RESUMO
Both strigolactones (SLs) and abscisic acid (ABA) biosynthetically originate from carotenoids. Considering their common origin, the interaction of these two hormones at the biosynthetic and/or regulatory level may be anticipated. Here we show that, in rice, drought simultaneously induces SL production in the root, and ABA production and the expression of SL biosynthetic genes in the shoot. Under control conditions, the ABA concentration was higher in shoots of the SL biosynthetic rice mutants dwarf10 (d10) and d17 than in wild-type plants, while a similar trend was observed for the SL perception mutant d3. These differences were enhanced under drought. However, drought did not result in an increase in leaf ABA content in the rice mutant line d27, carrying a mutation in the gene encoding the first committed enzyme in SL biosynthesis, to the same extent as in the other SL mutants and the wild type. Accordingly, d10, d17, and d3 lines were more drought tolerant than wild-type plants, whereas d27 displayed decreased tolerance. Finally, overexpression of OsD27 in rice resulted in increased levels of ABA when compared with wild-type plants. We conclude that the SL and ABA pathways are connected with each other through D27, which plays a crucial role in determining ABA and SL content in rice.
Assuntos
Ácido Abscísico/metabolismo , Secas , Lactonas/metabolismo , Oryza/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Reguladores de Crescimento de Plantas/genética , Folhas de Planta/fisiologia , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Estresse FisiológicoRESUMO
Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis-configured ß-carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used 13 C and 18 O-labeling to shed light on the reactions catalyzed by CCD8. The characterization of the resulting carlactone by LC-MS and NMR, and the identification of the assumed, less accessible second product allowed us to formulate a minimal reaction mechanism for carlactone generation.
Assuntos
Carotenoides/química , Dioxigenases/química , Lactonas/síntese química , Reguladores de Crescimento de Plantas/síntese química , Proteínas de Plantas/química , beta Caroteno/química , Biocatálise , Isótopos de Carbono , Dioxigenases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Isótopos de Oxigênio , Pisum sativum/química , Pisum sativum/enzimologia , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The Arabidopsis carotenoid cleavage dioxygenase 4 (AtCCD4) is a negative regulator of the carotenoid content of seeds and has recently been suggested as a candidate for the generation of retrograde signals that are thought to derive from the cleavage of poly-cis-configured carotene desaturation intermediates. In this work, we investigated the activity of AtCCD4 in vitro and used dynamic modeling to determine its substrate preference. Our results document strict regional specificity for cleavage at the C9-C10 double bond in carotenoids and apocarotenoids, with preference for carotenoid substrates and an obstructing effect on hydroxyl functions, and demonstrate the specificity for all-trans-configured carotenes and xanthophylls. AtCCD4 cleaved substrates with at least one ionone ring and did not convert acyclic carotene desaturation intermediates, independent of their isomeric states. These results do not support a direct involvement of AtCCD4 in generating the supposed regulatory metabolites. In contrast, the strigolactone biosynthetic enzyme AtCCD7 converted 9-cis-configured acyclic carotenes, such as 9-cis-ζ-carotene, 9'-cis-neurosporene, and 9-cis-lycopene, yielding 9-cis-configured products and indicating that AtCCD7, rather than AtCCD4, is the candidate for forming acyclic retrograde signals.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Dioxigenases/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Carotenoides/metabolismo , Cromatografia Líquida de Alta Pressão , Dioxigenases/metabolismo , Espectrometria de Massas , Especificidade por Substrato , Xantofilas/metabolismoRESUMO
MAIN CONCLUSION: The ß-carotene isomerase OsDWARF27 is stereo- and double bond-specific. It converts bicyclic carotenoids with at least one unsubstituted ß-ionone ring. OsDWARF27 may contribute to the formation of α-carotene-based strigolactone-like compounds. Strigolactones (SLs) are synthesized from all-trans-ß-carotene via a pathway involving the ß-carotene isomerase DWARF27, the carotenoid cleavage dioxygenases 7 and 8 (CCD7, CCD8), and cytochrome P450 enzymes from the 711 clade (MAX1 in Arabidopsis). The rice enzyme DWARF27 was shown to catalyze the reversible isomerization of all-trans- into 9-cis-ß-carotene in vitro. ß-carotene occurs in different cis-isomeric forms, and plants accumulate other carotenoids, which may be substrates of DWARF27. Here, we investigated the stereo and substrate specificity of the rice enzyme DWARF27 in carotenoid-accumulating E. coli strains and in in vitro assays performed with heterologously expressed and purified enzyme. Our results suggest that OsDWARF27 is strictly double bond-specific, solely targeting the C9-C10 double bond. OsDWARF27 did not introduce a 9-cis-double bond in 13-cis- or 15-cis-ß-carotene. Substrates isomerized by OsDWARF27 are bicyclic carotenoids, including ß-, α-carotene and ß,ß-cryptoxanthin, that contain at least one unsubstituted ß-ionone ring. Accordingly, OsDWARF27 did not produce the abscisic acid precursors 9-cis-violaxanthin or -neoxanthin from the corresponding all-trans-isomers, excluding a direct role in the formation of this carotenoid derived hormone. The conversion of all-trans-α-carotene yielded two different isomers, including 9'-cis-α-carotene that might be the precursor of strigolactones with an ε-ionone ring, such as the recently identified heliolactone.
Assuntos
Isomerases/fisiologia , Lactonas/metabolismo , Oryza/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Vias Biossintéticas , Carotenoides/química , Carotenoides/metabolismo , Cromatografia Líquida de Alta Pressão , Isomerases/química , Isomerases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Especificidade por SubstratoRESUMO
Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-ß-carotene at the C9'-C10' double bond, leading to ß-ionone and all-trans-ß-apo-10'-carotenal, both in vivo and in vitro. The enzyme also cleaved ß,ß-cryptoxanthin, zeaxanthin and lutein either at the C9'-C10' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-ß-carotene, which led to 9-cis-ß-apo-10'-carotenal. Our data indicate that all-trans-ß-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.
Assuntos
Biocatálise , Dioxigenases/metabolismo , Norisoprenoides/química , Solanum tuberosum/enzimologia , Xantofilas/metabolismo , beta Caroteno/química , beta Caroteno/metabolismo , Xantofilas/químicaRESUMO
Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7',8' double bonds adjacent to a 3-OH-ß-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-ß-apo-8'-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-ß-apo-8'-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the ß-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.
Assuntos
Carotenoides/biossíntese , Crocus/metabolismo , Dioxigenases/metabolismo , Biocatálise , Crocus/enzimologia , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents.
RESUMO
Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-ß-carotene to form a supposedly 9-cis-configured ß-apo-10'-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the ß-apo-10'-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10'-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-ß-carotene.
Assuntos
Arabidopsis/enzimologia , Dioxigenases/química , Pisum sativum/enzimologia , Proteínas de Arabidopsis , Carotenoides/química , Cinética , Espectroscopia de Ressonância Magnética , Estereoisomerismo , Especificidade por SubstratoRESUMO
Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ß-citraurin (3-hydroxy-ß-apo-8'-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and Mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ß-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ß-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ß-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7',8' double bond in zeaxanthin and ß-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7',8' double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.
Assuntos
Carotenoides/biossíntese , Carotenoides/metabolismo , Citrus/metabolismo , Sequência de Aminoácidos , Carotenoides/química , Cromatografia Líquida de Alta Pressão , Citrus/efeitos dos fármacos , Citrus/genética , Etilenos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Temperatura Alta , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Especificidade de Órgãos/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/genética , Alinhamento de SequênciaRESUMO
Strigolactones, phytohormones with diverse signaling activities, have a common structure consisting of two lactones connected by an enol-ether bridge. Strigolactones derive from carotenoids via a pathway involving the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8) and the iron-binding protein D27. We show that D27 is a ß-carotene isomerase that converts all-trans-ß-carotene into 9-cis-ß-carotene, which is cleaved by CCD7 into a 9-cis-configured aldehyde. CCD8 incorporates three oxygens into 9-cis-ß-apo-10'-carotenal and performs molecular rearrangement, linking carotenoids with strigolactones and producing carlactone, a compound with strigolactone-like biological activities. Knowledge of the structure of carlactone will be crucial for understanding the biology of strigolactones and may have applications in combating parasitic weeds.