RESUMO
Anthraquinones (AQs) constitute the largest group of natural quinones, which are used as safe natural dyes and have many pharmaceutical applications. In plants, AQs are biosynthesized through two main routes: the polyketide pathway and the shikimate pathway. The latter primarily forms alizarin-type AQs, and the prenylation of 1,4-dihydroxy-2-naphthoic acid (DHNA) is the first pathway-specific step. However, the prenyltransferase (PT) responsible for this key step remains uncharacterized. In this study, the cell suspension culture of Madder (Rubia cordifolia), a plant rich in alizarin-type AQs, was discovered to be capable of prenylating DHNA to form 2-carboxyl-3-prenyl-1,4-naphthoquinone and 3-prenyl-1,4-naphthoquinone. Then, a candidate gene belonging to the UbiA superfamily, R. cordifoliadimethylallyltransferase 1 (RcDT1), was shown to account for the prenylation activity. Substrate specificity studies revealed that the recombinant RcDT1 recognized naphthoic acids primarily, followed by 4-hydroxyl benzoic acids. The prenylation activity was strongly inhibited by 1,2- and 1,4-dihydroxynaphthalene. RcDT1 RNA interference significantly reduced the AQs content in R. cordifolia callus cultures, demonstrating that RcDT1 is required for alizarin-type AQs biosynthesis. The plastid localization and root-specific expression further confirmed the participation of RcDT1 in anthraquinone biosynthesis. The phylogenetic analyses of RcDT1 and functional validation of its rubiaceous homologs indicated that DHNA-prenylation activity evolved convergently in Rubiaceae via recruitment from the ubiquinone biosynthetic pathway. Our results demonstrate that RcDT1 catalyzes the first pathway-specific step of alizarin-type AQs biosynthesis in R. cordifolia. These findings will have profound implications for understanding the biosynthetic process of the anthraquinone ring derived from the shikimate pathway.
Assuntos
Antraquinonas , Dimetilaliltranstransferase , Rubia , Antraquinonas/metabolismo , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/genética , Rubia/metabolismo , Rubia/genética , Rubia/enzimologia , Especificidade por Substrato , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Naftóis/metabolismo , Naftoquinonas/metabolismo , Prenilação , Regulação da Expressão Gênica de PlantasRESUMO
Rubia yunnanensis Diels (R. yunnanensis), a Chinese perennial plant, is well-known for its medicinal values such as rheumatism, contusion, and anemia. It is rich in bioactive anthraquinones, but the biosynthetic pathways of anthraquinones in R. yunnanensis remain unknown. To investigate genes involved in anthraquinone biosynthesis in R. yunnanensis, we generated a de novo transcriptome of R. yunnanensis using the Illumina HiSeq 2500 sequencing platform. A total of 636,198 transcripts were obtained, in which 140,078 transcripts were successfully annotated. A differential gene expression analysis identified 15 putative genes involved in anthraquinone biosynthesis. Additionally, the hairy roots of R. yunnanensis were treated with 200 µM Methyl Jasmonate (MeJA). The contents of six bioactive anthraquinones and gene expression levels of 15 putative genes were measured using ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The results showed that the expressions levels for 11 of the 15 genes and the contents of two of six anthraquinones significantly increased by MeJA treatment. Pearson's correlation analyses indicated that the expressions of 4 of the 15 putative genes were positively correlated with the contents of rubiquinone (Q3) and rubiquinone-3-O-ß-d-xylopranosyl-(1â6)-ß-d-glucopyranoside (Q20). This study reported the first de novo transcriptome of R. yunnanensis and shed light on the anthraquinone biosynthesis and genetic information for R. yunnanensis.
Assuntos
Rubia , Antraquinonas/metabolismo , Cromatografia Líquida , Perfilação da Expressão Gênica/métodos , Rubia/genética , Rubia/metabolismo , Espectrometria de Massas em TandemRESUMO
Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.
Assuntos
Arabidopsis , Rubia , Tolerância ao Sal , Termotolerância , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Domínios Proteicos/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rubia/genética , Rubia/metabolismo , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Termotolerância/genéticaRESUMO
Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.
Assuntos
Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas , Reação em Cadeia da Polimerase em Tempo Real/normas , Rubia , Estresse Fisiológico/fisiologia , Transcriptoma/fisiologia , Padrões de Referência , Rubia/genética , Rubia/metabolismoRESUMO
Salinity which covers vast areas of the world is increasing every year. But some plants like madder can grow in these areas. Madder (Rubia tinctorum) is a perennial plant species from the Rubiaceae family. In this study, madder plants were first treated by different concentration of NaCl (100, 200, 300, and 400 mM). Then gene expression of salinity stress was studied. For gene study, vacuolar H+-pyrophosphatase pump (AVP) and tonoplast Na+/H+ antiporters (NHX) from madder plant were isolated and sequenced. Analyzing protein sequences of these genes demonstrated that the protein sequences have high similarity with the same genes in other plants. Constructing phylogenetic trees based on the protein sequences of the AVP and NHX genes, we found high similarity with Coffea arabica and Capsicum annuum, respectively. Studying gene expression of the AVP and NHX under the condition of salt stress revealed that the genes were up-regulated, which continues up to 400 mM of salt concentration.
Assuntos
Rubia/metabolismo , Tolerância ao Sal/genética , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos/genética , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Pirofosfatase Inorgânica/metabolismo , Filogenia , Folhas de Planta/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Rubia/genética , Salinidade , Estresse Salino , Vacúolos/metabolismoRESUMO
The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200â¯mg/l to 874â¯mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.
Assuntos
Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Proteínas Oncogênicas/genética , Rubia/genética , Rubia/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Linhagem Celular , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas Oncogênicas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Rubia/citologia , Rubia/crescimento & desenvolvimentoRESUMO
Rubia cordifolia L., is an industrially viable medicinal crop and is widely exploited for the therapeutic potential of its bioactive metabolite, purpurin. The present investigation aimed to explore the chemotypic and molecular variability in seven wild populations of R. cordifolia from South Eastern Ghats region of India. Thirty-eight individuals were assessed for molecular fingerprinting (ISSR markers) and densitometric quantification of purpurin and alizarin. The populations of Yelagiri Hills and Shervaroy Hills contained the highest levels of alizarin (0.115 and 0.093%, respectively) while Pachamalai and Kolli Hills revealed the highest purpurin content (0.284 and 0.280%, respectively). Genetic diversity was generally higher in the same populations that produced higher metabolite content, with the exception of Pachamalai, suggesting a highly prioritized conservation concern. The study revealed a Nei's total gene diversity at species level of 0.266 and of 0.187 at population level, with an average population genetic differentiation of 0.28. No clear genetic or chemical structure was retrieved between the studied populations, with individuals from different locations clustering together, and no significant correlation was obtained between metabolites and genetic diversity or between these and the populations' geographic distances.
Assuntos
Antraquinonas/metabolismo , Repetições de Microssatélites/genética , Rubia/genética , Rubia/metabolismo , Cromatografia Líquida de Alta Pressão , Marcadores Genéticos/genética , Índia , Polimorfismo GenéticoRESUMO
KEY MESSAGE: Overexpression of both native and mutant forms of AtCPK1 in Rubia cordifolia cells increased anthraquinone production and transcript abundance of the RcIPPI, RcOSBL, RcOSBS , and RcICS genes to different extents. Calcium-dependent protein kinases (CDPKs) are involved in various cell processes and are regulated by a calcium signal system. CDPKs also function in plant defense against stress factors such as pathogens, temperature, and salinity. In this study, we compared the effect of heterologous expression of two forms of the Arabidopsis AtCPK1 gene, native and constitutively active (Ca(2+)-independent), on anthraquinone production in transgenic Rubia cordifolia cells. Significant qualitative and quantitative differences were found in the content of anthraquinone derivatives in control and AtCPK1-transgenic calli. Expression of the AtCPK1 gene increased anthraquinone production by 3 and 12 times for native and constitutively active forms, respectively, compared with control cells. In addition, we identified and quantified the expression of genes encoding key enzymes of the anthraquinone biosynthesis pathway, including isochorismate synthase (ICS), o-succinylbenzoate synthase (OSBS), o-succinylbenzoate ligase (OSBL), and isopentenyl diphosphate isomerase (IPPi). In all AtCPK1-transgenic cell lines, expression of ICS, OSBS, OSBL, and IPPi increased considerably at 14-15 days of subculture and decreased at the end of cultivation (30 days). The results suggest that both native and constitutively active AtCPK1 forms induced anthraquinone accumulation at the logarithmic growth stage via enhancement of expression of genes involved in the metabolism of anthraquinones or their regulatory mechanisms.
Assuntos
Antraquinonas/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes de Plantas , Proteínas Quinases/genética , Rubia/genética , Rubia/metabolismo , Transformação Genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica de Plantas , Espectrometria de Massas , Fenótipo , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Rubia/crescimento & desenvolvimentoRESUMO
Expression of rol genes from Agrobacterium rhizogenes induces reprogramming of transformed plant cells and provokes pleiotropic effects on primary and secondary metabolism. We have previously established that the rolB and rolC genes impair reactive oxygen species (ROS) generation in transformed cells of Rubia cordifolia and Arabidopsis thaliana. In the present investigation, we tested whether this effect is associated with changes in the expression levels of NADPH oxidases, which are considered to be the primary source of ROS during plant-microbe interactions. We identified two full-length NADPH oxidase genes from R. cordifolia and examined their expression in non-transformed and rolB-transformed calli. In addition, we examined the expression of their homologous genes from A. thaliana in non-transformed and rolB-expressing cells. The expression of Rboh isoforms was 3- to 7-fold higher in both R. cordifolia and A. thaliana rolB-transformed cells compared with non-transformed cells. Our results for the first time show that Agrobacterium rolB gene regulates particular NADPH oxidase isoforms.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , NADPH Oxidases/genética , Rubia/genética , Transformação Genética , beta-Glucosidase/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Rubia/enzimologia , Alinhamento de Sequência , Estresse Fisiológico/genéticaRESUMO
Agrobacterium rhizogenes genetically transform plant cells naturally via horizontal gene transfer by the introduction of T-DNA from the Ri plasmid into genomic DNA to create favorable conditions for successful colonization. An intriguing feature of pRiA4-transformed cells is their recently discovered enhanced tolerance to abiotic stress stimuli and activation of antioxidant enzyme expression. The mechanism by which A. rhizogenes modulates the defense responses of transformed cells remains unclear. It has been established that calcium-dependent protein kinase (CDPK) genes mediate crosstalk of signaling pathways in plants, and these genes have been implicated in biotic and abiotic stress signaling. In this study, we identified fourteen CDPK genes from Rubia cordifolia and examined their expression in aerial plant organs as well as in non-transformed and A. rhizogenes A4-transformed calli. Expression of RcCDPK4, RcCDPK5, RcCDPK7, and RcCDPK10 was 1.2- to 3.9-fold higher in pRiA4-transformed cells than in non-transformed cells, whereas expression of RcCDPK1, RcCDPK9, RcCDPK11, and RcCDPK14 was 1.2- to 1.9-fold lower. Agrobacterium transformation substantially modified the transcriptional responses of specific RcCDPK isoforms in pRiA4-transformed cells under conditions of temperature- and salinity-induced stress. On the basis of the results, we suggest that A. rhizogenes T-DNA genes exert their diverse biological functions by altering the expression of various CDPK genes.
Assuntos
Proteínas de Bactérias/genética , Regulação da Expressão Gênica de Plantas , Proteínas Quinases/genética , Rubia/genética , Estresse Fisiológico , Agrobacterium/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos/genética , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Rubia/efeitos dos fármacos , Rubia/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , Temperatura , TranscriptomaRESUMO
The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.
Assuntos
Apoptose/genética , Expressão Gênica , Oncogenes , Células Vegetais/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Rubia/genética , Transformação Genética , Agrobacterium , Genes de Plantas , Microscopia Confocal , Necrose , Rubia/fisiologiaRESUMO
The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.
Assuntos
Agrobacterium/genética , Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Células Vegetais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , beta-Glucosidase/metabolismo , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Proteínas de Bactérias/genética , Morte Celular , Sobrevivência Celular , Meios de Cultura/metabolismo , Glutationa/metabolismo , Luz , Estresse Oxidativo , Panax/citologia , Panax/efeitos dos fármacos , Panax/genética , Panax/metabolismo , Paraquat/farmacologia , Células Vegetais/efeitos dos fármacos , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Rubia/efeitos dos fármacos , Rubia/genética , Rubia/metabolismo , Plantas Tolerantes a Sal/citologia , Plantas Tolerantes a Sal/efeitos dos fármacos , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Cloreto de Sódio/farmacologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Vitamina K 3/farmacologia , beta-Glucosidase/genéticaRESUMO
Here, seven new class III peroxidase genes of Rubia cordifolia L., RcPrx01-RcPrx07, were isolated and characterized. Expression of the Prx genes was studied in R. cordifolia aerial organs as well as in cells transformed with the rolB and rolC genes of Agrobacterium rhizogenes and cells transformed with the wild-type A. rhizogenes A4 strain. In rolC- and rolB-transformed cells, the rol genes were expressed under the control of the 35S promoter, whereas in A. rhizogenes A4-transformed cells the rol genes were expressed under the control of their native promoters. All studied peroxidase genes were greatly upregulated in rolB-overexpressing cells. In contrast, overexpression of the rolC gene and expression of the rol genes under the control of their native promoters had little effect on the abundance of peroxidase transcripts. In accordance with this observation, peroxidase activity was substantially increased in rolB cells and was slightly affected in other transformed cells. Our results indicate that rolB strictly affects the regulation of a set of seven R. cordifolia class III peroxidases.
Assuntos
Agrobacterium/genética , Proteínas de Bactérias/genética , Peroxidases/biossíntese , Peroxidases/genética , Rubia/genética , Rubia/microbiologia , Técnicas de Cultura de Tecidos , beta-Glucosidase/genética , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Ágar , Indução Enzimática , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes Bacterianos/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Funções Verossimilhança , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Peroxidases/química , Filogenia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Rubia/enzimologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transformação GenéticaRESUMO
Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.
Assuntos
Antraquinonas/metabolismo , Proteínas de Arabidopsis , Arabidopsis/genética , Biotecnologia/métodos , Plantas Geneticamente Modificadas/genética , Proteínas Quinases , Rubia/enzimologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/análise , Microscopia Confocal , Plantas Geneticamente Modificadas/enzimologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rubia/genética , Análise de Célula ÚnicaRESUMO
Calcium-dependent protein kinases (CDPKs) play an important role in plant cell responses to stress and pathogenic attack. In this study, we investigated the effect of heterologous expression of the Arabidopsis CDPK gene, AtCPK1, on anthraquinone production in transgenic Rubia cordifolia cells. AtCPK1 variants (a constitutively active, Ca(2+) -independent form and a non-active form used as a negative control) were transferred to callus cells by agrobacterial transformation. Overexpression of the constitutively active, Ca(2+) -independent form in R. cordifolia cells caused a 10-fold increase in anthraquinone content compared with non-transformed control cells, while the non-active form of AtCPK1 had no effect on anthraquinone production. Real-time PCR measurements showed that the activation of anthraquinone biosynthesis in transgenic calli correlated with the activation of isochorismate synthase gene expression. The activator effect of AtCPK1 was stable during prolonged periods of transgenic cell cultivation (more than 3 years) and the transgenic cultures exhibited high growth. Our results provide the first evidence that a CDPK gene can be used for the engineering of secondary metabolism in plant cells.
Assuntos
Antraquinonas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Quinases/metabolismo , Rubia/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , DNA de Plantas/química , DNA de Plantas/genética , Expressão Gênica , Técnicas de Transferência de Genes , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Proteínas Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rubia/genética , Análise de Sequência de DNA , Transformação GenéticaRESUMO
Microbe-plant interactions often lead to a decrease in the reactive oxygen species (ROS) level of plant cells, which allows pathogen survival through the suppression of plant immune responses. In the present investigation, we tested whether transformation of Rubia cordifolia cells by Agrobacterium rhizogenes had a similar effect. We isolated partial cDNA sequences of ascorbate peroxidase, catalase and Cu/Zn superoxide dismutase genes (RcApx1, RcApx2, RcApx3, RcCAT1, RcCAT2, RcCSD1, RcCSD2 and RcCSD3) from plant tissues, as well as pRiA4-transformed and normal calli of Rubia cordifolia, and studied their expression by real-time PCR. Transcription profiling revealed that ascorbate peroxidase (RcApx1) and Cu/Zn superoxide dismutase (RcCSD1) were the most abundant transcripts present in both plant tissues and non-transformed calli. Catalase genes were weakly expressed in these samples. The pRiA4-transformed calli showed enhanced expression of several genes encoding ROS-detoxifying enzymes. Confocal microscopy imaging revealed decreased ROS level in pRiA4-transformed calli compared to the control. These results demonstrate that A. rhizogenes, like other plant pathogens, uses a strategy aimed at decreasing ROS levels in host cells through the general upregulation of its antioxidant genes.
Assuntos
Antioxidantes/metabolismo , Regulação da Expressão Gênica de Plantas , Espécies Reativas de Oxigênio/metabolismo , Rhizobium/genética , Rubia/enzimologia , Rubia/microbiologia , Ascorbato Peroxidases , Catalase/genética , Catalase/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Temperatura Alta , Microscopia Confocal , Peroxidases/genética , Peroxidases/metabolismo , Filogenia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rubia/efeitos dos fármacos , Rubia/genética , Cloreto de Sódio/farmacologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transformação Genética/genéticaRESUMO
Among the different methods currently used to improve yields of secondary metabolites in cultured plant cells, the method involving transformation by rol genes represents an example of relatively new technology. These genes, isolated from plasmids of the plant pathogen Agrobacterium rhizogenes, are potential activators of secondary metabolism in transformed cells from the Solanaceae, Araliaceae, Rubiaceae, Vitaceae, and Rosaceae families. In some cases, the activator effect of individual rol genes was sufficient to overcome the inability of cultured plant cells to produce large amounts of secondary metabolites. Stimulation of production characteristics of cultured plant cells mediated by the rol genes was shown to be remarkably stable over long-term cultivation. In this chapter, we describe transformation of Rubia cordifolia L. cells with the rol genes as an example of metabolic engineering of secondary metabolites.
Assuntos
Proteínas de Bactérias/genética , Engenharia Genética/métodos , Rubia/genética , Rubia/metabolismo , Transformação Genética , Agrobacterium tumefaciens/genética , Antraquinonas/metabolismo , Proliferação de Células , Células Cultivadas , Rubia/citologiaRESUMO
It is known that expression of the Agrobacterium rhizogenes rolC gene in transformed plant cells causes defense-like reactions, such as increased phytoalexin production and expression of pathogenesis-related proteins. In the present study, we examined whether this phenomenon is associated with increased production of reactive oxygen species (ROS). Single-cell assays based on confocal microscopy and fluorogenic dyes (2,7-dichlorofluorescein diacetate and dihydrorhodamine 123) showed reduced steady-state levels of ROS in rolC-expressing Rubia cordifolia cells as compared with normal cells. Paraquat, a ROS inducer, caused significant ROS elevation in normal cells but had little effect on rolC-transformed cells. Likewise, ROS elevation triggered by a light stress was suppressed in transformed cells. Our results indicate that the rolC gene acts as a ROS suppressor in unstressed cells and its expression prevents stress-induced ROS elevations. We detected a two- to threefold increase in tolerance of rolC-transformed cells to salt, heat, and cold treatments. Simultaneously, rolC-transformed cells maintained permanently active defensive status, as found by measuring isochorismate synthase gene expression and anthraquinone production. Thus, the oncogene provoked multiple effects in which ROS production and phytoalexin production were clearly dissociated.
Assuntos
Proteínas de Bactérias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rubia/metabolismo , Estresse Fisiológico , Terpenos/metabolismo , Agrobacterium tumefaciens/genética , Análise de Variância , Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Células Cultivadas , Fluorometria , Regulação da Expressão Gênica de Plantas , Genes Bacterianos , Transferases Intramoleculares/metabolismo , Microscopia Confocal , Paraquat/farmacologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rubia/efeitos dos fármacos , Rubia/genética , Ácido Salicílico/metabolismo , Sesquiterpenos , FitoalexinasRESUMO
It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins. Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca2+ channel blockers and calcium deficiency. The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment. The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells. Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca2+ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine. These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca2+-dependent NADPH oxidase pathway.