Carotenoid Cleavage Dioxygenase Genes Research Articles

A Comprehensive Analysis of Carotenoid Cleavage Dioxygenases Genes in Solanum Lycopersicum

Carotenoid cleavage dioxygenases (CCDs) in plant species is one of the most important enzymes in the carotenoid metabolism. In this study, we performed a comprehensive analysis for the CCDs family in Solanum lycopersicum based on the whole tomato genome sequences and explored their expression pattern. At least seven CCD genes were discovered in the tomato genome sequence. Two pairs of them were arranged in tandem. The tandem duplication events could be dating to approximately 14 and 21 Mya, and the tandem duplication genes experienced a purifying selection during the course of evolution after diversification. Additionally, subcellular localization revealed that four members were predicted to be cytoplasm-localized and the three remaining members plastids-localized. Subsequently, a number of cis-regulatory elements, which were involved in light responsiveness, hormone regulation, and abiotic and biotic stresses, were identified in the promoter sequences of SlCCD genes. Phylogenetic tree revealed that the CCDs from Solanaceae crops have a closer genetic relationship. The difference in abundance and distinct expression patterns during the vegetative and reproductive development suggests different functions for these seven SlCCDs. Our findings suggest that SlCCDs family play important roles throughout the whole life course and will lay the foundation for further elaborating the regulatory mechanism of each member in tomato.

Distinct Carotenoid and Flavonoid Accumulation in a Spontaneous Mutant of Ponkan (Citrus reticulata Blanco) Results in Yellowish Fruit and Enhanced Postharvest Resistance.

As the most important fresh fruit worldwide, citrus is often subjected to huge postharvest losses caused by abiotic and biotic stresses. As a promising strategy to reduce postharvest losses, enhancing natural defense by potential metabolism reprogramming in citrus mutants has rarely been reported. The yellowish spontaneous mutant of Ponkan (Citrus reticulata Blanco) (YP) was used to investigate the influence of metabolism reprogramming on postharvest performance. Our results show that reduced xanthophyll accumulation is the cause of yellowish coloring of YP and might be attributed to the reduced carotenoid sequestration capacity and upregulated expression of carotenoid cleavage dioxygenase genes. Constantly higher levels of polymethoxylated flavones (PMFs) during the infection and the storage stage might make significant contribution to the more strongly induced resistance against Penicillium digitatum and lower rotting rate. The present study demonstrates the feasibility of applying bud mutants to improve the postharvest performance of citrus fruits.

Carotenoids and volatile profiles of yellow- and red-fleshed papaya fruit in relation to the expression of carotenoid cleavage dioxygenase genes

Fruit of two different papaya cultivars (‘Sui huang’, yellow flesh, and ‘Sui hong’, red flesh) were characterized in terms of carotenoid and volatile profiles throughout storage at 25°C. The results showed that ‘Sui huang’ cultivar exhibited significantly lower carotenoid contents than ‘Sui hong’, which accounted for the different color characteristic. The analyses of HPLC coupled to mass spectrometry showed that zeaxanthin, β-cryptoxanthin and and β-carotene were identified as the most abundant carotenoids in ‘Sui huang’ fruit while the most abundant carotenoids were lycopene, followed by β-carotene, zeaxanthin and β-cryptoxant hin in ‘Sui hong’ fruit. The high expression of carotenoid cleavage dioxygenase gene (CpCCD1) in ‘Sui hong’ papaya fruit might stimulate the degradation of β-carotene and lycopene and then form the specific volatiles of 6-methyl-5-hepten-2-one and β-ionone.

Production of β-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae.

BackgroundApocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants—like raspberries and roses—by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid β-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP.ResultsIntegration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of β-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased β-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of β-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase.ConclusionsAn efficient β-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene—the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0273-x) contains supplementary material, which is available to authorized users.

Expression Profile of Carotenoid Cleavage Dioxygenase Genes in Summer Squash (Cucurbita pepo L.).

Carotenoids are important dietary components that can be found in vegetable crops. The accumulation of these compounds in fruit and vegetables is altered by the activity of carotenoid cleavage dioxygenases (CCDs) enzymes that produce their degradation. The aim of this work was to study the possible implication of CCD genes in preventing carotenoid storage in the horticultural crop summer squash (Cucurbita pepo L.). The relationship between the presence of these compounds and gene expression for CCDs was studied in three varieties showing different peel and flesh colour. Expression analysis for the CCD genes CpNCED1, CpNCED2, CpNCED3, CpNCED9, CpCCD1, CpCCD4a, CpCCD4b and CpCCD8 was carried out on different organs and at several fruit developmental stages. The results showed that the CpCCD4a and CpCCD4b genes were highly expressed in the variety with lowest carotenoid content suggesting a putative role in carotenoid accumulation pattern in summer squash fruit.

Differential spatio-temporal expression of carotenoid cleavage dioxygenases regulates apocarotenoid fluxes during AM symbiosis

Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis.

New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus.

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC-DAD and the volatile products by GC/MS. β-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9',10') bonds and generating β-ionone, although β-cyclocitral resulting from a 7,8 (7',8') cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.

Comparison of a compatible and an incompatible pepper-tobamovirus interaction by biochemical and non-invasive techniques: Chlorophyll a fluorescence, isothermal calorimetry and FT-Raman spectroscopy

Leaves of a pepper cultivar harboring the L3 resistance gene were inoculated with Obuda pepper virus (ObPV), which led to the appearance of hypersensitive necrotic lesions approx. 72 h post-inoculation (hpi) (incompatible interaction), or with Pepper mild mottle virus (PMMoV) that caused no visible symptoms on the inoculated leaves (compatible interaction). ObPV inoculation of leaves resulted in ion leakage already 18 hpi, up-regulation of a pepper carotenoid cleavage dioxygenase (CCD) gene from 24 hpi, heat emission and declining chlorophyll a content from 48 hpi, and partial desiccation from 72 hpi. After the appearance of necrotic lesions a strong inhibition of photochemical energy conversion was observed, which led to photochemically inactive leaf areas 96 hpi. However, leaf tissues adjacent to these inactive areas showed elevated ΦPSII and Fv/Fm values proving the advantage of chlorophyll a imaging technique. PMMoV inoculation also led to a significant rise of ion leakage and heat emission, to the up-regulation of the pepper CCD gene as well as to decreased PSII efficiency, but these responses were much weaker than in the case of ObPV inoculation. Chlorophyll b and total carotenoid contents as measured by spectrophotometric methods were not significantly influenced by any virus inoculations when these pigment contents were calculated on leaf surface basis. On the other hand, near-infrared FT-Raman spectroscopy showed an increase of carotenoid content in ObPV-inoculated leaves suggesting that the two techniques detect different sets of compounds.

Phylogenetic analysis and evolutionary studies of plant carotenoid cleavage dioxygenase gene.

The oxidative breakdown of carotenoid evidences the formation of apocarotenoids through carotenoid cleavage dioxygenases (CCDs). Numerous CCDs and apocarotenoids have been identified and characterized in plants. Using available sequence data, a study was performed to investigate the phylogenetic relationship among CCD genes and to statistically estimate the sequence conservation and functional divergence. In total, 77 genes were identified from 39 species belonging to 21 families. Our result of phylogenetic analysis indicated the existence of well-conserved subfamilies. Moreover, comparative genomic analysis showed that the gene structures of the CCDs were highly conserved across some different lineage species. Through functional divergence analysis, a substantial divergence was found between CCD subfamilies. In addition, examination of the site-specific profile revealed the critical amino acid residues accounting for functional divergence. This study mainly focused on the evolution of CCD genes and their functional divergence which may deliver an initial step for further experimental verifications.

Functional characterisation of three members of the Vitis viniferaL. carotenoid cleavage dioxygenase gene family

BackgroundIn plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the “varietal character”. Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown.ResultsThree grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine.ConclusionsTaken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids.

Identifying a Carotenoid Cleavage Dioxygenase (ccd4) Gene Controlling Yellow/White Fruit Flesh Color of Peach

Peach flesh color is a monogenic trait with the white phenotype being dominant over the yellow; its expression has been reported to be determined by a carotenoid degradative enzyme. In the present study, a carotenoid cleavage dioxygenase (ccd4) gene was analyzed to test whether it can be responsible for the flesh color determinism. The analysis was conducted on chimeric mutants with white and yellow sectors of the fruit mesocarp; it was then extended to a pool of cultivars and a segregating F1 population. A ccd4 functional allele is consistently associated with the ancestral white flesh color; on the other hand, the yellow phenotype originated from at least three independent mutations disrupting ccd4 function, thus preventing carotenoid degradation. In addition, retro-mutations recovering ccd4 function and re-establishing the ancestral white flesh color were detected. Our results show that ccd4 is the gene controlling flesh color in peach; its expression results in the degradation of carotenoids in white-fleshed genotypes, while the yellow color arises as a consequence of its inactivation.

Comparative study of putative 9-cis-epoxycarotenoid dioxygenase and abscisic acid accumulation in the responses of Sunki mandarin and Rangpur lime to water deficit

Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.

CDNA cloning of the phytoene synthase (PSY) and expression analysis of PSY and carotenoid cleavage dioxygenase genes in Osmanthus fragrans

In the carotenoid biosynthesis pathway, phytoene synthase (PSY) catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate (GGPP) to phytoene and has been shown to be a rate-limiting enzyme for the synthesis of carotenoids. In this study, a PCR approach was used to isolate a phytoene synthase cDNA from flower of Osmanthus fragrans. It contained a 1314 bp CDS. The predicted protein (49.4 kDa) displayed a sequence of 438 amino acid residues. Phylogenetic analysis demonstrated that the O. fragrans PSY gene (OfPSY) clustered with Gardenia jasminoides, Coffea canephora, Lycium barbarum and Mespilus germanica. Moreover, the OfPSY sequence relates closely with other PSY sequences of higher plants. We detected flower’s carotenoid content and analysed OfPSY and carotenoid cleavage dioxygenase genes (OfCCD1 and OfCCD4) in two cultivars of O. fragrans with varied coloration, including O. fragrans “Yingui” (light yellow) and “Dangui” (orange) at different floral stages. We observed that “Dangui” can accumulate carotenoids when OfPSY transcript was already constant, and this showed that the OfPSY does not play a role in the accumulation of total carotenoid in this cultivar. However, the transcripts of OfCCD4 in “Yingui” flower petals were clearly higher than that in “Dangui”. This was consistent with the ionone content in “Yingui” flower petals, which was significantly higher than that in “Dangui”. This response could contribute to explain the different accumulation of carotenoids in petals of the 2 cultivars.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia)

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress.

Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin.

Vascular-specific activity of the Arabidopsis carotenoid cleavage dioxygenase 7 gene promoter

Carotenoid cleavage dioxygenases (CCDs) are involved in the production of diverse apocarotenoids including phytohormones, the visual molecules and the aromatic volatile compounds derived from carotenoids. Here, we examined the spatial expression of four of the CCD genes (AtCcd1, 4, 7 and 8) among the nine members of this family in Arabidopsis by RT-PCR. We found that the AtCcd7 gene showed strong expression in seeds. However, the promoter activity of the 1,867-bp 5'-upstream region of this gene exhibited a vascular specificity at all developmental stages throughout the transgenic Arabidopsis plants tested. The strength of the AtCcd7 promoter was also found to be lower than that of the 35S promoter by about 60%. The whole body expression of the β-glucuronidase (GUS) reporter gene driven by the AtCcd7 promoter in Arabidopsis plants was confirmed in different organs by RT-PCR and GUS enzymatic assays. Histochemical GUS staining further revealed that the AtCcd7 promoter has utility in limiting the expression of target genes to the vascular tissues in all plant organs such as the leaf, stem, root, flower and seed.

Molecular evolution of two consecutive carotenoid cleavage dioxygenase genes in strigolactone biosynthesis in plants

Strigolactones are newly discovered plant hormones that perform various functions, from signaling in symbiotic interactions with arbuscular mycorrhizal fungi to controlling outgrowth of axillary buds. We examined the phylogenetic relationships of two carotenoid cleavage dioxygenase genes (CCD7 and CCD8) that are involved in consecutive upstream steps of the proposed strigolactone biosynthesis pathway. The CCD7 and CCD8 sequences from 11 model species, divided into two clades, correspond to sequences from monocotyledons and dicotyledons. However, the sequences from the primitive moss, Physcomitrella patens, appeared to be evolutionarily distinct from those of the angiosperms. CCD7 and CCD8 are much conserved, since no significant positive selection was detected among these plants. Ks values indicated that CCD7 and CCD8 diverged about 290 to 430 million years ago. As essential genes in the strigolactone pathway, the divergence timing of the conserved CCD7 and CCD8 genes reflects the approximate time of generation of strigolactone as a regulatory substance. This timing calculation also coincides with initiation of symbiosis between plants and microorganisms, inferred from the fossil record. Molecular evolution analyses of genes in metabolic pathways can provide insight concerning gene evolution.

Modified CAROTENOID CLEAVAGE DIOXYGENASE8 expression correlates with altered branching in kiwifruit (Actinidia chinensis)

• CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants, including Arabidopsis, pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). • Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8. • Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence. • The CCD pathway for control of branch development is conserved across a wide range of species, including kiwifruit, a woody perennial.

Feedback-Regulation of Strigolactone Biosynthetic Genes and Strigolactone-Regulated Genes in Arabidopsis

Strigolactones (SLs) have recently been found to regulate shoot branching, but the functions of SLs at other stages of development and the regulation of SL-related gene expression are mostly unknown in Arabidopsis. In this study, we performed real-time reverse transcription-PCR (RT-PCR) and microarray analysis using wild-type plants and SL-deficient/insensitive mutants to understand the molecular mechanisms underlying SL biosynthesis and signaling. We found that there is responsiveness to SL in the gene expression of Arabidopsis seedlings, which includes feedback regulation of two carotenoid cleavage dioxygenase genes. Microarray analysis revealed that exogenously applied SL regulated the expression of several genes, including light signaling-related genes and auxin-inducible genes. We also found that MORE AXILLARY GROWTH (MAX)2 plays an important role in the expression of SL-regulated genes. Our data support previous studies indicating that SL might function at the seedling stage. Analysis of SL-responsive and MAX2 downstream gene candidates provides new opportunities to broaden our understanding of SL signaling.

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 is involved in the production of negative and positive branching signals in petunia.

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.