Carotenoid Cleavage Dioxygenase Research Articles

Multi-omics analyses provide insights into the genomic basis of differentiation among four sweet osmanthus groups.

Sweet osmanthus (Osmanthus fragrans) is famous in China for its flowers and contains four groups: Albus, Luteus, Aurantiacus, and Asiaticus. Understanding the relationships among these groups and the genetic mechanisms of flower color and aroma biosynthesis are of tremendous interest. In this study, we sequenced representative varieties from two of the four sweet osmanthus groups. Multi-omic and phylogenetic analyses of varieties from each of the four groups showed that Asiaticus split first within the species, followed by Aurantiacus and the sister groups Albus and Luteus. We show that the difference in flower color between Aurantiacus and the other three groups was caused by a 4-bp deletion in the promoter region of carotenoid cleavage dioxygenase 4 (OfCCD4) that leads to expression decrease. In addition, we identified 44 gene pairs exhibiting significant structural differences between the multi-seasonal flowering variety 'Rixianggui' in the Asiaticus group and other autumn flowering varieties. Through correlation analysis between intermediate products of aromatic components and gene expression, we identified eight genes associated with the linalool, α- and β-ionone biosynthesis pathways. Overall, our study offers valuable genetic resources for sweet osmanthus, while also providing genetic clues for improving the flower color and multi-season flowering of osmanthus and other flowers.

Accumulation of β-citraurin and other carotenoids in relation to the expression of PSY1 and CCD4b1 in peel of twelve red-peeled citrus cultivars

Peel color in citrus is largely genetically controlled. Red peel is favored by customers and is important, therefore, for fruit external quality. In order to understand the factors affecting distribution of carotenoids and color intensity of red-peeled citrus, we compared carotenoid accumulation and gene expression in the peel of twelve fully ripe red-peeled citrus cultivars with different genetic backgrounds. The results showed that β-citraurin, a red C30 carotenoid, was the second or third most abundant colored carotenoid in the peel of all twelve red-peeled citrus, ranging from 9.4 % to 26.7 % of total carotenoids. Statistical analysis of carotenoid composition and content showed that of the main carotenoids β-citraurin had the highest correlation with red appearance of cultivars. In addition, β-cryptoxanthin, which accounted for 3.6 % to 16.4 % of the total carotenoids, were also significantly positively correlated with citrus color index (CCI). The expression of two key genes, phytoene synthase 1 (PSY1) and carotenoid cleavage dioxygenase 4b1 (CCD4b1), differed among cultivars with a range of 3.5- and 48.8-fold, respectively. Surprisingly, accumulation of total carotenoids and β-citraurin was not correlated with the expression of PSY1 and CCD4b1, respectively, implying that diverse factors influence high carotenoid accumulation in red-peeled citrus.

Carotenoid cleavage enzymes evolved convergently to generate the visual chromophore.

The retinal light response in animals originates from the photoisomerization of an opsin-coupled 11-cis-retinaldehyde chromophore. This visual chromophore is enzymatically produced through the action of carotenoid cleavage dioxygenases. Vertebrates require two carotenoid cleavage dioxygenases, β-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65), to form 11-cis-retinaldehyde from carotenoid substrates, whereas invertebrates such as insects use a single enzyme known as Neither Inactivation Nor Afterpotential B (NinaB). RPE65 and NinaB couple trans-cis isomerization with hydrolysis and oxygenation, respectively, but the mechanistic relationship of their isomerase activities remains unknown. Here we report the structure of NinaB, revealing details of its active site architecture and mode of membrane binding. Structure-guided mutagenesis studies identify a residue cluster deep within the NinaB substrate-binding cleft that controls its isomerization activity. Our data demonstrate that isomerization activity is mediated by distinct active site regions in NinaB and RPE65-an evolutionary convergence that deepens our understanding of visual system diversity.

Integration of rice apocarotenoid profile and expression pattern of Carotenoid Cleavage Dioxygenases reveals a positive effect of β-ionone on mycorrhization

Carotenoids are susceptible to degrading processes initiated by oxidative cleavage reactions mediated by Carotenoid Cleavage Dioxygenases that break their backbone, leading to products called apocarotenoids. These carotenoid-derived metabolites include the phytohormones abscisic acid and strigolactones, and different signaling molecules and growth regulators, which are utilized by plants to coordinate many aspects of their life. Several apocarotenoids have been recruited for the communication between plants and arbuscular mycorrhizal (AM) fungi and as regulators of the establishment of AM symbiosis. However, our knowledge on their biosynthetic pathways and the regulation of their pattern during AM symbiosis is still limited. In this study, we generated a qualitative and quantitative profile of apocarotenoids in roots and shoots of rice plants exposed to high/low phosphate concentrations, and upon AM symbiosis in a time course experiment covering different stages of growth and AM development. To get deeper insights in the biology of apocarotenoids during this plant-fungal symbiosis, we complemented the metabolic profiles by determining the expression pattern of CCD genes, taking advantage of chemometric tools. This analysis revealed the specific profiles of CCD genes and apocarotenoids across different stages of AM symbiosis and phosphate supply conditions, identifying novel reliable markers at both local and systemic levels and indicating a promoting role of β-ionone in AM symbiosis establishment.

Functional identification of Medicago truncatulaMtRAV1 in regulating growth and development1

Related to ABI3 and VP1 (RAV) transcription factors belong to the AP2 and B3 superfamily. RAVs genes have been reported to be involved in plant growth and development regulation. This study screened three RAV genes from Medicago truncatula and named one of them MtRAV1. The MtRAV1 overexpressing plants exhibits traits such as plant dwarfing, delayed flowering, reduced leaf and floral organs, increased branching, and reduced pods and seeds. Gene expression analysis results showed that overexpression of MtRAV1 inhibited the expression of Flowering Locus T (MtFTa1), Suppressor of Overexpression of CO 1 (MtSOC1), GA3-oxidase1 (MtGA3OX1), DWARF14 (MtD14) and Carotenoid Cleavage Dioxygenase 7 (MtCCD7). To further investigate the regulation pathway involved by MtRAV1, RNA-sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) analysis were conducted. RNA-seq results indicated that MtRAV1 might affect plant growth and development by regulating some genes in photosynthesis, circadian rhythm and plant hormone signaling pathways, especially the auxin signaling pathway. Conjoint analysis of DAP-seq and RNA-seq revealed that MtRAV1 might inhibit the expression of Ferredoxin (MtFd-l3), Light-harvesting Chlorophyll a/b Binding Protein 1 (MtLhcb-l2) and Small Auxin Up-regulated RNA (MtSAUR-l), which related to photosystem II and auxin signaling pathway. Summarily, MtRAV1 was preliminarily proven to be a key growth inhibitory factor in M. truncatula.

Crocus genome reveals the evolutionary origin of crocin biosynthesis

Crocus sativus (saffron) is a globally autumn-flowering plant, and its stigmas are the most expensive spice and valuable herb medicine. Crocus specialized metabolites, crocins, are biosynthesized in distant species, Gardenia (eudicot) and Crocus (monocot), and the evolution of crocin biosynthesis remains poorly understood. With the chromosome-level Crocus genome assembly, we revealed that two rounds of lineage-specific whole genome triplication occurred, contributing important roles in the production of carotenoids and apocarotenoids. According to the kingdom-wide identification, phylogenetic analysis, and functional assays of carotenoid cleavage dioxygenases (CCDs), we deduced that the duplication, site positive selection, and neofunctionalization of Crocus-specific CCD2 from CCD1 members are responsible for the crocin biosynthesis. In addition, site mutation of CsCCD2 revealed the key amino acids, including I143, L146, R161, E181, T259, and S292 related to the catalytic activity of zeaxanthin cleavage. Our study provides important insights into the origin and evolution of plant specialized metabolites, which are derived by duplication events of biosynthetic genes.

Carotenoid Cleavage Dioxygenase 1 and Its Application for the Production of C13-Apocarotenoids in Microbial Cell Factories: A Review.

C13-apocarotenoids are naturally derived from the C9-C10 (C9'-C10') double-bond cleavage of carotenoids by carotenoid cleavage dioxygenases (CCDs). As high-value flavors and fragrances in the food and cosmetic industries, the sustainable production of C13-apocarotenoids is emerging in microbial cell factories by the carotenoid cleavage dioxygenase 1 (CCD1) subfamily. However, the commercialization of microbial-based C13-apocarotenoids is still limited by the poor performance of CCD1, which severely constrains its conversion efficiency from precursor carotenoids. This review focuses on the classification of CCDs and their cleavage modes for carotenoids to generate corresponding apocarotenoids. We then emphatically discuss the advances for C13-apocarotenoid biosynthesis in microbial cell factories with various strategies, including optimization of CCD1 expression, improvement of CCD1's catalytic activity and substrate specificity, strengthening of substrate channeling, and development of oleaginous microbial hosts, which have been verified to increase the conversion rate from carotenoids. Lastly, the current challenges and future directions will be discussed to enhance CCDs' application for C13-apocarotenoids biomanufacturing.

Genome-wide analysis of carotenoid cleavage oxygenases and identification of ripening-associated DzNCED5a in durian (Durio zibethinus) fruit

Durian (Durio zibethinus L.), popularly known as the “King of fruits,” holds significant economic importance in Southeast Asia, including Thailand. During its ripening process, the phytohormone abscisic acid (ABA) content has been reported to increase. However, a comprehensive understanding of ABA's specific role in durian fruit ripening remains elusive. Furthermore, little is known about the molecular aspects of the carotenoid cleavage pathway in this iconic fruit. Therefore, we performed genome-wide identification of the carotenoid cleavage oxygenase (CCO) family in durian. This family includes the nine-cis-epoxycarotenoid dioxygenases (NCEDs) responsible for ABA production and the carotenoid cleavage dioxygenases exhibiting diverse substrate specificities. Through phylogenetic analysis, we classified 14 CCOs in durian into 8 distinct subfamilies. Notably, each DzCCO subfamily displayed a conserved motif composition. Cis-acting element prediction showed that cis-elements related to plant hormones and environmental stress responses were distributed in the DzCCO promoter. In addition, transcriptome analysis was performed to examine the expression pattern during the fruit development and ripening stages. Interestingly, DzNCED5a, a ripening-associated gene, exhibited the highest expression level at the ripe stage, outperforming other CCOs. Its expression markedly correlated with increased ABA contents during the ripening stages of both the “Monthong” variety and other durian cultivars. Transiently expressed DzNCED5a in Nicotiana benthamiana leaves confirmed its function in ABA biosynthesis. These findings highlight the involvement of DzNCED5a in ABA production and its potential importance in durian fruit ripening. Overall, this study provides insights into the significance of CCOs in durian fruit ripening.

Carotenoid gene expression and metabolic changes in the pericarp of Mandarin ‘Shatangju’ under net shed shading during growth

Fruit colour is mostly determined by the composition and content of carotenoids. In this study, a metabolic transcription analysis of carotenoids in the pericarp of ‘Shatangju’ at three different growth periods under two cultivation methods, open-air and netting, was performed. The results showed that the lightness value, red-green value and yellow-blue value indices of the pericarp varied greatly. Fourteen carotenoids were detected in the citrus pericarp. The content of lycopene was 3.21 μg/g and 3.43 μg/g, and the content of β-citraurin was 6 μg/g and 7.27 μg/g under open-air and net shed cultivation, respectively. Shading affected the synthesis of key genes such as phytoene synthase (PSY), carotenoid cleavage dioxygenase 4 (CCD4), and zeaxanthin epoxidase (ZEP), thereby inhibiting the accumulation of lycopene and β-citraurin. Studies have shown that the degreening of the ‘Shatangju’ pericarp was accelerated, but the accumulation of carotenoid biosynthesis genes was inhibited, which was not conducive to the synthesis of carotenoids.

ERF5.1 modulates carotenoid accumulation by interacting with CCD4.1 in Lycium.

Carotenoids are important natural pigments and have medical and health functions for humans. Carotenoid cleavage dioxygenase 4 (CCD4) and ethylene responsive factor (ERF) participate in carotenoid metabolism, but their roles in Lycium have not been discovered. Here, we annotated LbCCDs from the Lycium reference genome and found that LbCCD4.1 expression was significantly correlated with the carotenoid metabolites during Lycium five fruit developmental stages. Over-expression of LbCCD4.1 in NQ's leaves resulted in a series of significantly lower contents of carotenoid metabolites, including β-carotene and β-cryptoxanthin. Moreover, LbERF5.1, a transcription factor belonging to the ERF family that was located in the nucleus, was isolated. Significant reductions in the carotenoids, especially lutein, violaxanthin and their derivatives, were observed in over-expressing ERF5.1 transgenic NQ's leaves. Over-expression or virus-induced gene silencing of LbERF5.1 in NQ's leaves induced a consistent up- or down-expression, respectively, of LbCCD4.1. Furthermore, yeast one-hybrid and dual-luciferase reporter assays showed that ERF5.1 interacted with the promoter of CCD4.1 to increase its expression, and LbERF5.1 could bind to any one of the three predicted binding sites in the promoter of LbCCD4.1. A transcriptome analysis of LbERF5.1 and LbCCD4.1 over-expressed lines showed similar global transcript expression, and geranylgeranyl diphosphate synthase, phytoene synthase, lycopene δ-cyclase cytochrome, cytochrome P450-type monooxygenase 97A, cytochrome P450-type monooxygenase 97C, and zeaxanthin epoxidase in the carotenoid biosynthesis pathway were differentially expressed. In summary, we uncovered a novel molecular mechanism of carotenoid accumulation that involved an interaction between ERF5.1 and CCD4.1, which may be used to enhance carotenoid in Lycium.

Transcriptome analysis of Lantana camara flower petals reveals candidate anthocyanin biosynthesis genes mediating red flower color development.

Flower color plays a crucial role in the appeal and selection of ornamental plants, directly influencing breeding strategies and the broader horticulture industry. Lantana camara, a widely favored flowering shrub, presents a rich palette of flower colors. Yet, the intricate molecular mechanisms governing this color variation in the species have remained largely unidentified. With the aim of filling this gap, this study embarked on a comprehensive de novo transcriptome assembly and differential gene expression analysis across 3 distinct lantana accessions, each showcasing a unique flower color. By harnessing the capabilities of both PacBio and Illumina sequencing platforms, a robust transcriptome assembly, encompassing 123,492 gene clusters and boasting 94.2% BUSCO completeness, was developed. The differential expression analysis unveiled 72,862 unique gene clusters that exhibited varied expression across different flower stages. A pronounced upregulation of 8 candidate core anthocyanin biosynthesis genes in the red-flowered accession was uncovered. This was further complemented by an upregulation of candidate MYB75 (PAP1) and bHLH42 (TT8) transcription factors. A candidate carotenoid cleavage dioxygenase (CCD4a) gene cluster also manifested a marked upregulation in white flowers. The study unveils the molecular groundwork of lantana's flower color variation, offering insights for future research and potential applications in breeding ornamental plants with desired color traits.

Far-red light inhibits lateral bud growth mainly through enhancing apical dominance independently of strigolactone synthesis in tomato.

The ratio of red light to far-red light (R:FR) is perceived by light receptors and consequently regulates plant architecture. Regulation of shoot branching by R:FR ratio involves plant hormones. However, the roles of strigolactone (SL), the key shoot branching hormoneand the interplay of different hormones in the light regulation of shoot branching in tomato (Solanum lycopersicum) are elusive. Here, we found that defects in SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 in tomato resulted in more lateral bud growth but failed to reverse the FR inhibition of lateral bud growth, which was associated with increased auxin synthesis and decreased synthesis of cytokinin (CK) and brassinosteroid (BR). Treatment of auxin also inhibited shoot branching in ccd mutants. However, CK released the FR inhibition of lateral bud growth in ccd mutants, concomitant with the upregulation of BR synthesis genes. Furthermore, plants that overexpressed BR synthesis gene showed more lateral bud growth and the shoot branching was less sensitive to the low R:FR ratio. The results indicate that SL synthesis is dispensable for light regulation of shoot branching in tomato. Auxin mediates the response to R:FR ratio to regulate shoot branching by suppressing CK and BR synthesis.

Genome-wide identification and in-silico expression analysis of carotenoid cleavage oxygenases gene family in Oryza sativa (rice) in response to abiotic stress.

Rice constitutes a foundational cereal and plays a vital role in the culinary sector. However, the detriments of abiotic stress on rice quality and productivity are noteworthy. Carotenoid cleavage oxygenases (CCO) hold vital importance as they enable the particular breakdown of carotenoids and significantly contribute towards the growth and response to abiotic stress in rice. Due to the insufficient information regarding rice CCOs and their potential role in abiotic stress, their utilization in stress-resistant genetic breeding remains limited. The current research identified 16 CCO genes within the Oryza sativa japonica group. These OsCCO genes can be bifurcated into three categories based on their conserved sequences: NCEDs (9-Cis-epoxycarotenoid dioxygenases), CCDs (Carotenoid cleavage dioxygenases) and CCD-like (Carotenoid cleavage dioxygenases-like). Conserved motifs were found in the OsCCO gene sequence via MEME analysis and multiple sequence alignment. Stress-related cis-elements were detected in the promoter regions of OsCCOs genes, indicating their involvement in stress response. Additionally, the promoters of these genes had various components related to plant light, development, and hormone responsiveness, suggesting they may be responsive to plant hormones and involved in developmental processes. MicroRNAs play a pivotal role in the regulation of these 16 genes, underscoring their significance in rice gene regulation. Transcriptome data analysis suggests a tissue-specific expression pattern for rice CCOs. Only OsNCED6 and OsNCED10 significantly up-regulated during salt stress, as per RNA seq analyses. CCD7 and CCD8 levels were also higher in the CCD group during the inflorescence growth stage. This provides insight into the function of rice CCOs in abiotic stress response and identifies possible genes that could be beneficial for stress-resistant breeding.

The Functional Characteristics and Soluble Expression of Saffron CsCCD2.

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.

Integrative Analysis of Metabolome and Transcriptome Revealed Lutein Metabolism Contributed to Yellow Flower Formation in Prunus mume.

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.

Comparison of carotenoid, chlorophyll concentrations and their biosynthetic transcript levels in different coloured cauliflower

Carotenoids and chlorophylls are among the most widely distributed pigments in nature that play essential roles in the photosynthetic apparatus and confer diverse colours in plants. Among all vegetables, cauliflower (Brassica oleracea L. ssp. var. botrytis) is rich in phytochemicals and is an important crop grown all over the world. This study investigates carotenoid and chlorophyll concentrations in differently pigmented cultivars and elucidates the role of transcriptional regulation of carotenoid accumulation including lutein and β-carotene. Here, we characterised changes in pigments by UHPLC-DAD-ToF-MS and changes in transcript levels of carotenoid metabolic genes by qRT-PCR in florets and leaves of orange (‘Jaffa’ and ‘Sunset’), purple (‘Di Sicilia Violetto’ and ‘Graffiti’), green (‘Trevi’) and white (‘Clapton’) cultivars. Transcript levels of all carotenoid metabolic genes showed different transcript level patterns in the leaves and florets. Compared to the other cultivars, the orange cultivars had the highest levels of β-carotene in the florets and lutein in the leaves resulting in changes lutein/β-carotene ratios. In the green cultivar, higher transcript levels were also found, especially for phytoene synthase and phytoene desaturase genes of the core biosynthesis pathway. However, no increased carotenoid concentrations were observed, possibly due to a higher carotenoid turnover induced by the carotenoid cleavage dioxygenase 4 in the green cultivar. In the white (‘Clapton’) and purple (‘Di Sicilia Violetto’ and ‘Graffiti’) cultivars the phytoene desaturase transcript levels as well as carotenoid concentrations were low. Chlorophyll concentrations changed in trend comparable to the carotenoid concentrations and were only significantly lower in the leaves of the orange cultivar ‘Jaffa’. Also, the chlorophyll a/b ratio changed in ‘Jaffa’. In florets the highest chlorophylls concentrations were observed for the green cultivar (‘Trevi’) and the purple cultivar (‘Di Sicilia Violetto’). Taken together, the study demonstrates the complex source-sink relationship of carotenoid accumulation in different coloured cauliflower.

Global hypermethylation of the N6-methyladenosine RNA modification associated with apple heterografting.

Grafting can facilitate better scion performance and is widely used in plants. Numerous studies have studied the involvement of mRNAs, small RNAs, and epigenetic regulations in the grafting process. However, it remains unclear whether the mRNA N6-methyladenosine (m6A) modification participates in the apple (Malus x domestica Borkh.) grafting process. Here, we decoded the landscape of m6A modification profiles in 'Golden delicious' (a cultivar, Gd) and Malus prunifolia 'Fupingqiuzi' (a unique rootstock with resistance to environmental stresses, Mp), as well as their heterografted and self-grafted plants. Interestingly, global hypermethylation of m6A occurred in both heterografted scion and rootstock compared with their self-grafting controls. Gene Ontology (GO) term enrichment analysis showed that grafting-induced differentially m6A-modified genes were mainly involved in RNA processing, epigenetic regulation, stress response, and development. Differentially m6A-modified genes harboring expression alterations were mainly involved in various stress responses and fatty acid metabolism. Furthermore, grafting-induced mobile mRNAs with m6A and gene expression alterations mainly participated in ABA synthesis and transport (e.g. carotenoid cleavage dioxygenase 1 [CCD1] and ATP-binding cassette G22 [ABCG22]) and abiotic and biotic stress responses, which might contribute to the better performance of heterografted plants. Additionally, the DNA methylome analysis also demonstrated the DNA methylation alterations during grafting. Downregulated expression of m6A methyltransferase gene MdMTA (ortholog of METTL3) in apples induced the global m6A hypomethylation and distinctly activated the expression level of DNA demethylase gene MdROS1 (REPRESSOR OF SILENCING 1) showing the possible association between m6A and 5mC methylation in apples. Our results reveal the m6A modification profiles in the apple grafting process and enhance our understanding of the m6A regulatory mechanism in plant biological processes.

Multiple loss-of-function mutations of carotenoid cleavage dioxygenase 4 reveal its major role in both carotenoid level and apocarotenoid composition in flue-cured mature tobacco leaves

Apocarotenoid volatiles contribute to the flavor of many agricultural products. In many flowering plants, carotenoid cleavage dioxygenase 4 (CCD4) is involved in the decomposition of carotenoids and resultant production of C13-apocarotenoids, such as β-ionone derived from β-carotene. To understand the possible role of tobacco CCD4 genes (NtCCD4-S, NtCCD4-T1 and NtCCD4-T2) in these processes, we analyzed loss-of-function phenotypes. RNA interference transgenic plants showed yellow color in mature (senescent) leaves. Mature leaves of chemically induced double-mutant plants showed a stronger yellow color, and those of triple-mutant plants showed a pronounced yellow color. Carotenoid analysis of the leaves from mutants showed that lutein and β-carotene increased in line with the degree of color change compared to wild type, whereas there was little change in green color in their young leaves. This result indicates that CCD4s are important for the decomposition of carotenoids in the tobacco leaf maturation process. Analysis of apocarotenoids in flue-cured leaves of the multiple-mutant plants showed that many compounds, including megastigmatrienones, were decreased in comparison to wild type, whereas intriguingly β-ionone and dihydroactinidiolide were increased. Our results suggest that CCD4s play a key role in both carotenoid level and apocarotenoid composition in flue-cured mature tobacco leaves.

Expression profile of the NCED/CCD genes in chickpea and lentil during abiotic stress reveals a positive correlation with increased plant tolerance

Carotenoid cleavage dioxygenase (CCD) gene family is organized in two subfamilies: (i) 9-cis epoxycarotenoid dioxygenase (NCED) genes and (ii) CCD genes. NCED genes are essential for catalyzing the first step of the abscisic-acid (ABA) biosynthesis, while CCD genes produce precursors of the strigolactones hormone. The functional characterization of these gene subfamilies has not been yet performed in chickpea and lentil. Herein, were identified and systematically characterized two NCED and five CCD genes in the chickpea and two NCED and six CCD genes in lentil. After in silico sequence analysis and phylogeny, the expression profile of the NCED/CCD genes was determined by meta-analysis and real-time PCR in plants under different stress conditions. Sequence data revealed that NCED/CCD genes are highly conserved between chickpea and lentil. This conservation was observed both at gene and protein sequence levels and phylogenetic relationships. Analysis of the promoter sequences revealed that all NCED/CCD genes have a considerable number of cis-regulatory elements responsive to biotic and abiotic stress. Protein sequence analysis evidenced that NCED/CCD genes share several conserved motifs and that they have a highly interconnected interaction network. Furthermore, the three-dimensional structure of these proteins was determined and indicated that some proteins have structures with considerable similarity. The meta-analysis revealed that NCED/CCD genes are dynamically modulated in different organs and under different stress conditions, but they have a positive correlation with plant tolerance. In accordance, real-time PCR data showed that both NCED and CCD genes are differentially modulated in plants under drought stress. In particular, CaNCED2, CaCCD5, LcNCED2, LcCCD1, and LcCCD2 genes have a positive correlation with improved plant tolerance to drought stress. Therefore, this study presented a detailed characterization of the chickpea and lentil NCED/CCD genes and provided new insights to improve abiotic stress tolerance in these two important crops.

The transcription factor SPL13 mediates strigolactone suppression of shoot branching by inhibiting cytokinin synthesis in Solanum lycopersicum.

Plant architecture imposes a large impact on crop yield. IDEAL PLANT ARCHITECTURE 1 (IPA1), which encodes a SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor, is a target of molecular design for improving grain yield. However, the roles of SPL transcription factors in regulating tomato (Solanum lycopersicum) plant architecture are unclear. Here, we show that the expression of SPL13 is down-regulated in the lateral buds of strigolactone (SL)-deficient ccd mutants and is induced by GR24 (a synthetic analog of SL). Knockout of SPL13 by CRISPR/Cas9 resulted in higher levels of cytokinins (CKs) and transcripts of the CK synthesis gene ISOPENTENYL TRANSFERASES 1 (IPT1) in the stem nodes, and more growth of lateral buds. GR24 suppresses CK synthesis and lateral bud growth in ccd mutants, but is not effective in spl13 mutants. On the other hand, silencing of the IPT1 gene inhibited bud growth of spl13 mutants. Interestingly, SL levels in root extracts and exudates are significantly increased in spl13 mutants. Molecular studies indicated that SPL13 directly represses the transcription of IPT1 and the SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and MORE AXILLARY GROWTH 1 (MAX1). The results demonstrate that SPL13 acts downstream of SL to suppress lateral bud growth by inhibiting CK synthesis in tomato. Tuning the expression of SPL13 is a potential approach for decreasing the number of lateral shoots in tomato.