Resumo Novas abordagens para o desenvolvimento de organismos geneticamente modificados, resistentes a doenças, incluem alvos específicos de expressão gênica, visando a aumentar as chances de controle do patógeno. A expressão gênica, induzida por promotores de genes de Citrus sinensis, derivados do floema, poderia ser avaliada e comparada com aquela induzida por um promotor constitutivo no mesmo tecido, levando à produção de laranjas-doces transgênicas, potencialmente mais resistentes a doenças causadas por bactérias de floema. Plantas de citrange ‘Carrizo’ [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] foram transformadas, via Agrobacterium tumefaciens, com o vetor binário pCAMBIA2301 contendo o gene uidA (ß-glucuronidase), dirigido pelo promotor constitutivo CaMV35S (CaMV35S :: uidA) ou pelos promotores de citros CsPP2.B1 (CsPP2 .B1 :: uidA) ou CsVTE2 (CsVTE2 :: uidA). Brotos regenerados in vitro foram enxertados em limão ‘Cravo’ (C. limonia Osbeck). A transformação genética foi confirmada por análises de Southern blot. A expressão do gene uidA foi avaliada por RT-qPCR, e a histolocalização do gene, dirigida por esses três promotores, foi acessada por seções de ramos tratadas com X-GLUC. A expressão do gene uidA, exibida por promotores específicos de tecido, foi, em geral, menor do que a do promotor constitutivo CaMV35; no entanto, as construções dirigidas por promotores específicos de tecido podem levar à expressão em tecidos restritos. Os promotores CsPP2.B1 e CsVTE2 podem ser considerados adequados para utilização em construções gênicas, visando à resistência a doenças.

Abstract New approaches for developing disease-resistant genetically modified organisms have included specific targets for gene expression to enhance the chances for pathogen control. Gene expression driven by phloem-derived Citrus sinensis gene promoters could be evaluated and compared with the expression induced by a strong constitutive promoter in the same tissue, leading to the production of transgenic sweet oranges potentially more resistant to diseases caused by phloem-limited bacteria. ‘Carrizo’ citrange [ (Poncirus trifoliataL.) Raf. x Citrus sinensis (L.) Osbeck] was transformed, via Agrobacterium tumefaciens, with the binary vector pCAMBIA2301 bearing the uidA gene (ß-glucuronidase) driven by the CaMV35S constitutive promoter (CaMV35S::uidA) or by the CsPP2.B1 (CsPP2.B1::uidA) or by the CsVTE2 (CsVTE2::uidA) citrus promoters. In vitro regenerated shoots were grafted onto ‘Rangpur’ lime (C. limonia Osbeck). The genetic transformation was confirmed by Southern blot analyses. uidA gene expression was evaluated by RT-qPCR, and gene histolocalization controlled by these three promoters was accessed by X-GLUC treated stem sections. uidA gene expression exhibited by tissue-specific promoters was overall lower than from the constitutive promoter CaMV35; however, constructs driven by tissue-specific promoters may lead to expression in restricted tissues. CsPP2.B1 and CsVTE2 promoters can be considered adequate for the utilization in gene constructs aiming disease resistance.

ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV.

O Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura vem gerando híbridos para utilização como porta-enxertos, que necessitam ser avaliados em relação ao comportamento frente à infecção natural por isolados locais de Citrus tristeza virus (CTV) e à presença de sintomas de descamamento eruptivo (BahiaBarkScaling disease - BBS). Este trabalho apresenta resultados da avaliação do comportamento de 141 híbridos (sob a forma de pés-francos ou enxertados) estabelecidos na área experimental da Embrapa Mandioca e Fruticultura, no Recôncavo Sul da Bahia. Foram avaliadas a presença e a severidade de sintomas de caneluras e descamamento por meio de escala de notas. Para detectar a presença do CTV, foi utilizado o método sorológico de ELISA indireto e RT-PCR. Os híbridos avaliados foram classificados como imunes, tolerantes e intolerantes ao CTV. A maioria dos híbridos que apresentaram sintomas de BBS tem uma tangerineira como parental.

The Citrus Breeding Program of Embrapa Cassava & Fruits have been generating hybrids for using as rootstocks, which need to be evaluated in relation to behavior in the natural infection with local strains of Citrus tristeza virus, CTV and the presence of symptoms of flaking eruptive (Bahia Bark Scaling disease - BBS). This study presents the results of the evaluation of 141 hybrids (nucelar seedlings and grafted in rootstocks) stated in the experimental area of Embrapa Cassava & Fruits in the Southern Recôncavo of the states of Bahia. Severity of stem pitting of CTV and BBS symptoms were evaluated by using a rating scale. It was used serological method of ELISA (indirect method) to detect the presence of CTV and RT-PCR. The hybrids evaluated were classified as immunes, tolerant and intolerant to CTV Most of the hybrids that showed symptoms of BBS had a mandarin as parental.

Citrus leprosis (CL) is a serious threat to the citrus industry, especially for sweet oranges. For a long time, Citrus spp. were considered the only susceptible hosts. However, other plant species were also found either experimentally or naturally to be susceptible to Citrus leprosis virus C (CiLV-C). To assess the experimental host range of CiLV-C, a large number of plant species were inoculated with Brevipalpus phoenicis, viruliferous to CiLV-C, under experimental conditions. Out of the 140 tested species (43 families), 59 species (24 families) developed localized chlorotic and/or necrotic lesions upon inoculation of leaves with viruliferous mites, and 40 species (18 families) of them yielded positive results for CiLV-C detection in at least one of the following assays: ELISA, RT-PCR, transmission electron microscopy and immunfluorescence. For those that developed lesions and yielded negative results in CiLV-C detection assays, the results may be attributed to the small number of lesions and their necrotic state with very little viral material. The fact that a considerable number of plant species are susceptible to the virus after mite inoculation brings up implications for the epidemiology, quarantine and evolution of the citrus leprosis pathosystem.

Citrus leprosis (CL) was first described in South America in the 1920's. It is considered similar to a disease first observed back to 1860 in Florida. It is a destructive disease characterized by localized lesions on the leaves, fruits and stems, which may lead to the death of the affected plant if left untreated. Around 1940, CL was demonstrated to be transmitted by Brevipalpus mites in Argentina. Because little information exists on the status of CL pathosystem in Argentina and Paraguay, a survey was made in several citrus growing areas of these countries from 2009 to 2011, to evaluate its presence and relevance as well as the identification of the virus and the mite vector. CL was found in most of the sweet orange and/or mandarin orchards in Paraguay (Departamentos de Boquerón, Concepción, San Pedro, Cordillera, Alto Paraná, Itapúa) and Argentina (Provincias de Misiones, Corrientes, Entre Ríos). Incidence was usually low. The causal virus was identified as Citrus leprosis virus C (CiLV-C) by RT-PCR, electron microscopy and immunofluorescence. In all the visited regions in Paraguay and the region of Montecarlo, Argentina, the mites collected in plants infected by CiLV-C were identified as B. phoenicis.

This work reports the in vitro expression of Citrus leprosis virus C (CiLV-C) putative coat protein (p29) and the production of a polyclonal antibody to be used as a tool for serological diagnosis of citrus leprosis. The ORF2/RNA1, corresponding to p29, was cloned in pET28a and transformed into Escherichia coli cells (BL21). Expression of p29 was induced in vitro and the protein was purified and used for immunization of rabbits to produce the polyclonal antibody. The anti p29 serum was shown to be highly specific to CiLV-C detection by immunological methods (Western blot, PTA-ELISA, tissue blot and in situ immunolocalization), without cross reaction with healthy citrus plants or other cytoplasmic and nuclear viruses transmitted by Brevipalpus mites. These results demonstrate that the antibody against CiLV-C p29 protein is highly specific for CiLV-C detection. In situ immunogold labeling assays on thin sections of sweet orange leaf cells infected by CiLV-C demonstrated that short, bacilliform particles present within cisternae of the endoplasmic reticulum were specifically labeled, confirming their viral nature. The dense cytoplasmic viroplasm was also heavily labeled indicating that it represents a site of p29 accumulation.

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Em 2005 houve um relato da presença da leprose dos citros no Estado de Chiapas, ao sul do México, baseado em sintomatologia. Subsequentemente a doença foi constatada em pomares de laranjeiras doces no Estado de Tabasco, nas localidades de Huimanguillo e Cunduacan. Amostras de folhas com lesões lepróticas de laranjeiras Valencia ou Hamlin foram coletadas e posteriormente examinadas ao microscópio eletrônico e submetidas a ensaios de detecção molecular por RT-PCR usando primers específicos. Estudos ultraestruturais revelaram alterações citopatológicas típicas causadas pelo CiLV-C (partículas baciliformes curtas no lúmen do retículo endoplasmático e a presença de viroplasmas vacuolados e elétron-densos no citoplasma) e hipertrofia em algumas células do parênquima lacunoso. Ensaios de RT-PCR resultaram na amplificação, na maioria das amostras de folhas dessecadas, de fragmento de DNA de tamanho esperado. Ácaros Brevipalpus coletados nos pomares foram identificados como B. phoenicis. Ensaios de transmissão com este ácaro confirmaram seu papel como vetor do CiLV-C para laranja doce, mas não houve transmissão para limão, limão Volkameriana, citrange C35 e citrange Carrizo. Todos estes dados considerados em conjunto confirmam que CiLV-C está presente em Tabasco, México e que a leprose dos citros descrita em Chiapas é causada pelo CiLV-C.

Citrus leprosis was detected in sweet oranges in Chiapas, Mexico, in 2005 based on symptoms. The disease was soon after observed in sweet orange orchards at Huimanguillo and Cunduacan, state of Tabasco. Leaf samples of leprosis-affected Valencia or Hamlin sweet oranges were collected and subjected to ultrastructural examination and molecular detection of CiLV-C by RT-PCR. Cytopathic effects typical of CiLV-C infection as presence of short, baciliform particles in the endoplasmic reticulum cisternae and electron dense, vacuolated viroplasma in the cytoplasm, as well as hypertrophy of some spongy parenchyma cells were observed in the tissues of the leaf lesions. RT-PCR using specific primers for CiLV-C produced DNA fragments of the expected size. Brevipalpus mites were present in these orchards and identified as B. phoenicis, the known vector for CiLV-C. Transmission tests with these mites confirmed their role as leprosis vectors to sweet orange but not to lemon, Volkamerian lemon, C35 citrange and Carrizo citrange. These data confirm the presence of CiLV-C in Tabasco, Mexico and that the citrus leprosis found in Chiapas was caused by CiLV-C.

A doença leprose dos citros tem etiologia viral sendo o citrus leprosis virus seu agente etiológico. Demonstrou-se que há dois vírus distintos que causam sintomas de leprose em ci-tros: citoplasmático (CiLV-C) e o nuclear (CiLV-N). O objetivo desse estudo foi comparar as diferenças morfo-anatômicas nas lesões causadas por CiLV-C e por CiLV-N em laranjeira doce (Citrus sinensis (L.) Osbeck) 'Pêra'. As lesões foliares e dos frutos foram coletadas em Piracicaba/SP (tipo citoplas-mático) e em Monte Alegre do Sul/SP e Amparo/SP (tipo nuclear). As lesões foram fotografadas e em seguida fixadas em solução Karnovsky, desidratadas em série etílica, incluídas em historesina e secionadas em micrótomo rotativo. As lâminas foram coradas, analisadas e fotografadas. As lesões foliares e do fruto causadas pelos dois vírus eram morfologicamente distintas. Somente a lesão causada por CiLV-N apresentou três regiões bem definidas. Em ambas as lesões ocorreu acúmulo de substâncias lipídicas nas áreas necrosadas que se achavam envoltas por células com conteúdo protéico amorfo ou em gotas. Somente as lesões da folha e do fruto causadas pelo CiLV-N exibiram ductos traumáticos gomosos nos feixes vasculares.

The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 μm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.

O objetivo deste trabalho foi buscar novas fontes de resistência à leprose-dos-citros, no Banco Ativo de Germoplasma do Centro APTA Citros Sylvio Moreira, Instituto Agronômico, em Cordeirópolis, SP. Foram utilizadas plantas obtidas por sementes de 26 acessos, infectadas com o vírus da leprose-dos-citros (Citrus leprosis virus - CiLV), por meio do seu vetor Brevipalpus phoenicis. O aparecimento de lesões, a partir de 21 dias após a inoculação, foi observado em 11 dos genótipos testados (42,3%). Quinze espécies, entre elas Citrus pennivesiculata e C. celebica, comportaram-se como altamente resistentes, enquanto outras, como C. keraji, foram mais suscetíveis que o padrão C. sinensis. Os dados mostraram grande variação de respostas de Citrus spp. à leprose, com elevado número de espécies resisentes, que podem ser utilizadas como fonte de resistência à doença em programas de melhoramento.

The objective of this work was to prospect for new sources of resistance to leprosis in the Citrus Germplasm Bank of Centro APTA Citros Sylvio Moreira, Instituto Agronômico, in Cordeirópolis, SP, Brazil. Plants obtained through seeds from 26 citrus accessions were mite-infected with Citrus leprosis virus (CiLV). Typical lesions started to appear in 11 genotypes (42.3% of the tested accessions) 21 days after inoculation. Fifteen species, among them Citrus pennivesiculata and C. celebica, behaved as highly resistant, while other genotypes, as C. keraji, were more susceptible than the standard C. sinensis. The data showed a wide variation in the response of Citrus spp. to leprosis, with a large number of resistant species, wich can be used as source of resistance to this disease, in breeding programs.

O objetivo deste trabalho foi apresentar a descrição anatômica dos tecidos lesionados pelo Citrus leprosis virus do tipo citoplasmático (CiLV-C), em ramos e folhas de laranjeira-doce (Citrus sinensis (L.) Osbeck) 'Pêra', e fornecer dados para a compreensão das interações entre o vírus e a planta hospedeira. Tecidos que apresentavam lesões foram seccionados em micrótomo rotativo (5-7 µm de espessura) e, posteriormente, as lâminas foram coradas e montadas em resina sintética. Nas análises realizadas no microscópio eletrônico de varredura, as amostras, depois de fixadas, foram imersas em glicerina 30% e, em seguida, criofraturadas, desidratadas em série etílica, secadas ao ponto crítico de CO2, e cobertas com uma delgada camada de ouro. As lesões foliares se iniciaram como pontuações necróticas, envolvidas por halos cloróticos que limitam o crescimento da lesão. Nos ramos, ocorreram dois tipos de lesões com fendas. Na região da fenda, houve intensa hiperplasia do parênquima cortical e floemático, que promoveu a obliteração das células condutoras do floema. A formação de ductos gomosos traumáticos nas lesões caulinares foi descrita. As alterações verificadas no floema podem explicar porque o vírus torna-se não-sistêmico.

The objective of this work was to present the anatomical description of tissues lesioned by Citrus leprosis virus cytoplasmatic type (CiLV-C), in leaves and twigs of 'Pêra' sweet orange (Citrus sinensis (L.) Osbeck) providing new data to understand the interactions between the virus and the plant host, in Brazil. Leprosis lesions were cut on a rotary microtome (5-7 µm thick), stained and mounted in synthetic resin. For scanning electron microscope analysis, fixed tissues were immersed in glycerin 30% and criofractured, dehydrated in a graded ethylic series, critical point drying and coated with gold. Foliar lesions began as necrotic points surrounded by a yellow halo that inhibited the lesion expansion. The twigs presented two different lesions: depressed ones and pustules with ruptures. There was hyperplasia of the cortical and phloematic parenchyma cells causing the obliteration of phloematic vessels. These histological alterations of the phloem tissue could explain why the virus does not become systemic even occasionally infecting phloem parenchyma cells. The development of gummosis traumatic ducts in twig lesions was described here.

Solano-violeta (Solanum violaefolium) é uma planta ornamental rasteira usada para cobrir solos de áreas sombreadas. Um vírus que induz manchas anelares nas folhas desta planta, tentativamente designado Solanum violaefolium ringspot virus - SvRSV, transmitido pelo ácaro Brevipalpus phoenicis (Acari: Tenuipalpidae) foi encontrado em Piracicaba, SP. Trata-se de um vírus baciliforme que se assemelha a outros vírus do tipo citoplasmático transmitidos por Brevipalpus sp. Este trabalho teve como objetivo relatar propriedades biológicas e estabelecer uma caracterização molecular parcial do SvRSV. O vírus pode ser transmitido mecanicamente a várias outras espécies botânicas, causando lesões localizadas. Entre as espécies avaliadas, Datura stramonium mostrou-se a melhor hospedeira experimental. Observou-se também a manifestação de sintomas nestas plantas após infestação das mesmas por B. obovatus previamente alimentado em lesões de SvRSV, confirmando esta outra espécie de ácaro como vetor do vírus. Suas propriedades físicas in vitro foram: temperatura de inativação 40-45 ºC; ponto final de diluição 10-3-10-4; longevidade in vitro 12 dias. Em secções ultrafinas, as partículas do SvRSV mostraram-se levemente mais delgadas e mais longas que as de outros vírus do mesmo grupo. A partir do dsRNA do SvRSV foi construída uma biblioteca de cDNA e foram identificadas duas possíveis regiões codificadoras das proteínas de movimento e replicase viral. Baseado nestas regiões foram desenhados "primers" para amplificação do RNA do SvRSV por RT-PCR. Sondas baseadas nas seqüências obtidas hibridizaram com ss- e dsRNA de D. stramonium infectadas pelo vírus. Ensaios preliminares de RT-PCR e hibridização não resultaram em reação com o vírus da leprose dos citros, tipo citoplasmático (CiLV-C).

Solanum violaefolium is an ornamental plant, with prostrate, trailing growth habit and is cultivated in shaded areas. A virus that causes ringspot symptoms on its leaves, tentatively named as Solanum violaefolium ringspot virus (SvRSV) and transmitted by Brevipalpus phoenicis (Acari: Tenuipalpidae) was found in Piracicaba city, São Paulo State. It is a bacilliform virus that resembles other cytoplasmic viruses transmitted by Brevipalpus sp. The objective of this work is to describe the biological properties and establish partial molecular characterization of the SvRSV. The SvRSV can be transmitted mechanically to several plant species causing local lesions. Among the tested species, Datura stramonium was proved to be the best experimental host. It was observed that S. violaefolium plants were infested by B. obovatus that also transmitted SvRSV in preliminary assays. Its in vitro physical properties were: thermal inactivation point: 40-45 ºC; dilution end point: 10-3-10-4 and in vitro longevity: 12 days. Ultrathin sections revealed that SvRSV particles are slightly thinner and longer than other cytoplasmic-type viruses transmitted by Brevipalpus sp. A cDNA library from dsRNA molecules extracted from SvRSV was constructed and two regions, which putatively code for the replicase and movement proteins were identified. Based on these sequences, primer pairs were designed for SvRSV´s RNA amplification by RT-PCR. Indeed, probes based on such sequences hybridized with ss- and dsRNA from infected D. stramonium plants. Preliminary molecular assays using primers and probes of SvRSV did not detect Citrus leprosis virus, cytoplasmic type (CiLV-C), another cytoplasmic type Brevipalpus-transmitted viruses.

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.br sebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.

RNA silencing mechanisms are conserved throughout eukaryotic evolution, possibly due to their importance in viral resistance and other aspects of cell biology. Here, we explored the Citrus EST (CitEST) database in search of sequences related to the most important known genes involved in RNA silencing. Transcripts strongly matching Argonaute (AGO), Dicer-like (DCL), Hua enhancer (HEN), and RNA-dependent RNA Polymerase (RdRP) were found in many of the citrus libraries. The reads were clustered and quantified. This shows that post-transcriptional gene silencing apparatus is active in citrus. It seems plausible that a better understanding of the players of RNA silencing in Citrus spp. and related genera may help create new tools to defeat the viral diseases that affect the citrus industry. Functional analyses of these citrus genes would enable the pursuit of this hypothesis.

Citrus species are known by their high content of phenolic compounds, including a wide range of flavonoids. In plants, these compounds are involved in protection against biotic and abiotic stresses, cell structure, UV protection, attraction of pollinators and seed dispersal. In humans, flavonoid consumption has been related to increasing overall health and fighting some important diseases. The goals of this study were to identify expressed sequence tags (EST) in Citrus sinensis (L.) Osbeck corresponding to genes involved in general phenylpropanoid biosynthesis and the key genes involved in the main flavonoids pathways (flavanones, flavones, flavonols, leucoanthocyanidins, anthocyanins and isoflavonoids). A thorough analysis of all related putative genes from the Citrus EST (CitEST) database revealed several interesting aspects associated to these pathways and brought novel information with promising usefulness for both basic and biotechnological applications.