are controlled by lycopene ε-cyclase (LCYE) and β-cyclase (LCYB), which leave an important trace in Earth's oxygenation. (2) Objectives: This work intends to disclose the underlying molecular evolutionary mechanism of Earth's oxygenation in shaping green algal carotenogensis with a special focus on lycopene cyclases. (3) Methods: The two kinds of cyclases were analyzed by site-directed mutagenesis, phylogeny, divergence time and functional divergence. (4) Results: Green lineage LCYEs appeared at ~1.5 Ga after the first significant appearance and accumulation of atmospheric oxygen, the so-called Great Oxygenation Event (GOE), from which LCYBs diverged by gene duplication. Bacterial β-bicyclases evolved from β-monocyclase. Enhanced catalytic activity accompanied evolutionary transformation from ε-/β-monocyclase to β-bicyclase. Strong positive selection occurred in green lineage LCYEs after the GOE and in algal LCYBs during the second oxidation, the Neoproterozoic Oxygenation Event (NOE). Positively selected sites in the catalytic cavities of the enzymes controlled the mono-/bicyclase activity, respectively. Carotenoid profiling revealed that oxidative adaptation has been wildly preserved in evolution. (5) Conclusions: the functionalization of the two enzymes is a result of primary to secondary adaptations to Earth's oxygenation. [ABSTRACT FROM AUTHOR]

Physalis pubescens and Physalis alkekengi, members of the Physalis genus, are valued for their delicious and medicinal fruits as well as their different ripened fruit colors—golden for P. pubescens and scarlet for P. alkekengi. This study aimed to elucidate the pigment composition and genetic mechanisms during fruit maturation in these species. Fruit samples were collected at four development stages, analyzed using spectrophotometry and high-performance liquid chromatography (HPLC), and complemented with transcriptome sequencing to assess gene expression related to pigment biosynthesis. β-carotene was identified as the dominant pigment in P. pubescens, contrasting with P. alkekengi, which contained both lycopene and β-carotene. The carotenoid biosynthesis pathway was central to fruit pigmentation in both species. Key genes pf02G043370 and pf06G178980 in P. pubescens, and TRINITY_DN20150_c1_g3, TRINITY_DN10183_c0_g1, and TRINITY_DN23805_c0_g3 in P. alkekengi were associated with carotenoid production. Notably, the MYB-related and bHLH transcription factors (TFs) regulated zeta-carotene isomerase and β-hydroxylase activities in P. pubescens with the MYB-related TF showing dual regulatory roles. In P. alkekengi, six TF families—bHLH, HSF, WRKY, M-type MADS, AP2, and NAC—were implicated in controlling carotenoid synthesis enzymes. Our findings highlight the intricate regulatory network governing pigmentation and provide insights into Physalis germplasm’s genetic improvement and conservation.

The plant circadian system, as a result of adaptive evolution, is closely related to sensitivity to photoperiod. Zea mays L. originally belongs to the short-day species, while modern cultivated maize accessions are considered neutral with respect to photoperiod. The work analyzes the impact of changing the long-day regime to ultrashort days and long nights on the level of transcripts of key circadian rhythm (GIGZ1A, GIGZ1B), photosystem I (psaA), photosystem II (psbA), and carotenoid biosynthesis (ZmPSY1, ZmPSY2, ZmLCYE, ORANGE-GREEN) genes in the leaves of two mid-late ripening inbred lines of corn (L-5580-1 and L-5739), similar in morphophysiological characteristics. In the same leaves, the content of chlorophylls and carotenoids was determined. It was found that the studied lines differ significantly in the dynamics of the gene transcript level and pigment content in response to a change in photoperiod. GIGZ1a and GIGZ1b expression increases 1 h after the end of the ultrashort day in both lines, and is then characterized by an increase or decrease depending on the line. Expression of photosystem psaA and psbA genes differs between lines, both in the level and dynamics of the response to a change in photoperiod. Activity of carotenogenesis genes ZmPSY1, ZmPSY2, ZmLCYE, and ORANGE-GREEN increases in both lines when day changes to night (except ZmPSY2 in L‑5580-1) and decreases to varying degrees depending on the line towards the end of the night period. The content of pigments 1 h after changing the regime increases in L-5580-1 and does not change in L-5739, and it decreases in both lines by the end of the night. The obtained data indicate the preservation of the circadian rhythm in L-5580-1 and increased adaptability of L-5739 and can be used to search for donors of the trait of high adaptability to changes in photoperiod among maize accessions. [ABSTRACT FROM AUTHOR]

The loquat (Eriobotrya japonica L.) is a special evergreen tree, and its fruit is of high medical and health value as well as having stable market demand around the world. In recent years, research on the accumulation of nutrients in loquat fruit, such as carotenoids, flavonoids, and terpenoids, has become a hotspot. The SBP-box gene family encodes transcription factors involved in plant growth and development. However, there has been no report on the SBP-box gene family in the loquat genome and their functions in carotenoid biosynthesis and fruit ripening. In this study, we identified 28 EjSBP genes in the loquat genome, which were unevenly distributed on 12 chromosomes. We also systematically investigated the phylogenetic relationship, collinearity, gene structure, conserved motifs, and cis-elements of EjSBP proteins. Most EjSBP genes showed high expression in the root, stem, leaf, and inflorescence, while only five EjSBP genes were highly expressed in the fruit. Gene expression analysis revealed eight differentially expressed EjSBP genes between yellow- and white-fleshed fruits, suggesting that the EjSBP genes play important roles in loquat fruit development at the breaker stage. Notably, EjSBP01 and EjSBP19 exhibited completely opposite expression patterns between white- and yellow-fleshed fruits during fruit development, and showed a close relationship with SlCnr involved in carotenoid biosynthesis and fruit ripening, indicating that these two genes may participate in the synthesis and accumulation of carotenoids in loquat fruit. In summary, this study provides comprehensive information about the SBP-box gene family in the loquat, and identified two EjSBP genes as candidates involved in carotenoid synthesis and accumulation during loquat fruit development. [ABSTRACT FROM AUTHOR]

Abstract Early spring cold spells can lead to leaf chlorosis during the rice seedling greening process. However, the physiological and molecular mechanisms underlying the rice greening process under low-temperature conditions remain unknown. In this study, comparative transcriptome and morphophysiological analyses were performed to investigate the mechanisms mediating the responses of the Koshihikari (Kos) and Kasalath (Kas) rice cultivars to chilling stress. According to their growth-related traits, electrolyte leakage, and chlorophyll fluorescence parameters, Kos was more tolerant to low-temperature stress than Kas. Moreover, chloroplast morphology was more normal (e.g., oval) in Kos than in Kas at 17 °C. The comparative transcriptome analysis revealed 610 up-regulated differentially expressed genes that were common to all four comparisons. Furthermore, carotenoid biosynthesis was identified as a critical pathway for the Kos response to chilling stress. The genes in the carotenoid biosynthesis pathway were expressed at higher levels in Kos than in Kas at 17 °C, which was in accordance with the higher leaf carotenoid content in Kos than in Kas. The lycopene β-cyclase and lycopene ε-cyclase activities increased more in Kos than in Kas. Additionally, the increases in the violaxanthin de-epoxidase and carotenoid hydroxylase activities in Kos seedlings resulted in the accumulation of zeaxanthin and lutein and mitigated the effects of chilling stress on chloroplasts. These findings have clarified the molecular mechanisms underlying the chilling tolerance of rice seedlings during the greening process.

Early spring cold spells can lead to leaf chlorosis during the rice seedling greening process. However, the physiological and molecular mechanisms underlying the rice greening process under low-temperature conditions remain unknown. In this study, comparative transcriptome and morphophysiological analyses were performed to investigate the mechanisms mediating the responses of the Koshihikari (Kos) and Kasalath (Kas) rice cultivars to chilling stress. According to their growth-related traits, electrolyte leakage, and chlorophyll fluorescence parameters, Kos was more tolerant to low-temperature stress than Kas. Moreover, chloroplast morphology was more normal (e.g., oval) in Kos than in Kas at 17 °C. The comparative transcriptome analysis revealed 610 up-regulated differentially expressed genes that were common to all four comparisons. Furthermore, carotenoid biosynthesis was identified as a critical pathway for the Kos response to chilling stress. The genes in the carotenoid biosynthesis pathway were expressed at higher levels in Kos than in Kas at 17 °C, which was in accordance with the higher leaf carotenoid content in Kos than in Kas. The lycopene β-cyclase and lycopene ε-cyclase activities increased more in Kos than in Kas. Additionally, the increases in the violaxanthin de-epoxidase and carotenoid hydroxylase activities in Kos seedlings resulted in the accumulation of zeaxanthin and lutein and mitigated the effects of chilling stress on chloroplasts. These findings have clarified the molecular mechanisms underlying the chilling tolerance of rice seedlings during the greening process. [ABSTRACT FROM AUTHOR]

In this work, Raman spectroscopy and DFT calculations have been used to identify pigments biosynthesized during the ripening of tomatoes. In situ analysis of the exocarp from tomatoes at six stages of maturation showed Raman bands attributed mainly to carotenes and flavonoids. The Raman spectra of the exocarp in earlier stages (green and breaker) are rich in carotenoids with nine conjugated double bonds, while later stages (light‐red and red) are dominated by ones with 11 conjugated unsaturation. The Raman structural characterization of carotenoids biosynthesized from the phytoene, phytofluene, to β‐carotene is reported for the first time. Changes in carotenoid composition were observed along with the maturation; however, flavonoids/polyphenols that are concomitantly biosynthesized since the green stage presented the same spectral pattern along the process. Here, we present some clues based on Raman spectroscopy of the chemical synergism between carotenoids and flavonoids in coloring tomatoes. [ABSTRACT FROM AUTHOR]

The Chinese plum (Prunus salicina L.) is a fruit tree belonging to the Rosaceae family, native to south-eastern China and widely cultivated throughout the world. Fruit sugar metabolism and color change is an important physiological behavior that directly determines flavor and aroma. Our study analyzed six stages of fruit growth and development using RNA-seq, yielding a total of 14,973 DEGs, and further evaluation of key DEGs revealed a focus on sugar metabolism, flavonoid biosynthesis, carotenoid biosynthesis, and photosynthesis. Using GO and KEGG to enrich differential genes in the pathway, we selected 107 differential genes and obtained 49 significant differential genes related to glucose metabolism. The results of the correlation analyses indicated that two genes of the SWEET family, evm.TU.Chr1.3663 (PsSWEET9) and evm.TU.Chr4.676 (PsSWEET2), could be closely related to the composition of soluble sugars, which was also confirmed in the ethylene treatment experiments. In addition, analysis of the TOP 20 pathways between different growth stages and the green stage, as well as transient overexpression in chili, suggested that capsanthin/capsorubin synthase (PsCCS) of the carotenoid biosynthetic pathway contributed to the color change of plum fruit. These findings provide an insight into the molecular mechanisms involved in the ripening and color change of plum fruit. [ABSTRACT FROM AUTHOR]

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between 'HJH' with yellow flowers and 'ZLE' with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of 'HJH'. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in 'ZLE' than that in 'HJH'. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the 'HJH' petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color. [ABSTRACT FROM AUTHOR]

The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis—a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.

Ilex × attenuata ‘Sunny Foster’ represents a yellow leaf mutant originating from I. × attenuata ‘Foster#2’, a popular ornamental woody cultivar. However, the molecular mechanisms underlying this leaf color mutation remain unclear. Using a comprehensive approach encompassing cytological, physiological, and transcriptomic methodologies, notable distinctions were discerned between the mutant specimen and its wild type. The mutant phenotype displayed aberrant chloroplast morphology, diminished chlorophyll content, heightened carotenoid/chlorophyll ratios, and a decelerated rate of plant development. Transcriptome analysis identified differentially expressed genes (DEGs) related to chlorophyll metabolism, carotenoid biosynthesis and photosynthesis. The up-regulation of CHLD and CHLI subunits leads to decreased magnesium chelatase activity, while the up-regulation of COX10 increases heme biosynthesis—both impair chlorophyll synthesis. Conversely, the down-regulation of HEMD hindered chlorophyll synthesis, and the up-regulation of SGR enhanced chlorophyll degradation, resulting in reduced chlorophyll content. Additionally, genes linked to carotenoid biosynthesis, flavonoid metabolism, and photosynthesis were significantly down-regulated. We also identified 311 putative differentially expressed transcription factors, including bHLHs and GLKs. These findings shed light on the molecular mechanisms underlying leaf color mutation in I. × attenuata ‘Sunny Foster’ and provide a substantial gene reservoir for enhancing leaf color through breeding techniques.

(1) Background: Oxygen has exerted a great effect in shaping the environment and driving biological diversity in Earth’s history. Green lineage has evolved primary and secondary carotenoid biosynthetic systems to adapt to Earth’s oxygenation, e.g., Haematococcus lacustris, which accumulates the highest amount of secondary astaxanthin under stresses. The two systems are controlled by lycopene ε-cyclase (LCYE) and β-cyclase (LCYB), which leave an important trace in Earth’s oxygenation. (2) Objectives: This work intends to disclose the underlying molecular evolutionary mechanism of Earth’s oxygenation in shaping green algal carotenogensis with a special focus on lycopene cyclases. (3) Methods: The two kinds of cyclases were analyzed by site-directed mutagenesis, phylogeny, divergence time and functional divergence. (4) Results: Green lineage LCYEs appeared at ~1.5 Ga after the first significant appearance and accumulation of atmospheric oxygen, the so-called Great Oxygenation Event (GOE), from which LCYBs diverged by gene duplication. Bacterial β-bicyclases evolved from β-monocyclase. Enhanced catalytic activity accompanied evolutionary transformation from ε-/β-monocyclase to β-bicyclase. Strong positive selection occurred in green lineage LCYEs after the GOE and in algal LCYBs during the second oxidation, the Neoproterozoic Oxygenation Event (NOE). Positively selected sites in the catalytic cavities of the enzymes controlled the mono-/bicyclase activity, respectively. Carotenoid profiling revealed that oxidative adaptation has been wildly preserved in evolution. (5) Conclusions: the functionalization of the two enzymes is a result of primary to secondary adaptations to Earth’s oxygenation.

Carotenoids are secondary metabolites that are synthesized and stored in all types of plant plastids. These pigments play a significant role in protection against oxidative stress, as well as in the coloration of flowers and sink organs. The tubers of potato Solanum tuberosum L. synthesize carotenoids, including during post-harvest storage. In this study, we analyze the expression of carotenoid biogenesis pathway genes (PSY1, PSY2, PSY3, PDS, ZDS, Z-ISO, CRTISO, LCYB1, LCYB2, LCYE, VDE, ZEP, NSY, NCED1, NCED2, and NCED6), as well as genes putatively involved in the initiation of chromoplast differentiation (OR1 and OR2) in the dynamics of the long-term cold storage (September, February, April) of tubers of potato cultivars Barin, Utro, Krasavchik, Severnoe siyanie, and Nadezhda. It is shown that OR1 and OR2 mRNAs are present in tubers of all cultivars at all stages of storage. The expression profile of all analyzed carotenoid biosynthesis genes during tuber storage is characterized by a significant decrease in transcript levels in February compared to September, with some exceptions. During the period from February to April, the level of gene transcripts changes insignificantly. Biochemical analysis of the carotenoid content in the dynamics of cold storage shows that at the time of harvesting, the highest carotenoid content is in tubers of the cv. Utro; tubers of other cultivars are characterized by a similar amount of carotenoids. During storage from September to April, the total carotenoids changed in a genotype-dependent manner without any trend common to all cultivars. In general, the activity of most genes of the carotenoid metabolism pathway during the long-term cold storage of tubers is characterized for the first time, which significantly complements the known data on the expression response of these genes to abiotic stress. [ABSTRACT FROM AUTHOR]

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C 5 isopentenyl building blocks formed from mevalonic acid into PACs, including β-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and β-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial β-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops. This study demonstrates the functionality of the transiently and stably transformed, complete fungal, C 40 carotenoid biosynthetic pathway and its capability to produce provitamin A in the cytosol of different plant species and tissues. It shows the advantages of using the cytosol as an additional compartment for plant metabolic engineering of carotenes and reveals cytosolic lipid droplets as a novel sequestering sink for storing provitamin A outside of plastids. [ABSTRACT FROM AUTHOR]

Carotenoids are powerful antioxidants that mediate transfer of electrons, directly affect abiotic stress responses in plants through regulating activity of antioxidant enzymes. ζ-Carotene desaturase (ZDS) is a key enzyme in carotenoid biosynthesis pathway, which can catalyze ζ-carotene to form lycopene to regulate carotenoid biosynthesis and accumulation. However, the mechanism of its regulation of saline-alkali stress remains unclear. In this research, based on transcriptomic analysis of Malus halliana with a apple rootstock, we screened out ZDS gene (LOC103451012), with significantly high expression by saline-alkali stress, whose expression in the leaves was 10.8-fold than that of the control (0 h) under 48 h of stress. Subsequently, the MhZDS gene was isolated from M. halliana, and transgenic Arabidopsis thaliana, tobacco, and apple calli were successfully obtained through agrobacterium-mediated genetic transformation. We found that overexpression of MhZDS enhanced the tolerance of A. thaliana, tobacco and apple calli under saline-alkali stress and caused a variety of physiological and biochemical changes: compared with wild-type, transgenic plants grew better under saline stress and MhZDS-OE lines showed higher chlorophyll content, POD, SOD, CAT activities and proline content, lower electrical conductivity and MDA content. These results indicate that MhZDS plays an important role in plant resistance to saline-alkali stress, providing excellent resistance genes for the regulatory network of salinity stress response in apples and provide a theoretical basis for the breeding of apple varieties with strong saline-alkali resistance. [ABSTRACT FROM AUTHOR]

Alternative splicing refers to the process of producing different splicing isoforms from the same pre-mRNA through different alternative splicing events, which almost participates in all stages of plant growth and development. In order to understand its role in the fruit development of Osmanthus fragrans, transcriptome sequencing and alternative splicing analysis was carried out on three stages of O. fragrans fruit (O. fragrans "Zi Yingui"). The results showed that the proportion of skipping exon events was the highest in all three periods, followed by a retained intron, and the proportion of mutually exclusive exon events was the lowest and most of the alternative splicing events occurred in the first two periods. The results of enrichment analysis of differentially expressed genes and differentially expressed isoforms showed that alpha-Linolenic acid metabolism, flavonoid biosynthesis, carotenoid biosynthesis, photosynthesis, and photosynthetic-antenna protein pathways were significantly enriched, which may play an important role in the fruit development of O. fragrans. The results of this study lay the foundation for further study of the development and maturation of O. fragrans fruit and further ideas for controlling fruit color and improving fruit quality and appearance. [ABSTRACT FROM AUTHOR]

Carotenoids are isoprenoid compounds that play a role as pigments in photosynthetic and non-photosynthetic organisms. Carotenoids are essential in biological systems by acting as antioxidants. Thus, carotenoids have numerous applications in the environment, food, and medical sectors. Presently, most of the commercially available carotenoids are either extracted from different plants or produced synthetically using chemicals. The need to develop naturally derived pigments made it indispensable to discover alternate sources of carotenoids. Microbes have immense potential to produce carotenoids and thus, act as promising substitutes for chemically synthesized carotenoids. Microbial carotenoid production offers advantages, such as controlled cultivation and minimized production period. Moreover, the capability of microbes to utilize lignocellulosic waste as a substrate for the synthesis of carotenoids makes the overall process more sustainable. This review highlights the production of carotenoids from bacterial, fungal, and algal sources, followed by genetic modification of carotenogenic and non-carotenogenic species. Lastly, the review summarizes the extraction process and the applications of carotenoids in different fields. • Carotenoids are well-recognized for their antioxidant potential. • Microbial carotenoids are promising substitutes to synthetic carotenoids. • Lignocellulosic waste acts as cheap carbon source for carotenoid production. • Gene manipulation enhances the overall production process. [ABSTRACT FROM AUTHOR]

Tomato Solanum lycopersicum L. is an important agricultural crop and, at the same time, a model for studying the ontogeny of the succulent fruit. The decisive role in the ripening of the fruit is played by abscisic acid, which is formed as a result of the oxidative cleavage of epoxycarotenoids 9-cis-epoxycarotenoid dioxygenases NCED. Gene-expression profiles of SINCED1 and SINCED2 and the content of carotenoids in fruits at different stages of development were determined in three varieties of tomato with different color of ripe fruit. It was shown that transcripts of both genes are present in all organs. Transcript level of SINCED1 was approximately four to six times higher than the level of SINCED2 transcripts; peak activity of SINCED1 occurs in the late stages of ripening, while that of SINCED2 is at the initial stage. Ripe fruits are characterized by the highest amount of carotenoids; lycopene was found only in the fruits of late stages in red-fruited varieties, the highest content of β-carotene was found in ripe fruits of the yellow-fruited variety. The precursor of abscisic acid, violaxanthin, is present only in the immature fruit; the other precursor, neoxanthin, decreases with ripening and is absent at the ripeness stage. In red-fruited varieties, a correlation was found between the level of SINCED1 and SINCED2 transcripts with the content of β-carotene. Findings suggest the coparticipation of SINCED1 and SINCED2 in the biosynthesis of abscisic acid during the development and ripening of tomato fruit. In this case, the key role belongs to the gene SINCED1, the peak of activity of which falls on the stage of changing the color of the fruit. Lower levels of SINCED2 transcripts and its peak activity in the early stages of fruit development suggests a division of NCED functions between the two enzymes. [ABSTRACT FROM AUTHOR]

The color of the maize (Zea mays L.) grain is determined by the content and composition of carotenoids, including provitamin A, which is a product of the β-β (β-carotene, β-cryptoxanthin) and β-ɛ (α-carotene) branches of carotenogenesis. The ratio of the fluxes of the branches depends on the activity of the lycopene-ɛ-cyclase LcyE, which determines the β-ɛ branch. In this work, we analyzed allelic variants of the LcyE gene, which are potentially effective for increasing the biosynthesis of β-carotene, in 20 maize inbred lines of domestic selection, which differ in grain color. The 5'-UTR regions of the LcyE gene were amplified and sequenced. Fragment analysis showed the presence of allele 2 in four lines and the new allele 5 in 16 lines. The polymorphism of the new allele 5 was characterized—four single nucleotide polymorphisms and two deletions. The comparison of cis-regulatory elements in the analyzed 5'-UTR region in alleles 2 and 5 revealed a difference in binding sites with transcription factors. Expression of the LcyE gene was determined in the leaves of two lines with allele 2 and three lines with allele 5. A direct relationship was shown between the presence of allele 5 and a decrease in gene expression: the level of gene transcription in the case of allele 2 was 10–15 times higher than in the case of the allele 5. It has been suggested that the presence of allele 5 of the LcyE gene in the maize genome correlates with a decrease or suppression of the LcyE expression and, with stable activity of other carotenogenesis enzymes, with grain color. The use of allele 5 donors in combination with a known dark yellow or orange color of the grain can be used in the breeding of maize with increased synthesis of provitamin A in the grain. [ABSTRACT FROM AUTHOR]

The unique color and type characteristics of watermelon fruits are regulated by many molecular mechanisms. However, it still needs to be combined with more abundant genetic data to fine-tune the positioning. We assembled genomes of two Korean inbred watermelon lines (cv. 242-1 and 159-1) with unique color and fruit-type characteristics and identified 23,921 and 24,451 protein-coding genes in the two genomes, respectively. To obtain more precise results for further study, we resequenced one individual of each parental line and an F2 population composed of 87 individuals. This identified 1,539 single-nucleotide polymorphisms (SNPs) and 80 InDel markers that provided a high-density genetic linkage map with a total length of 3,036.9 cM. Quantitative trait locus mapping identified 15 QTLs for watermelon fruit quality-related traits, including b-carotene and lycopene content in fruit flesh, fruit shape index, skin thickness, flesh color, and rind color. By investigating the mapping intervals, we identified 33 candidate genes containing variants in the coding sequence. Among them, Cla97C01G008760 was annotated as a phytoene synthase with a singlenucleotide variant (A!G) in the first exon at 9,539,129 bp of chromosome 1 that resulted in the conversion of a lysine to glutamic acid, indicating that this gene might regulate flesh color changes at the protein level. These findings not only prove the importance of a phytoene synthase gene in pigmentation but also explain an important reason for the color change of watermelon flesh. [ABSTRACT FROM AUTHOR]

This document is a corrigendum for an article titled "Identification of interacting proteins of transcription factor DpAP2 related to carotenoid biosynthesis from marine microalga Dunaliella parva." The corrigendum addresses an error in the funding statement of the original article, where the grant number for the National Training Program of Innovation and Entrepreneurship for Undergraduates was incorrect. The correct grant number is provided, and the authors state that this error does not affect the scientific conclusions of the article. The corrigendum also includes the contact information for the corresponding author and the publication details of the original article. [Extracted from the article]

Microalgae are considered as ideal cell factories for producing natural carotenoids which display favorable biological activities. As the most important abiotic factor, light not only provides energy for photosynthetic metabolism, but also regulates numerous biological processes. Blue light is the main wavelength of light that can travel through water. Previous studies have shown that blue light triggered carotenoid accumulation in several microalgae species, but the molecular mechanism remains unclear. Cryptochromes were blue-light-absorbing photoreceptors that have been found in all studied algal genomes. In this study, several different types of cryptochrome genes were cloned from Haematococcus pluvialis and Phaeodactylum tricornutum. Among them, cryptochrome genes HpCRY4 from H. pluvialis and PtCPF1 from P. tricornutum were upregulated under blue light treatment, in correlation with the increase of astaxanthin and fucoxanthin contents. Besides, heterologous expression and gene knockout was performed to verify the function of HpCRY4 and PtCPF1 in regulating carotenoid biosynthesis in microalgae. These results indicate that carotenoid biosynthesis in microalgae promoted by blue light was mediated by cryptochromes as photoreceptors. [ABSTRACT FROM AUTHOR]

Glycyrrhiza uralensis Fisch. is often cultivated in arid, semi-arid, and salt-affected regions that suffer from drought stress, which leads to the accumulation of reactive oxygen species (ROS), thus causing oxidative stress. Plant growth-promoting bacteria (PGPB) and silicon (Si) have been widely reported to be beneficial in improving the tolerance of plants to drought stress by maintaining plant ROS homeostasis. Herein, combining physiological, transcriptomic, and metabolomic analyses, we investigated the response of the antioxidant system of G. uralensis seedlings under drought stress to Bacillus pumilus (G5) and/or Si treatment. The results showed that drought stress caused the overproduction of ROS, accompanied by the low efficiency of antioxidants [i.e., superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), the ascorbate (AsA)–glutathione (GSH) pool, total carotenoids, and total flavonoids]. Inversely, supplementation with G5 and/or Si enhanced the antioxidant defense system in drought-stressed G. uralensis seedlings, and the complex regulation of the combination of G5 and Si differed from that of G5 or Si alone. The combination of G5 and Si enhanced the antioxidant enzyme system, accelerated the AsA–GSH cycle, and triggered the carotenoid and flavonoid metabolism, which acted in combination via different pathways to eliminate the excess ROS induced by drought stress, thereby alleviating oxidative stress. These findings provide new insights into the comparative and synergistic roles of PGPB and Si in the antioxidant system of plants exposed to drought and a guide for the application of PGPB combined with Si to modulate the tolerance of plants to stress. [ABSTRACT FROM AUTHOR]

Abstract Background The genus Planococcus is comprised of halophilic bacteria generally reported for the production of carotenoid pigments and biosurfactants. In previous work, we showed that the culturing of the orange-pigmented Planococcus sp. CP5-4 isolate increased the evaporation rate of industrial wastewater brine effluent, which we attributed to the orange pigment. This demonstrated the potential application of this bacterium for industrial brine effluent management in evaporation ponds for inland desalination plants. Here we identified a C30-carotenoid biosynthetic gene cluster responsible for pigment biosynthesis in Planococcus sp. CP5-4 through isolation of mutants and genome sequencing. We further compare the core genes of the carotenoid biosynthetic gene clusters identified from different Planococcus species’ genomes which grouped into gene cluster families containing BGCs linked to different carotenoid product chemotypes. Lastly, LC–MS analysis of saponified and unsaponified pigment extracts obtained from cultures of Planococcus sp. CP5-4, revealed the structure of the main (predominant) glucosylated C30-carotenoid fatty acid ester produced by Planococcus sp. CP5-4. Results Genome sequence comparisons of isolated mutant strains of Planococcus sp. CP5-4 showed deletions of 146 Kb and 3 Kb for the non-pigmented and “yellow” mutants respectively. Eight candidate genes, likely responsible for C30-carotenoid biosynthesis, were identified on the wild-type genome region corresponding to the deleted segment in the non-pigmented mutant. Six of the eight candidate genes formed a biosynthetic gene cluster. A truncation of crtP was responsible for the “yellow” mutant phenotype. Genome annotation revealed that the genes encoded 4,4′-diapolycopene oxygenase (CrtNb), 4,4′- diapolycopen-4-al dehydrogenase (CrtNc), 4,4′-diapophytoene desaturase (CrtN), 4,4′- diaponeurosporene oxygenase (CrtP), glycerol acyltransferase (Agpat), family 2 glucosyl transferase 2 (Gtf2), phytoene/squalene synthase (CrtM), and cytochrome P450 hydroxylase enzymes. Carotenoid analysis showed that a glucosylated C30-carotenoid fatty acid ester, methyl 5-(6-C17:3)-glucosyl-5, 6′-dihydro-apo-4, 4′-lycopenoate was the main carotenoid compound produced by Planococcus sp. CP5-4. Conclusion We identified and characterized the carotenoid biosynthetic gene cluster and the C30-carotenoid compound produced by Planococcus sp. CP5-4. Mass-spectrometry guided analysis of the saponified and unsaponified pigment extracts showed that methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate esterified to heptadecatrienoic acid (C17:3). Furthermore, through phylogenetic analysis of the core carotenoid BGCs of Planococcus species we show that various C30-carotenoid product chemotypes, apart from methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate and 5-glucosyl-4, 4-diaponeurosporen-4′-ol-4-oic acid, may be produced that could offer opportunities for a variety of applications.

Carotenoids play a pivotal role in plant. Tagetes erecta , commonly called marigold, has increasing nutritional and economic value due to its high level of carotenoids in flower. However, the functional genes in the carotenoid biosynthesis of T. erecta have not been studied. In this work, three T. erecta varieties with flowers of yellow, yellow-orange and orange color, respectively, were examined for carotenoids composition and corresponding expression profiling of biosynthetic genes at four developmental stages. The results indicated that the varieties with higher lutein content, orange-flower 'Juwang' and yellow-orange 'Taishan', exhibited significant upregulation of genes in the upstream biosynthesis pathway, especially PDS (phytoene desaturase), PSY (phytoene synthase) and ZDS (zeta-carotene desaturase), whereas downstream carotenoid cleavage genes CCD (carotenoid cleavage dioxygenase) were markedly downregulated throughout flower development in the highest lutein containing variety 'Juwang'. Furthermore, marigold TePDS , TePSYS3 and TeZDS were isolated and transformed into tomato. Overexpression of TePDS or TeZDS resulted in the promotion of fruit ripening and accumulation of carotenoids in the transgenic lines. On the other hand, marigold TePSYS3 showed multiple effects, not only on fruit carotenogenesis but also on pigmentation patterns in vegetative tissues and plant growth. Taken together, the variations in expression profiles of the biosynthetic genes contribute to dynamic change in carotenoid levels and diversity of flower coloration in T. erecta. These functional genes of T. erecta were verified in tomato and provide targets for genetic improvement of fruit carotenoids accumulation. • Carotenoids composition indicate carotenogenesis variances among different color flowers of T. erecta. • Differentially expressed TePSYs , TePDS TeZDS and TeCCDs contribute to carotenoids accumulation in T. erecta. • TePDS and TeZDS improve fruit ripening and carotenoids accumulation in tomato. • TePSY3 effects pigmentation pattern in fruit and vegetative tissues. [ABSTRACT FROM AUTHOR]

The Chinese plum (Prunus salicina L.) is a fruit tree belonging to the Rosaceae family, native to south-eastern China and widely cultivated throughout the world. Fruit sugar metabolism and color change is an important physiological behavior that directly determines flavor and aroma. Our study analyzed six stages of fruit growth and development using RNA-seq, yielding a total of 14,973 DEGs, and further evaluation of key DEGs revealed a focus on sugar metabolism, flavonoid biosynthesis, carotenoid biosynthesis, and photosynthesis. Using GO and KEGG to enrich differential genes in the pathway, we selected 107 differential genes and obtained 49 significant differential genes related to glucose metabolism. The results of the correlation analyses indicated that two genes of the SWEET family, evm.TU.Chr1.3663 (PsSWEET9) and evm.TU.Chr4.676 (PsSWEET2), could be closely related to the composition of soluble sugars, which was also confirmed in the ethylene treatment experiments. In addition, analysis of the TOP 20 pathways between different growth stages and the green stage, as well as transient overexpression in chili, suggested that capsanthin/capsorubin synthase (PsCCS) of the carotenoid biosynthetic pathway contributed to the color change of plum fruit. These findings provide an insight into the molecular mechanisms involved in the ripening and color change of plum fruit.

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between ‘HJH’ with yellow flowers and ‘ZLE’ with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of ‘HJH’. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in ‘ZLE’ than that in ‘HJH’. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the ‘HJH’ petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color.

Abstract Background Rice is highly sensitive to chilling stress during the seedling stage. However, the adaptable photo-thermo sensitive genic male sterile (PTGMS) rice line, Yu17S, exhibits tolerance to low temperatures. Currently, the molecular characteristics of Yu17S are unclear. Results To evaluate the molecular mechanisms behind cold responses in rice seedlings, a comparative transcriptome analysis was performed in Yu17S during seedling development under normal temperature and low temperature conditions. In total, 9317 differentially expressed genes were detected. Gene ontology and pathway analyses revealed that these genes were involved mostly in photosynthesis, carotenoid biosynthesis, carbohydrate metabolism and plant hormone signal transduction. An integrated analysis of specific pathways combined with physiological data indicated that rice seedlings improved the performance of photosystem II when exposed to cold conditions. Genes involved in starch degradation and sucrose metabolism were activated in rice plants exposed to cold stress treatments, which was accompanied by the accumulation of soluble sugar, trehalose, raffinose and galactinol. Furthermore, chilling stress induced the expression of phytoene desaturase, 15-cis-ζ-carotene isomerase, ζ-carotene desaturase, carotenoid isomerase and β-carotene hydroxylase; this was coupled with the activation of carotenoid synthase activity and increases in abscisic acid (ABA) levels in rice seedlings. Conclusions Our results suggest that Yu17S exhibited better tolerance to cold stress with the activation of carotenoid synthase activity and increasing of ABA levels, and as well as the expression of photosynthesis-related genes under cold condition in rice seedlings.

α- and β-carotenes belong to the most essential carotenoids in the human body and display remarkable pharmacological value for health due to their beneficial antioxidant activities. Distinct high α-/β-carotene stoichiometries have gained increasing attention for their effective preventions of Alzheimer’s disease, cardiovascular disease, and cancer. However, it is extremely difficult to obtain α-carotene in nature, impeding the accumulations of high α-/β-carotene stoichiometries and excavation of their antioxidant activities. Herein, we developed a dynamically operable strategy based on lycopene cyclases (LCYB and LCYE) for concurrently enriching α- and β-carotenes along with high stoichiometries in E. coli. Membrane-targeted and promoter-centered approaches were firstly implemented to spatially enhance catalytic efficiency and temporally boost expression of TeLCYE to address its low competitivity at the starting stage. Dynamically temperature-dependent regulation of TeLCYE and TeLCYB was then performed to finally achieve α-/β-carotene stoichiometries of 4.71 at 37 °C, 1.65 at 30 °C, and 1.06 at 25 °C, respectively. In the meantime, these α-/β-carotene ratios were confirmed to result in diverse antioxidative activities. According to our knowledge, this is the first time that both the widest range and antioxidant activities of high α/β-carotene stoichiometries were reported in any organism. Our work provides attractive potentials for obtaining natural products with competitivity and a new insight on the protective potentials of α-/β-carotenes with high ratios for health supply. [ABSTRACT FROM AUTHOR]

Simple Summary: Cordyceps militaris produce a wide variety of bioactive components, such as cordycepic acid, cordycepin, polysaccharides, pentostatin, ergosterol, and carotenoids. In particular, natural carotenoids from C. militaris are attracting increasing attention in human healthy and food coloring. Investigating the genetic regulatory mechanism of carotenoid biosynthesis will help to increase the carotenoid content of C. militaris through genetically engineering. This study focuses on the role of a putative Zn2Cys6 fungal transcription factor Cmcrf1 on carotenoid biosynthesis and fruiting body formation. Deletion of Cmcrf1 exhibited drastically reduced carotenoid biosynthesis and failed to generate fruiting bodies. In addition, the ΔCmcrf1 mutant exhibited significantly increased conidiation and increased hypersensitivity to cell-wall-perturbing agents. This study is helpful to deepen our knowledge of the regulatory mechanism of carotenoid biosynthesis in C. militaris. Cordyceps militaris is a high-value medicinal and edible fungus that produces many bioactive compounds, including carotenoid, and thus, improving the carotenoid productivity of C. militaris will increase its commercial value. However, little is known about the genetic regulatory mechanism of carotenoid biosynthesis in C. militaris. To further understanding the regulatory mechanism of carotenoid biosynthesis, we performed a large-scale screen of T-DNA insertional mutant library and identified a defective mutant, denoted T111, whose colonies did not change color from white to yellow upon exposure to light. Mutation analysis confirmed that a single T-DNA insertion occurred in the gene encoding a 695-amino-acid putative fungal-specific transcription factor with a predicted Zn2Cys6 binuclear cluster DNA-binding domain found uniquely in fungi. Targeted deletion of this gene, denoted C. militaris carotenogenesis regulatory factor 1 (Cmcrf1), generated the ΔCmcrf1 mutant that exhibited drastically reduced carotenoid biosynthesis and failed to generate fruiting bodies. In addition, the ΔCmcrf1 mutant showed significantly increased conidiation and increased hypersensitivity to cell-wall-perturbing agents compared with the wild-type strain. However, the Cmcrf1 gene did not have an impact on the mycelia growth of C. militaris. These results show that Cmcrf1 is involved in carotenoid biosynthesis and is required for conidiation and fruiting body formation in C. militaris. [ABSTRACT FROM AUTHOR]

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Chromoplasts are specialized plastids in plants involved in carotenoid synthesis, accumulation, and stress resistance. In tomatoes (Solanum lycopersicum), the Chromoplast-associated carotenoid binding protein (CHRC) regulates chromoplast development and carotenoid accumulation, although its precise mechanisms are not yet fully understood. To investigate the role of SlCHRC in carotenoid biosynthesis, we generated transgenic tomatoes using overexpression (oe-SlCHRC) and CRISPR/Cas9-mediated gene editing (cr-SlCHRC) techniques. The results demonstrated inhibited fruit ripening and delayed onset of color break in both transgenic lines. The oe-SlCHRC lines exhibited increased carotenoid accumulation, particularly (E/Z)-phytoene, lycopene, and γ-carotene, with abundant plastoglobules and carotenoid crystals observed via TEM. In contrast, cr-SlCHRC mutants showed a greener phenotype, reduced carotenoid content, and fewer plastoglobules at the BK + 10 stage. Transcriptome analysis indicated that SlCHRC influences key genes in carotenoid biosynthesis, such as SlNCED2, as well as genes related to chloroplast development, photosynthesis, and plastoglobule formation. Additionally, SlCHRC enhances heat stress tolerance in tomato fruits by upregulating heat shock proteins (HSPs), antioxidants, and proline accumulation. These findings indicate that SlCHRC plays a crucial role in improving tomato fruit quality under heat stress conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Carotenoids are a large class of isoprenoid compounds which are biosynthesized by plants, algae, along with certain fungi, bacteria and insects. In plants, carotenoids provide crucial functions in photosynthesis and photoprotection. Furthermore, carotenoids also serve as precursors to apocarotenoids, which are derived through enzymatic and non-enzymatic cleavage reactions. Apocarotenoids encompass a diverse set of compounds, including hormones, growth regulators, and signaling molecules which play vital roles in pathways associated with plant development, stress responses, and plant-organismic interactions. Regulation of carotenoid biosynthesis indirectly influences the formation of apocarotenoids and bioactive effects on target pathways. Recent discovery of a plethora of new bioactive apocarotenoids across kingdoms has increased interest in expanding knowledge of the breadth of apocarotenoid function and regulation. In this review, we provide insights into the regulation of carotenogenesis, specifically linked to the biosynthesis of apocarotenoid precursors. We highlight plant studies, including useful heterologous platforms and synthetic biology tools, which hold great value in expanding discoveries, knowledge and application of bioactive apocarotenoids for crop improvement and human health. Moreover, we discuss how this field has recently flourished with the discovery of diverse functions of apocarotenoids, thereby prompting us to propose new directions for future research., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract Background Polygonatum kingianum Coll. et Hemsl. is an important plant in Traditional Chinese Medicine. The extracts from its tubers are rich in polysaccharides and other metabolites such as saponins. It is a well-known concept that growing medicinal plants in semi-arid (or drought stress) increases their natural compounds concentrations. This study was conducted to explore the morpho-physiological responses of P. kingianum plants and transcriptomic signatures of P. kingianum tubers exposed to mild, moderate, and severe drought and rewatering. Results The stress effects on the morpho-physiological parameters were dependent on the intensity of the drought stress. The leaf area, relative water content, chlorophyll content, and shoot fresh weight decreased whereas electrolyte leakage increased with increase in drought stress intensity. A total of 53,081 unigenes were obtained; 59% of which were annotated. We observed that 1352 and 350 core genes were differentially expressed in drought and rewatering, respectively. Drought stress driven differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, and stilbenoid diarylheptanoid and gingerol biosynthesis, and carotenoid biosynthesis pathways. Pathways such as plant-pathogen interaction and galactose metabolism were differentially regulated between severe drought and rewatering. Drought reduced the expression of lignin, gingerol, and flavonoid biosynthesis related genes and rewatering recovered the tubers from stress by increasing the expression of the genes. Increased expression of carotenoid biosynthesis pathway related genes under drought suggested their important role in stress endurance. An increase in starch and sucrose biosynthesis was evident from transcriptomic changes under drought stress. Rewatering recovered the drought affected tubers as evident from the contrasting expression profiles of genes related to these pathways. P. kingianum tuber experiences an increased biosynthesis of sucrose, starch, and carotenoid under drought stress. Drought decreases the flavonoids, phenylpropanoids, gingerol, and lignin biosynthesis. These changes can be reversed by rewatering the P. kingianum plants. Conclusions These results provide a transcriptome resource for P. kingianum and expands the knowledge on the effect of drought and rewatering on important pathways. This study also provides a large number of candidate genes that could be manipulated for drought stress tolerance and managing the polysaccharide and secondary metabolites’ contents in P. kingianum.