RESUMEN
Optogenetic tools to control gene expression have many advantages over the classical chemically inducible systems, overcoming intrinsic limitations of chemical inducers such as solubility, diffusion, and cell toxicity. They offer an unmatched spatiotemporal resolution and permit quantitative and noninvasive control of the gene expression. Here we describe a protocol of a synthetic light-inducible system for the targeted control of gene expression in plants based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). The synthetic toggle switch system is in the ON state when plant protoplasts are illuminated with red light (660 nm) and can be returned to the OFF state by subsequent illumination with far-red light (760 nm). In this protocol, the implementation of a red light-inducible expression system in plants using Light-Emitting Diode (LED) illumination boxes is described, including the isolation and transient transformation of plant protoplasts from Arabidopsis thaliana and Nicotiana tabacum.
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Nicotiana/genética , Optogenética/métodos , Fitocromo B/genética , Proteínas de Plantas/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Mediciones Luminiscentes/métodos , Fitocromo B/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Protoplastos/efectos de la radiación , Nicotiana/metabolismo , Nicotiana/efectos de la radiaciónRESUMEN
In plants, type I and II S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology-based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56-58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 microm) and furfuryl thiol (Km 60 microm) were the best substrates (6-7-fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5-fold lower than with thiols. The enzyme was named C. roseus S-methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 so far is the only recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S-methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups.
Asunto(s)
Catharanthus/enzimología , Catharanthus/genética , Metiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformación Proteica , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.
Asunto(s)
Catharanthus/enzimología , Flavanonas/metabolismo , Flavonoides/metabolismo , Metiltransferasas/metabolismo , Oxigenasas/metabolismo , Secuencia de Aminoácidos , ADN Complementario/genética , Intrones/genética , Isoenzimas , Ácidos Cetoglutáricos/metabolismo , Metilación , Metiltransferasas/genética , Datos de Secuencia Molecular , Oxigenasas/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase (OMT) activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis. This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose. SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38-43 kDa). Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs. Homology-based RT-PCR identified cDNAs for four new OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT. The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases. The enzyme functions were investigated with purified recombinant proteins after cDNA expression in Escherichia coli. Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT. It represented a new type of OMT that performs two sequential methylations at the 3'- and 5'-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for C. roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.
Asunto(s)
Catharanthus/enzimología , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Catharanthus/genética , Catharanthus/metabolismo , Células Cultivadas , Regulación de la Expresión Génica de las Plantas , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Estructura Molecular , Filogenia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The molecular mechanisms underlying the onset of Fe-deficiency chlorosis and the maintenance of photosynthetic function in chlorotic chloroplasts are relevant to global photosynthetic productivity. We describe a series of graded responses of the photosynthetic apparatus to Fe-deficiency, including a novel response that occurs prior to the onset of chlorosis, namely the disconnection of the LHCI antenna from photosystem I (PSI). We propose that disconnection is mediated by a change in the physical properties of PSI-K in PSI in response to a change in plastid Fe content, which is sensed through the occupancy, and hence activity, of the Fe-containing active site in Crd1. We show further that progression of the response involves remodeling of the antenna complexes-specific degradation of existing proteins coupled to the synthesis of new ones, and establishment of a new steady state with decreased stoichiometry of electron transfer complexes. We suggest that these responses are typical of a dynamic photosynthetic apparatus where photosynthetic function is optimized and photooxidative damage is minimized in graduated responses to a combination of nutrients, light quantity and quality.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/fisiología , Deficiencias de Hierro , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis , Animales , Cloro/metabolismo , Clorofila/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica , Immunoblotting , Hierro/metabolismo , Espectrometría de Masas , Péptidos/química , Péptidos/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , Proteoma , ARN/metabolismo , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
Plant O-methyltransferases (OMTs) have important roles in secondary metabolite biosynthesis. Sequencing projects and homology-based cloning strategies yield sequences for proteins with similarities to known OMTs, but the identification of the physiological substrates is not trivial. We investigated with a cDNA cloned from Catharanthus roseus the possibilities for predicting the substrates of OMTs, using the information from previous work and two newly identified motifs that were based on information from the crystal structures of two plant OMTs. The results, confirmed by functional analysis of the recombinant protein, indicated that a careful analysis of the deduced protein sequence can provide clues for predicting the substrates of cloned OMTs.