Carotenoid Cleavage Dioxygenase Research Articles

Differential expression of CCD4(4B) drives natural variation in fruit carotenoid content in strawberry (Fragaria spp.).

Carotenoids are a diverse group of pigments imparting red, orange, and yellow hues to many horticultural plants, also enhancing their nutritional properties and health benefits. In strawberry, the genetic and molecular mechanisms regulating the natural variation of fruit carotenoid composition remain largely unexplored. In this study, we use a population segregating in yellow/white flesh to detect a major quantitative trait locus (QTL), qYellow Flesh-4B, located on chromosome 4B and accounting for 82% of total phenotypic variation. In the QTL interval, specific polymorphisms on the promoter of the carotenoid cleavage dioxygenase CCD4(4B) were associated with yellow flesh, down-regulation of CCD4(4B) during ripening, and increased carotenoid content. The role of CCD4(4B) in carotenoid turnover was further confirmed through transient overexpression in strawberry fruits, which resulted in decreased concentrations of the xanthophylls violaxanthin, lutein, and zeaxanthin. Notably, a -35 C>T single-nucleotide polymorphism (SNP) in the CCD4(4B) promoter was predictive of both CCD4(4B) expression and carotenoid content across a diverse collection of octoploid Fragaria species. These findings provide valuable genetic insights into the natural variation of carotenoid composition and accumulation in strawberry. A high-resolution melting (HRM) DNA test developed in this study offers a rapid and reliable method for predicting high carotenoid content in strawberry fruits, representing a valuable tool for breeding projects aimed at enhancing the nutritional value of this crop.

Genome-Wide Identification and In Silico Expression Analysis of CCO Gene Family in Citrus clementina (Citrus) in Response to Abiotic Stress.

The Citrus clementina (citrus) plant produces various phytohormones due to the significant involvement of the carotenoid cleavage oxygenase (CCO) gene family in its growth and development. CCO genes can be divided into two main categories: NCED (9-cis-epoxy carotenoid dioxygenase), responsible for abscisic acid (ABA) production, and CCD (carotenoid cleavage dioxygenase), involved in pigment and strigolactone formation. To better understand the roles and positions of CcCCO gene members in relation to these hormones, researchers analyzed the clementine genome. To identify their structural features, they employed phylogenetic analysis, protein interactions, localization, structure, miRNA targets, evolutionary analysis, and transcriptome studies. The study revealed the presence of 15 CcCCO genes, including 11 NCED and 4 CCD genes, scattered across various chromosomes, with the majority located in chloroplasts. Promoter sequencing analysis indicated the presence of different cis-regulatory elements that likely interacted with phytohormones, such as auxin and abscisic acid among others. Notably, two genes, CcNCED1 and CcNCED3, were significantly expressed among the CCO genes, and these were found to be expressed during stress and played a crucial role in enabling optimal plant development. Furthermore, a comprehensive genome-wide comparison of CCO genes in C. Clementine and Arabidopsis thaliana models was conducted to understand their functional characteristics. This research provides a solid foundation for further exploration of the unique attributes of the C. clementina plant, contributing to a deeper understanding of its growth and development processes.

VviWRKY24 promotes β-damascenone biosynthesis by targeting VviNCED1 to increase abscisic acid in grape berries

Abstract Norisoprenoids, which are produced by the cleavage of various carotenoids, are a class of volatile aroma compounds that widely distributed in plants. In wine, they represent a significant source of floral and fruity aromas. β-Damascenone is the most abundant and important norisoprenoid constituent in grape berries (Vitis vinifera L.) and wines. However, the regulatory mechanism of β-damascenone biosynthesis remains poorly understood. The present study has identified a WRKY transcription factor, VviWRKY24, as a key regulator of β-damascenone accumulation in grape berries. The results of overexpression and gene silencing assays in grape leaves, berries and calli demonstrated that VviWRKY24 altered the flow of norisoprenoid metabolism and influenced the composition ratio of norisoprenoids, particularly enhancing the levels of β-damascenone. The results of the RNA-seq, yeast one-hybrid, electrophoretic mobility shift and dual-luciferase assays provided confirmation that VviWRKY24 promoted abscisic acid (ABA) biosynthesis by directly upregulating the expression of VviNCED1. The increase in ABA content resulted in further induction of the expression of CAROTENOID CLEAVAGE DIOXYGENASE 4B (VviCCD4b) on β-damascenone metabolic pathway. These findings elucidate the upstream regulation of ABA and the promotion of ABA on the accumulation of β-damascenone in grapes. This study contributes to a novel understanding of the regulatory mechanisms of β-damascenone biosynthesis and provides a strategy for improving the aroma quality of grapes and wine.

Red peel regulator 1 links ethylene response factor 25 and β-citraurin biosynthetic genes to regulate ethylene-induced peel reddening in citrus.

The reddish apocarotenoid β-citraurin, produced by CAROTENOID CLEAVAGE DIOXYGENASE 4b (CsCCD4b), is responsible for peel reddening in citrus (Citrus spp.). Ethylene induces the characteristic red color of citrus peel, but the underlying molecular mechanism remains largely unclear. Here, we identified red peel regulator 1 (CsRP1), a trihelix transcriptional activator that regulates ethylene-induced peel reddening by directly binding to a key V-myb avian myeloblastosis viral oncogene homolog (MYB)-binding site in the CsCCD4b promoter, thus activating its transcription. Furthermore, 2 drought-responsive cis-elements in the CsRP1 promoter are bound by the ethylene response factor ethylene response factor 25 (CsERF25). We reconstructed the CsERF25-CsRP1-CsCCD4b transcriptional regulatory cascade through transient expression of CsERF25 and CsRP1 in citrus peel and via stable transformation of citrus calli. In this cascade, CsERF25 expression was induced by ethylene to activate CsRP1 expression, and then, CsRP1 directly induced CsCCD4b transcription to catalyze β-citraurin biosynthesis. CsRP1 and CsERF25 also bound to the promoters of other carotenogenic genes and induced their transcription, thereby promoting β-citraurin accumulation. Collectively, our findings reveal a complex regulatory network modulating ethylene-induced citrus peel reddening and provide innovative strategies for improving the nutritional and esthetic values of citrus and other fruit crops.

A favorable natural variation in CCD7 from orchardgrass confers enhanced tiller number.

Tiller number is a crucial determinant that significantly influences the productivity and reproductive capacity of forage. The regeneration potential, biomass production, and seed yield of perennial forage species are highly reliant on the development of tillering. Strigolactones (SLs) are recently discovered carotenoid-derived phytohormones that play a crucial role in the regulation of tillering in annual crops. However, the modulation of tiller growth in perennial forage by SLs remains insufficiently investigated. In this study, we identified two alleles of the SLs biosynthesis gene, DgCCD7A and DgCCD7D, which encode CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), from two distinct subspecies of orchardgrass (Dactylis glomerata) exhibiting contrasting tillering phenotype and SLs content. The functionality of the DgCCD7A allele derived from high-tillering phenotypic orchardgrass was found to be diminished compared to that of DgCCD7D from the low-tillering type in rescuing the increased branching phenotype of CCD7-defective mutants in Arabidopsis and rice (Oryza sativa). Notably, the introduction of DgCCD7A in rice resulted in an increase in tiller number without significantly compromising grain yield. Moreover, we demonstrated that the L309P variation in DgCCD7A is a rare natural variant exclusively found in orchardgrass. Our findings revealed that DgCCD7A, a rare favorable natural variation of CCD7 in orchardgrass, holds significant potential for breeding application in improving the plant architecture of perennial forage and crops.

Carotenoid Cleavage Dioxygenase Gene CCD4 Enhances Tanshinone Accumulation and Drought Resistance in Salvia miltiorrhiza.

Danshen (Salvia miltiorrhiza Bunge) is a perennial herbaceous plant of the Salvia genus in the family Lamiaceae. Its dry root is one of the important traditional Chinese herbal medicines with a long officinal history. The yield and quality of S. miltiorrhiza are influenced by various factors, among which drought is one of the most significant types of abiotic stress. Based on the transcriptome database of S. miltiorrhiza, our research group discovered a carotenoid cleavage dioxygenase gene, SmCCD4, belonging to the carotenoid cleavage oxygenase (CCO) gene family which is highly responsive to drought stress on the basis of our preceding work. Here, we identified 26 CCO genes according to the whole-genome database of S. miltiorrhiza. The expression pattern of SmCCD4 showed that this gene is strongly overexpressed in the aboveground tissue of S. miltiorrhiza. And by constructing SmCCD4 overexpression strains, it was shown that the overexpression of SmCCD4 not only promotes the synthesis of abscisic acid and increases plant antioxidant activity but also regulates the synthesis of the secondary metabolites tanshinone and phenolic acids in S. miltiorrhiza. In summary, this study is the first in-depth and systematic identification and investigation of the CCO gene family in S. miltiorrhiza. The results provide useful information for further systematic research on the function of CCO genes and provide a theoretical basis for improving the yield and quality of S. miltiorrhiza.

Genome-Wide Identification and Expression Analysis of Carotenoid Cleavage Dioxygenase Genes in Salvia miltiorrhiza.

In the process of catalyzing carotenoids into various apocarotenoids and other derivatives, carotenoid cleavage dioxygenases (CCDs) play key roles. However, little information on CCDs has been reported in regard to Salvia miltiorrhiza. In this study, a total of 21 CCD genes were identified in the whole genome of S. miltiorrhiza, mainly distributed between five chromosomes. Phylogenetic relationship analysis revealed that 21 SmCCD genes were classified into four subfamilies, including SmCCD4, 7, 8, and NCED; the members of the same subfamily show similar gene structures and tertiary structures. The interspecific collinearity with other plant species, such as Arabidopsis thaliana and Oryza sativa was analyzed. Cis-elements analysis demonstrated that the majority were stress response-, light response-, growth-, and development-related. The expression pattern of the SmCCD genes was expressed in the analyzed tissues. Furthermore, the majority of the SmCCD4 subfamily members varied in their expression levels under the treatment of MeJA, YE, and ABA, indicating the potential function of SmCCD4 in the metabolism process of S. miltiorrhiza. In general, this study provides a systematic analysis of SmCCD genes and lays the foundation for uncovering the regulation and function of SmCCD genes in S. miltiorrhiza.

Introducing CCD1 into isolated Rhodotorula strain enhances flavor production and improves cigar fermentation.

The fermentation process plays an important role in enhancing the quality of cigar tobacco leaves. Through fermentation, microbial metabolism can degrade aromatic precursors and macromolecules, which increases the content of aroma compounds and reduces irritancy of tobacco leaves. To further enhance the fermentation effect of cigar tobacco leaves, a Rhodotorula strain (Rh3), capable of producing carotenoids and improving fermentation quality, was isolated from cigar tobacco leaves. Subsequent genetic engineering techniques introduced the carotenoid cleavage dioxygenase 1 (CCD1) gene into the isolated Rh3. The modified Rh3 exhibits a significant increase in carotenoid degradation products compared with the original Rh3 in culture medium (from 0.29μg/mg to 15μg/mg). Subsequent cigar tobacco leaf fermentation experiments revealed that the modified Rh3 produced 65.9% more carotenoid degradation products compared to the control group, outperforming the original strain, which achieved a 41.4% increase. Furthermore, the modified strain preserves its ability to improve the intrinsic chemical composition of cigar tobacco leaves. We show here that modified Rh3 can increase the content of carotenoid degradation products, thereby enhancing the fermentation effect of cigar tobacco leaves. This study presents a beneficial exploration to improve the quality of cigar tobacco leaves for future use and offers a promising strategy for producing flavor compounds from discarded tobacco leaves.

Characterization of SUPPRESSOR OF MAX2 1-LIKE (SMXL) Genes in ‘duli’ (Pyrus betulifolia L.) and Expression Analysis of PbSMXLs in Response to Plant Growth Regulators and Salt Stress

SUPPRESSOR OF MAX2 1-LIKE (SMXL) proteins are negative regulators of strigolactone (SL) signal transduction that play an important role in regulating plant branching and responses to abiotic stress. Here, we studied the role of SMXL proteins in pear growth, development, and stress resistance. A total of 18 SMXL members were characterized in ‘duli’. All SMXL members were localized to chloroplasts. Chromosome mapping analysis showed that the members of this family were unevenly distributed on 14 chromosomes. Gene fragment replication analysis showed that there were no tandem repeat genes in PbSMXLs, and 12 pairs of homologous genes were fragment duplications. There were 30 pairs of homologous genes between ‘duli’ and apples, and 17 between ‘duli’ and Arabidopsis thaliana. Analysis of cis-acting elements showed that there was a large number of photo-effector elements, short-effector elements, hormone-responsive elements, and abiotic stress-responsive elements in the promoter sequences of this family. Analysis of enzyme activity and endogenous SL showed that β-carotenoid isomerase (D27), carotenoid cleavage dioxygenase 7 (CCD7), lateral branch oxidoreductase (LBO) levels, and SL content were higher in ‘duli’ roots and leaves compared in the control under exogenous GA3 (gibberellin 3), IAA (indole-3-acetic acid), GR24 (synthetic SL analog), and NaCl. Most SMXL genes in ‘duli’ were highly expressed in branches and axillary lobes, but their expression was low in fruits. qRT-PCR analysis revealed that eight PbSMXL genes were responsive to GA3, PAC (Paclobutrazol), IAA, ABA (abscisic acid), GR24, and Tis108 (SL biosynthesis inhibitor). PbSMXLs responded positively to salt stress. The expression of PbSMXL6 and PbSMXL15 was significantly induced under salt stress. The expression of PbSMXL7, PbSMXL10, and PbSMXL15 was significantly induced by Tis108 treatment. The results of this study enhance our understanding of the role of SMXL genes in the responses to plant growth regulators and salt stress. Our findings will also aid future studies of the functions of SMXL genes in ‘duli’.

ZmCCD8 regulates sugar and amino acid accumulation in maize kernels via strigolactone signalling.

How carbon (sucrose) and nitrogen (amino acid) accumulation is coordinatively controlled in cereal grains remains largely enigmatic. We found that overexpression of the strigolactone (SL) biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) resulted in greater ear diameter and enhanced sucrose and amino acid accumulation in maize kernels. Loss of ZmCCD8 function reduced kernel growth with lower sugar and amino acid concentrations. Transcriptomic analysis showed down-regulation of the transcription factors ZmMYB42 and ZmMYB63 in ZmCCD8 overexpression alleles and up-regulation in zmccd8 null alleles. Importantly, ZmMYB42 and ZmMYB63 were negatively regulated by the SL signalling component UNBRANCHED 3, and repressed expression of the sucrose transporters ZmSWEET10 and ZmSWEET13c and the lysine/histidine transporter ZmLHT14. Consequently, null alleles of ZmMYB42 or ZmMYB63 promoted accumulation of soluble sugars and free amino acids in maize kernels, whereas ZmLHT14 overexpression enhanced amino acid accumulation in kernels. Moreover, overexpression of the SL receptor DWARF 14B resulted in more sucrose and amino acid accumulation in kernels, down-regulation of ZmMYB42 and ZmMYB63 expression, and up-regulation of ZmSWEETs and ZmLHT14 transcription. Together, we uncover a distinct SL signalling pathway that regulates sucrose and amino acid accumulation in kernels. Significant association of two SNPs in the 5' upstream region of ZmCCD8 with ear and cob diameter implicates its potential in breeding toward higher yield and nitrogen efficiency.

Synthesis of β-ionone from xylose and lignocellulosic hydrolysate in genetically engineered oleaginous yeast Yarrowia lipolytica.

β-ionone, an apocarotenoid derived from a C40 terpenoid has an intense, woody smell and a low odor threshold that has been widely used in as an ingredient in food and cosmetics. Yarrowia lipolytica is a promising host for β-ionone production because of its oleaginous nature, its ability to produce high levels of acetyl-CoA (an important precursor for terpenoids), and the availability of synthetic biology tools to engineer the organism. In this study, β-carotene-producing Y. lipolytica strain XK17 was employed for β-ionone biosynthesis. First, we explored the effect of different sources of carotenoid cleavage dioxygenase (CCD) genes on β-ionone production. A high-yielding strain rUinO-D14 with 122mg/L of β-ionone was obtained by screening promoters combined with rDNA mediated multi-round iterative transformations to optimize the expression of the CCD gene of Osmanthus fragrans. Second, to further develop a high-level production strain for β-ionone, we optimized key genes in the mevalonate pathway by multi-round iterative transformations mediated by non-homologous end joining, combined with a protein tagging strategy. Finally, the introduction of a heterologous oxidoreductase pathway enabled the engineered Y. lipolytica strain to use xylose as a sole carbon source and produce β-ionone. In addition, the potential for use of lignocellulosic hydrolysate as the carbon source for β-ionone production showed that the NHA-A31 strain had a high β-ionone productivity level. This study demonstrates that engineered Y. lipolytica can be used for the efficient, green and sustainable production of β-ionone.

Molecular cloning, expression, and biochemical characterization of carotenoid cleavage dioxygenase 1 (LbCCD1) from Lycium barbarum

Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, which have significant industrial applications due to their aromatic and bioactive properties. This study focuses on the molecular cloning and biochemical characterization of the LbCCD1 from Lycium barbarum. The LbCCD1 gene was successfully cloned and heterologously expressed in Escherichia coli and several carotenoids were using as substrates for investigate its specificity by in vitro and in vivo experiment. The LbCCD1 protein was able to cleave a variety of carotenoids including β-carotene, zeaxanthin, astaxanthin, and β-apo-8′-carotenal, at the 9, 10 (9′, 10′) double bond to produce β-ionone, 3‑hydroxy-4-oxo-β-ionone, and 3‑hydroxy-β-ionone, respectively in vitro. LbCCD1 could also cleave zeaxanthin and β-carotene at the 9, 10 (9′, 10′) double bond to produce β-ionone, respectively, in E. coli accumulating carotenoids. Interestingly, LbCCD1 did not exhibit cleavage activity on lycopene either in vivo or in vitro unlike other CCD1 family enzymes.In the previous experiment, it was confirmed that LbCCD1 exhibits cleavage activity towards β-apo-8′-carotenal in vitro, so we used β-apo-8′-carotenal as the substrate for characterizing the enzymatic properties. The expression of LbCCD1 was optimized at such conditions (temperature 24 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of LbCCD1 revealed the optimal activities were at pH 9 and 55 °C. The addition of 10 % ethanol could increase enzyme activity to above 15 %. However, the concentration of Fe2+ has a minimal effect on enzyme activity. The Vmax for β-apo-8′-carotenal was 8.6 U/mg, while the Km was 0.27 mM. To preliminarily verify the potential of LbCCD1 as a biological component for β-ionone production. By introducing LbCCD1 into the β-carotene-high-producing chassis cell and optimizing the conditions (temperature 30 °C, IPTG 0.01 mM, Fe2+ concentration 0.05 mM), the β-ionone yield reached 21.45 mg/L. This study focused on one of the CCDs derived from woody plants, which have been relatively underexplored. It lays the groundwork for expanding the CCD enzyme library, identifying suitable CCDs, and investigating the structure-function relationship of CCDs. Furthermore, it sets the stage for engineering novel CCD genes and developing advanced applications of CCDs as biocatalysts and platforms for synthetic biology. These advancements will enable the efficient production of volatile aroma compounds from carotenoids.

Maximizing saffron apocarotenoid production in varied tomato fruit carotenoid contexts.

Saffron spice owes its commercial appreciation to its specific apocarotenoids: crocins, picrocrocin, and safranal. In Crocus sativus, these compounds are biosynthesized from zeaxanthin through oxidative cleavage by the carotenoid cleavage dioxygenase 2 (CCD2). Transgenic tomato plants expressing CsCCD2 in the fruit, named Tomaffron, accumulate high levels of saffron apocarotenoids despite the low substrate availability for CsCCD2. In the present study, CsCCD2 has been introduced into Xantomato; this tomato variety accumulates high levels of zeaxanthin and β-carotene in ripe fruit due to a combination of four mutant alleles. Xantomato and Tomaffron genotypes have been combined to optimize apocarotenoid production. The best transgenic lines accumulated 15 and 14 times more crocins and picrocrocin than Tomaffron, alongside a fourfold increase in β-carotene compared to Xantomato, albeit at a cost in fruit yield. Segregation of the four mutations has been carried out to find the best combination for obtaining high levels of saffron apocarotenoids without adverse effects on fruit yield. Plants harboring the high-pigmented 3 (hp3) and BETA (BSh) mutations accumulated 6 and 15 times more crocins and picrocrocin than Tomaffron, without observable pleiotropic effects. Additionally, those high levels of saffron apocarotenoids were obtained in fruit accumulating high levels of both lycopene and β-carotene independently or in combination, suggesting a regulatory role for the apocarotenoids produced and indicating that it is possible to increase the levels of both types of healthy promoting molecules simultaneously.

A structural and functional model for alkene dioxygenases

In this article, we report sterically-controlled iron sites based on non-chelating bulky imidazole ligands. Adding 6 equiv. of 1,2-dimethylimidazole (1,2-Me2Im) to Fe(OTf)2⋅2CH3CN affords the first example of a 5-coordinate imidazole‑iron complex ([Fe(1,2-Me2Im)5](OTf)2, 1). The structure is distorted square pyramidal (τ5 = 0.41). When an iPr group is substituted for the methyl group at the 2-position on the imidazole (2-iPr-1-MeIm), the 14-electron complex ([Fe(2-iPr-1-MeIm)4](OTf)2, 2) is obtained. This complex exhibits slightly distorted tetrahedral geometry (τ'4 = 0.93) with four N-donors and serves as a 4-His iron structural model complex for carotenoid cleavage dioxygenases (CCD). The electronic structure of 1 and 2 were characterized by Mössbauer spectroscopy. Reactions of 1 and 2 with model olefin substrates (1-R-4-(1-methoxyprop-1-en-2-yl)benzene; R = Me or Br) in the presence of oxygen result in olefin cleavage yielding ketone and aldehyde products, although 2 yields more products than 1. Support for a proposed reaction mechanism for 2 is offered from Density Functional Theory (DFT) calculations.

Transcriptome wide analysis of MADS box genes in Crocus sativus and interplay of CstMADS19-CstMADS26 in orchestrating apocarotenoid biosynthesis

Flowers of Crocus sativus L. are immensely important not only for arrangement of floral whorls but more because each floral organ is dominated by a different class of specialized compounds. Dried stigmas of C. sativus flowers form commercial saffron, and are known to accumulate unique apocarotenoids like crocin, picrocrocin and safranal. Inspite of being a high value crop, the molecular mechanism regulating flower development in Crocus remains largely unknown. Moreover, it would be very interesting to explore any co-regulatory mechanism which controls floral architecture and secondary metabolic pathways which exist in specific floral organs. Here we report transcriptome wide identification of MADS box genes in Crocus. A total of 39 full length MADS box genes were identified among which three belonged to type I and 36 to type II class. Phylogeny classified them into 11 sub-clusters. Expression pattern revealed some stigma up-regulated genes among which CstMADS19 encoding an AGAMOUS gene showed high expression. Transient over-expression of CstMADS19 in stigmas of Crocus resulted in increased crocin by enhancing expression of pathway genes. Yeast one hybrid assay demonstrated that CstMADS19 binds to promoters of phytoene synthase and carotenoid cleavage dioxygenase 2 genes. Yeast two hybrid and BiFC assays confirmed interaction of CstMADS19 with CstMADS26 which codes for a SEPALATA gene. Co-overexpression of CstMADS19 and CstMADS26 in Crocus stigmas enhanced crocin content more than was observed when genes were expressed individually. Collectively, these findings indicate that CstMADS19 functions as a positive regulator of stigma based apocarotenoid biosynthesis in Crocus.

Carotenoid biosynthesis profiling unveils the variance of flower coloration in Tagetes erecta and enhances fruit pigmentation in tomato

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.

Genome-wide analysis and identification of Carotenoid Cleavage Oxygenase (CCO) gene family in coffee (coffee arabica) under abiotic stress

The coffee industry holds importance, providing livelihoods for millions of farmers globally and playing a vital role in the economies of coffee-producing countries. Environmental conditions such as drought and temperature fluctuations can adversely affect the quality and yield of coffee crops.Carotenoid cleavage oxygenases (CCO) enzymes are essential for coffee plants as they help break down carotenoids contributing to growth and stress resistance. However, knowledge about the CCO gene family in Coffee arabica was limited. In this study identified 21 CCO genes in Coffee arabica (C. arabica) revealing two subfamilies carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxy carotenoid dioxygenases (NCED) through phylogenic analysis. These subfamilies exhibited distribution patterns in terms of gene structure, domains, and motifs. The 21 CaCCO genes, comprising 5 NCED and 16 CCD genes were found across chromosomes. Promoter sequencing analysis revealed cis-elements that likely interact with plant stress-responsive, growth-related, and phytohormones, like auxin and abscisic acid. A comprehensive genome-wide comparison, between C. arabica and A. thaliana was conducted to understand the characteristics of CCO genes. RTqPCR data indicated that CaNCED5, CaNCED6, CaNCED12, and CaNCED20 are target genes involved in the growth of drought coffee plants leading to increased crop yield, in a conditions, with limited water availability. This reveals the role of coffee CCOs in responding to abiotic stress and identifies potential genes useful for breeding stress-resistant coffee varieties.

Transcriptomic and Metabolomic Insights into ABA-Related Genes in Cerasus humilis under Drought Stress.

Cerasus humilis, a small shrub of the Cerasus genus within the Rosaceae family, is native to China and renowned for its highly nutritious and medicinal fruits, robust root system, and remarkable drought resistance. This study primarily employed association transcriptome and metabolome analyses to assess changes in abscisic acid (ABA) levels and identify key regulatory genes in C. humilis subjected to varying degrees of drought stress. Notably, we observed distinct alterations in transcription factors across different drought intensities. Specifically, our transcriptome data indicated noteworthy shifts in GATA, MYB, MYC, WRKY, C2H2, and bHLH transcription factor families. Furthermore, combined transcriptomic and metabolomic investigations demonstrated significant enrichment of metabolic pathways, such as 'Carbon metabolism', 'Biosynthesis of amino acids', 'Biosynthesis of cofactors', 'Phenylpropanoid biosynthesis', 'Starch and sucrose metabolism', and 'Plant hormone signal transduction' under moderate (Mod) or severe (Sev) drought conditions. A total of 11 candidate genes involved in ABA biosynthesis and signaling pathways were identified. The down-regulated genes included secoisolariciresinol dehydrogenase-like and PYL2. Conversely, genes including FAD-dependent urate hydroxylase-like, cytochrome P450 97B2, carotenoid cleavage dioxygenase 4 (CCD4), SnRK2.2, ABI 5-like protein 5, PP2C 51, and SnRK2.3, were up-regulated under Mod or Sev drought stress. This study lays the genetic foundation for ABA biosynthesis to enhance drought tolerance and provides genetic resources for plant genetic engineering and breeding efforts.

Structural complexity of the white flower locus in Chrysanthemum

ABSTRACT The white flower trait in Chrysanthemum is attributed to the carotenoid cleavage dioxygenase 4a gene (CCD4a), a key domestication gene that contributed to generation of various flower colours in modern chrysanthemum cultivars. This trait is believed to be controlled by a single dominant locus. We studied the structure of this CCD4a locus utilising the whole genome sequence information of Chrysanthemum makinoi, a diploid white flower species, and found six homologous sequences of CCD4a, two of which being functional, and arranged in tandem in a 586-kb region on Chromosome 7. A qPCR analysis disclosed that white flower Chrysanthemum species possess at least two and as many as eight copies of CCD4a homologous sequences per monoploid genome, indicating high polymorphism of the CCD4a region. A detailed analysis of the structural diversity of the CCD4a region in Chrysanthemum could provide significant insights into the domestication trajectory of the cultivated chrysanthemum.

Zaxinone Synthase overexpression modulates rice physiology and metabolism, enhancing nutrient uptake, growth and productivity.

The rice Zaxinone Synthase (ZAS) gene encodes a carotenoid cleavage dioxygenase (CCD) that forms the apocarotenoid growth regulator zaxinone in vitro. Here, we generated and characterized constitutive ZAS-overexpressing rice lines, to better understand ZAS role in determining zaxinone content and regulating growth and architecture. ZAS overexpression enhanced endogenous zaxinone level, promoted root growth and increased the number of productive tillers, leading to about 30% higher grain yield per plant. Hormone analysis revealed a decrease in strigolactone (SL) content, which we confirmed by rescuing the high-tillering phenotype through application of a SL analogue. Metabolomics analysis revealed that ZAS overexpressing plants accumulate higher amounts of monosaccharide sugars, in line with transcriptome analysis. Moreover, transgenic plants showed higher carbon (C) assimilation rate and elevated root phosphate, nitrate and sulphate level, enhancing the tolerance towards low phosphate (Pi). Our study confirms ZAS as an important determinant of rice growth and architecture and shows that ZAS regulates hormone homoeostasis and a combination of physiological processes to promote growth and grain yield, which makes this gene an excellent candidate for sustainable crop improvement.