Carrot AOX2a transcript profile responds to growth and chilling exposure

Alternative oxidase (AOX) is a key enzyme of the alternative respiration, known to be involved in plant development and in response to various stresses. To verify the role of DcAOX1 and DcAOX2a genes in carrot tap root growth and in response to cold stress, their expression was analyzed in two experiments: during root growth for 13 weeks and in response to a cold challenge trial of 7 days, in both cases using different carrot cultivars. Carrot root growth is initially characterized by an increase in length, followed by a strong increase in weight. DcAOX2a presented the highest expression levels during the initial stages of root growth for all cultivars, but DcAOX1 showed no particular trend in expression. Cold stress had a negative impact on root growth, and generally up-regulated DcAOX2a with no consistent effect on DcAOX1. The identification of cis-acting regulatory elements (CAREs) located at the promoters of both genes showed putative sequences involved in cold stress responsiveness, as well as growth. However, DcAOX2a promoter presented more CAREs related to hormonal pathways, including abscisic acid and gibberellins synthesis, than DcAOX1. These results point to a dual role of DcAOX2a on carrot tap root secondary growth and cold stress responseinfo:eu-repo/semantics/publishedVersio

Detection and Quantification of Fusarium spp. (F. oxysporum, F. verticillioides, F. graminearum) and Magnaporthiopsis maydis in Maize Using Real-Time PCR Targeting the ITS Region

Fusarium spp. and Magnaporthiopsis maydis are soil-inhabiting fungi and respectively the causal agents of fusarium ear rot and late wilt, two important diseases that can affect maize, one of the most important cereal crops worldwide. Here, we present two sensitive real-time PCR TaqMan MGB (Minor Groove Binder) assays that detect and discriminate several Fusarium spp. (F. oxysporum, F. verticillioides, and F. graminearum) from M. maydis. The method is based on selective real-time qPCR amplification of the internal transcribed spacer (ITS) region and allows the quantification of the fungi. The applicability of this newly developed TaqMan methodology was demonstrated in a field experiment through the screening of potentially infected maize roots, revealing a high specificity and proving to be a suitable tool to ascertain Fusarium spp. and M. maydis infection in maize. Its high sensitivity makes it very efficient for the early diagnosis of the diseases and also for certification purposes. Thus, qPCR through the use of TaqMan probes is here proposed as a promising tool for specific identification and quantification of these soil-borne fungal pathogens known to cause disease on a large number of crops

Transcription of alternative oxidase (AOX) and plastid terminal oxidase (PTOX) during stress-regulated root tissue growth in Daucus carota L. - An approach to identify functional marker candidates for breeding on carrot yield stability

A presente tese explora a hipótese de utilização dos genes da oxidase alternativa (AOX) e da oxidase terminal da plastoquinona (PTOX) como genes-alvo para o desenvolvimento de marcadores funcionais (MF) para avaliar a performance do crescimento em cenoura, fator determinante da produtividade. Para avaliar se os referidos genes estão associados com o crescimento da cenoura procedeu—se ao seu isolamento e posterior análise dos seus perfis de transcrição em diversos sistemas biológicos. O sistema in vitro selecionado, denominado sistema de culturas primárias, permitiu avaliar alterações na quantidade de transcritos desses genes durante os processos de reprogramação celular e crescimento. Ao nível da planta foi também estudado o efeito do frio na expressão precoce dos genes AOX. Ambos os genes DcAOX1 e DcAOX2a revelaram uma resposta rápida e um padrão semelhante apos stresse (inoculação in vitro e resposta ao frio). Foi igualmente verificado um incremento na expressão do gene DcPTOX durante a fase inicial do processo de reprogramação celular. Estudos de expressão dos genes AOX durante o desenvolvimento da raiz da cenoura revelaram que o gene DcAOX2a será potencialmente o gene mais envolvido neste processo. De modo a avaliar a hipótese de envolvimento do gene DcPTOX no crescimento da raíz procederam—se a estudos de expressão ao nível do tecido meristemático. Todavia, para um mais completo entendimento da ligação entre DcPTOX e o crescimento secundário e/ou acumulação de carotenos, a expressão do gene DcPTOX foi também avaliada em raízes de cenoura durante o desenvolvimento, utilizando cultivares caracterizadas por distintos conteúdos de carotenos. Os resultados obtidos demonstraram a associação do gene DcPTOX a ambos os processos. O envolvimento da PTOX no crescimento adaptativo da raiz foi analisado com um ensaio que permitiu identificar, no tecido meristemático, uma resposta precoce do gene DcPTOX face a uma diminuição da temperatura. Adicionalmente, foi efetuada a seleção de genes de referência para uma analise precisa da expressão génica por RT-qPCR em diversos sistemas biológicos de cenoura, e a importância do seu estudo ao nível do sistema biológico foi realçada. Os resultados desta tese são encorajadores para prosseguir os estudos de utilização dos genes AOX e PTOX como MF no melhoramento da performance do crescimento adaptativo em cenoura, fator determinante para a produtividade; ABSTRACT: This thesis explores the hypothesis of using the alternative oxidase (AOX) and theplastid terminal oxidase (PTOX) as target genes for functional marker (FM) development for yield-determining growth performance in carrot. To understand if these genes are associated to growth, different AOX gene family members and the single PTOX gene were isolated, and their expression patterns evaluated in diverse carrot plant systems. An in-vitro primary culture system was selected to study AOX and PTOX transcript changes during cell reprogramming and growth performance. At plant level, a putative early response of AOX to chilling was also evaluated. In fact, both DcAOXl and DcAOXZa were early responsive and showed similar patterns under stress conditions (in vitro inoculation and chilling). A role for DcPTOX during earliest events of cell reprogramming was also suggested. Next, the expression profiles of AOX gene family members during carrot tap root development were investigated. DcAOXZa was identified as the most responsive gene to root development. In order to evaluate if DcPTOX is associated with carrot tap root growth performance, DcPTOX transcript levels were measured in the central root meristem. To further understand whether DcPTOX is associated with secondary growth and/or carotenoids accumulation, DcPTOX expression was also studied in deveIOping carrot tap roots in cultivars with different carotenoids contents. The results indicated that DcPTOX associates to both carotenoid biosynthesis and secondary growth during storage root development. To obtain further insights into the involvement of PTOX on adaptive growth, the early effects of temperature decrease were explored in the root meristem, where a short—term early response in DcPTOX was found, probably associated with adaptive growth. Furthermore, a selection of the most suitable reference genes for accurate RT—qPCR analysis in several carrot experimental systems was performed and discussed. The present research provides the necessary toolbox for continuing studies in carrot AOX and PTOX genes as promising resources for FM candidates in order to assist breeding on yield—determining adaptive growth performance

Carrot alternative oxidase gene AOX2a demonstrates allelic and genotypic polymorphisms in intron 3

Single nucleotide polymorphisms (SNPs) and insertion–deletions (InDels) are becoming important genetic markers for major crop species. In this study, we focus on variations at genomic level of the Daucus carota L. AOX2a gene. The use of gene- specific primers designed in exon regions on the boundaries of introns permitted to recognize intron length polymorphism (ILP) in intron 3 AOX2a by simple polymerase chain reaction (PCR) assays. The length of intron 3 can vary in individual carrot plants. Thus, allelic variation can be used as a tool to discriminate between single plant genotypes. Using this approach, individual plants from cv. Rotin and from diverse breeding lines and cultivars were identified that showed genetic variability by AOX2a ILPs. Repetitive patterns of intron length variation have been observed which allows grouping of genotypes. Polymorphic and identical PCR fragments revealed underlying high levels of sequence polymorphism. Variability was due to InDel events and intron single nucleotide polymorphisms (ISNPs), with a repetitive deletion in intron 3 affecting a putative pre-miRNA site. The results suggest that high AOX2a gene diversity in D. carota can be explored for the development of functional markers related to agronomic traits

Fungal communities associated with peacock and cercospora leaf spots in olive

Venturia oleaginea and Pseudocercospora cladosporioides are two of the most important olive fungal pathogens causing leaf spots: peacock spot, and cercosporiosis, respectively. In the present study, fungal communities associated with the presence of these pathogens were investigated. Overall, 300 symptomatic and asymptomatic trees from different cultivars were sampled from Alentejo, Portugal. A total of 788 fungal isolates were obtained and classified into 21 OTUs; Ascomycota was clearly the predominant phylum (96.6%). Trees from cultivar ‘Galega vulgar’ showed a significant higher fungal richness when compared to ‘Cobrançosa’, which in turn showed significant higher values than ‘Picual’. Concerning plant health status, symptomatic plants showed significant higher fungal richness, mainly due to the high number of isolates of the pathogens V. oleaginea and P. cladosporioides. In terms of fungal diversity, there were two major groups: ca. 90% of the isolates found in symptomatic plants belonged to V. oleaginea, P. cladosporioides, Chalara sp., and Foliophoma sp. while ca. 90% of the isolates found in asymptomatic plants, belonged to Alternaria sp. and Epicoccum sp. This study highlights the existence of different fungal communities in olive trees, including potential antagonistic organisms that can have a significant impact on diseases and consequently on olive production.publishersversionpublishe

Alternative oxidase involvement in Daucus carota somatic embryogenesis

Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration.The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down- regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation

Identification of Fungi Responsible for Grapevine Trunk Disease in Alentejo Region

Grapevine trunk diseases (GTDs) are one of the most important fungal diseases affecting grapevine plants in all the major growing regions of the world (Pintos et al, 2018)1 with up to 133 fungal species belonging to 34 genera associated to this disease (Gemaje et al, 2018)2. Management of GTDs is difficult and its complete eradication is not possible. For these reasons, its control is meanly focused on disease prevention and mitigation (U´rbez-Torres 2011)3.The work here presented had the aim to identify molecularly the endophytic fungi present in different tissues of grapevines, and among them the fungi responsible for GTDs. Pure fungal cultures were isolated from diseased grapevines plants belonging to two cultivars of Alentejo region (‘Trincadeira’ and ‘Alicante Bouchet’). Mycelium was then harvested, gDNA was extracted (Doyle and Doyle, 1987), and for fungal genetic classification the ribosomal internal transcribed spacer (ITS) region of nuclear rDNA was amplified through PCR by using ITS1 and ITS4 primers (White et al. 1990)4. All the fungi sequences that showed resemblance of 98% were used to classify the fungus analysed. The most prevalent grapevine trunk disease fungi identified were Hormonema viticola, Stereum armeniaccum, Philophorafastigiata, Diaporthe, Truncatella angustata, Cytospora acaciae, Diplodia pseudoseriata, Neofusicocum parvum, and Pestalotiopsis sp

Spatial and temporal variation of fungal endophytic richness and diversity associated to the phyllosphere of olive cultivars

Fungal endophytes are micro-organisms that colonize healthy plant tissues without causing disease symptoms. They are described as plant growth and disease resistance promoters and have shown antimicrobial activity. The spatial-temporal distribution of endophytic communities in olive cultivars has been poorly explored. This study aims to investigate the richness and diversity of endophytic fungi in different seasons and sites, within the Alentejo region, Portugal. Additionally, and because the impact of some pathogenic fungi (e.g. Colletotrichum spp.) varies according to olive cultivars; three cultivars, Galega vulgar, Cobrançosa and Azeiteira, were sampled. 1868 fungal isolates were identified as belonging to 26 OTUs; 13 OTUs were identified to the genera level and 13 to species level. Cultivar Galega vulgar and season autumn showed significant higher values in terms of endophytic richness and diversity. At site level, Elvas showed the lowest fungal richness and diversity of fungal endophytes. This study reinforces the importance of exploring the combined spatio-temporal distribution of the endophytic biodiversity in different olive cultivars. Knowledge about endophytic communities may help to better understand their functions in plants hosts, such as their ecological dynamics with pathogenic fungi, which can be explored for their use as biocontrol agents

The gymnosperm Pinus pinea contains both AOX gene subfamilies, AOX1and AOX2

The gymnosperm Pinus pinea L. (stone pine) is a typical Mediterranean pine used for nuts and timber production,and as an ornamental around the world. Pine genomes are large in comparison to other species. The hypothesis that retrotransposons, such as gymny, made a large contribution to this alteration in genome size was recently confirmed. However, P. pinea is unique in other various aspects. P. pinea demonstrates a different pattern of gymny organization than other Pinus subgenera. Additionally, P. pinea has a highly recalcitrant behaviour in relation to standard conifer protocols for the induction of somatic embryogenesis or rooting. Because such types of cell reprogramming can be explained as a reaction of plant cells to external stress, it is of special interest to study sequence peculiarities in stress-inducible genes, such as the alternative oxidase (AOX). This is the first report containing molecular evidence for the existence of AOX in gymnosperms at the genetic level. P. pinea AOXs were isolated by a polymerase chain reaction (PCR) approach and three genes were identified. Two of the genes belong to the AOX1 subfamily and one belongs to the AOX2 subfamily. The existence of both AOX subfamilies in gymnosperms is reported here for the first time. This discovery supports the hypothesis that AOX1 and AOX2 subfamilies arose prior to the separation of gymnosperms and angiosperms, and indicates that the AOX2 is absent in monocots because of subsequent gene loss events. Polymorphic P. pinea AOX1 sequences from a selected genetic clone are presented indicating non-allelic, non-synonymous and synonymous translation products

Molecular basis of resistance/susceptibility of Vitis vinifera L. to wood infection by fungal trunk pathogens in Alentejo region

Grapevine (Vitis vinifera L.) is one of the most cultivated crops around the globe and has a high commercial value for fresh table grape, dried fruit and wine production. Grapevine is affected by several diseases among which grapevine trunk diseases (GTDs) are the most spread. These diseases are caused by several species of fungi that infect and damage the wood, causing chronic infections, affecting the longevity and productivity of grapevines in all major growing regions worldwide. Until now, no effective treatments are known. The recent increase of GTDs incidence is believed to be the consequence of several factors such as the grapevine planting ‘boom’ as occurred in Alentejo region, which increased the circulation of potentially contaminated propagating material, the drastic changes in production methods that greatly favor fungal infection, and finally because of the European laws concerning pesticide restrictions which have banned the use of effective chemical products available against GTDs fungi. The recently accepted PhD project that will be here presented intends to exploit the natural phenotypic expression of plants’ resistance/susceptibility to GTDs and to identify the gene(s) involved in these processes. Firstly, a preliminary study on the fungal community present will be performed in plants from selected cultivars from different vineyards in Alentejo region, with and without trunk diseases symptoms, using a strategy based on next generation sequence analysis (NGS). This study will give a better knowledge of the endophytic and pathogenic fungal communities and update the information on the diversity and abundance of the fungi responsible for trunk diseases. The antagonistic capacity of the identified endophytic fungi against the most frequently GTDs will also be tested. The information obtained on fungal communities, together with natural phenotypic expression of resistance/susceptibility to these diseases, will be the basis for the selection of plants for all transcriptome analyses. The comprehensive transcriptome data set will provide molecular insights into gene network responsible for activation of antifungal responses to trunk diseases, in resistant and susceptible grapevine cultivars, indicating novel candidates to be latter tested in strategies involving gene knockout and overexpression and contribute to the development of effective protective methods against these diseases