Browsing by Author "Oliveira, Maria Margarida"
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- Abiotic stress and induced DNA hypomethylation cause interphase chromatin structural changes in rice rDNA lociPublication . Santos, Ana Paula; Ferreira, Liliana J.; Maroco, João; Oliveira, Maria MargaridaGlobal climate change, i.e. higher and more variable temperatures, and a gain in soil salinity are increasing plant stress with direct consequences on crop yield and quality levels. Rice productivity is strongly affected by abiotic stress conditions. The regulation of chromatin structure in response to environmental stress is poorly understood. We investigated the interphase chromatin organization from rice plants in non-stress versus stress conditions. We have used a cytogenetic approach, based on fluorescence in situ hybridization (FISH) with 45S, 5S rDNA and centromeric probes on rice tissue sections. The abiotic stress conditions included cold, heat and mild salinity and were applied during seed germination. In contrast to cold, saline and heat stresses caused extensive decondensation of 45S rDNA chromatin and also an increase in the distance between the 2 homologous 5S rDNA loci. 5-Azacytidine (5-AC), a DNA hypomethylating drug, greatly increased 45S rDNA chromatin decondensation and interestingly was able to induce polarization of centromeres in rice interphase nuclei. The abiotic stresses tested did not perturb the spatial position of centromeres, typically with circular arrangement around the nucleolus. The results suggest a role for chromatin plasticity in a world of climate changes.
- Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)Publication . Marum, Liliana; Rocheta, Margarida; Maroco, João; Oliveira, Maria Margarida; Miguel, Célia M.Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.
- Bridging sd1 molecular knowledge with recent breeding strategies for the improvement of traditional rice varieties - a japonica case-studyPublication . Negrão, Sónia; Palaniappan, Jayamani; Maroco, João; Lourenço, Tiago; Mackill, David; Oliveira, Maria MargaridaThe rice semidwarfing gene, sd1, also known as the “green revolution gene”, has been studied intensively due to its contribution to the increase of crop production. Although sd1 breeding was extensively applied since the 1960s, the recent advances in the molecular basis of this gene allowed designing a more precise breeding strategy - marker assisted backcrossing (MAB) - to track sd1 introgression in two traditional rice varieties. For selection of sd1 plants we first confirmed the efficiency of specific markers based on Os200 x 2 gene sequence. Background selection was also performed with the help of microsatellites markers (SSR) and a total of 7 breeding lines were recovered containing a higher percentage of recurrent parent genome (RPG). Analysis of Covariance (ANCOVA) using mean progenitor plant height as covariate was performed to compare several agronomic and quality-related parameters in two different environments. The results suggest that plant height differs significantly between the two environments F(1, 220) = 155.336; p < 0.001. From the total variability of plant height we could conclude that 73% is due to the genotype, while 10.4% depends on the environment. In addition, the percentage of RPG seems negatively correlated with plant height (p < 0.005). MAB and background selection thus revealed as useful tools to assist breeding for semidwarfism in traditional rice varieties.
- Drought stress response in Jatropha curcas: Growth and physiologyPublication . Sapeta, Helena; Costa, J. Miguel; Lourenço, Tiago; Maroco, João; Van der Linde, Piet; Oliveira, Maria MargaridaTolerance to drought remains poorly described for Jatropha curcas accessions from different geographical and climatic origins. To address this issue we studied the response of two J. curcas accessions, one from Indonesia (wet tropical climate) and the other from Cape Verde islands (semi-arid climate). Potted seedlings (with 71 days) of both accessions were subjected to continuous well watered conditions (control) or to a drought stress period followed by re-watering. To mimic natural conditions in which drought stress develops gradually, stress was imposed progressively by reducing irrigation (10% reduction every 2 days, on a weight base), for a period of 28 days, until a field capacity of 15% (maximum stress) was achieved, followed by one week under well-watered conditions. We measured soil and plant water status, growth and biomass partitioning, leaf morphology, leaf gas exchange and chlorophyll a fluorescence. Both accessions maintained high leaf relative water content (70–80%) even at maximum stress. Net photosynthesis (An) was not affected by mild to moderate stress but it abruptly dropped at severe stress. This was due to reduced stomatal conductance, which showed earlier decline than An. Plant growth (stem elongation, leaf emergence and total leaf area) was reduced, minimizing water loss, but no significant differences were found between accessions. Drought stress did not reduce chlorophyll contents but led to reduced chlorophyll a/b. Both accessions showed fast recovery of both stomatal and photochemical parameters suggesting a good tolerance to water stress. Both J. curcas accessions showed a-dehydrationavoidant behaviour, presenting a typical water saving strategy due to strict stomatal regulation, regardless of their provenance.
- Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesisPublication . Gonçalves, Sónia; Cairney, John; Maroco, João; Oliveira, Maria Margarida; Miguel, Célia M.In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.
- Improved in vitro rooting of Prunus dulcis Mill. cultivarsPublication . Tereso, Susana; Miguel, Célia M.; Mascarenhas, M.; Roque, A.; Trindade, H.; Maroco, João; Oliveira, Maria MargaridaA highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47 - 100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm-3 gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous
- New allelic variants found in key rice salt-tolerance genes an association studyPublication . Negrao, Sonia; Almadanim, Maria Cecilia; Pires, Inês S.; Abreu, Isabel A.; Maroco, João Paulo; COURTOIS, Brigitte; Gregorio, Glenn B.; McNally, Kenneth; Oliveira, Maria MargaridaSalt stress is a complex physiological trait affecting plants by limiting growth and productivity. Rice, one of the most important food crops, is rated as salt-sensitive. High-throughput screening methods are required to exploit novel sources of genetic variation in rice and further improve salinity tolerance in breeding programmes. To search for genotypic differences related to salt stress, we genotyped 392 rice accessions by EcoTILLING. We targeted five key salt-related genes involved in mechanisms such as Na+/K+ ratio equilibrium, signalling cascade and stress protection, and we found 40 new allelic variants in coding sequences. By performing association analyses using both general and mixed linear models, we identified 11 significant SNPs related to salinity. We further evaluated the putative consequences of these SNPs at the protein level using bioinformatic tools. Amongst the five nonsynonymous SNPs significantly associated with saltstress traits, we found a T67K mutation that may cause the destabilization of one transmembrane domain in OsHKT1;5, and a P140A alteration that significantly increases the probability of OsHKT1;5 phosphorylation. The K24E mutation can putatively affect Salt interaction with other proteins thus impacting its function. Our results have uncovered allelic variants affecting salinity tolerance that may be important in breeding.
- Salt tolerant and sensitive rice varieties display differential methylome flexibility under salt stressPublication . Ferreira, Liliana J.; Azevedo, Vanessa; Maroco, João; Oliveira, Maria Margarida; Santos, Ana PaulaDNA methylation has been referred as an important player in plant genomic responses to environmental stresses but correlations between the methylome plasticity and specific traits of interest are still far from being understood. In this study, we inspected global DNA methylation levels in salt tolerant and sensitive rice varieties upon salt stress imposition. Global DNA methylation was quantified using the 5-methylcytosine (5mC) antibody and an ELISAbased technique, which is an affordable and quite pioneer assay in plants, and in situ imaging of methylation sites in interphase nuclei of tissue sections. Variations of global DNA methylation levels in response to salt stress were tissue- and genotype-dependent. We show a connection between a higher ability of DNA methylation adjustment levels and salt stress tolerance. The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. In spite of the same tendency for reduction of global methylation under salinity, in the salt-sensitive rice variety IR29 such reduction was not statistically supported. In ‘Pokkali’, the salt stress-induced demethylation may be linked to active demethylation due to increased expression of DNA demethylases under salt stress. In ‘IR29’, the induction of both DNA demethylases and methyltransferases may explain the lower plasticity of DNA methylation. We further show that mutations for epigenetic regulators affected specific phenotypic parameters related to salinity tolerance, such as the root length and biomass. This work emphasizes the role of differential methylome flexibility between salt tolerant and salt sensitive rice varieties as an important player in salt stress tolerance, reinforcing the need to better understand the connection between epigenetic networks and plant responses to environmental stresses.
- Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformationPublication . Tereso, Susana; Miguel, Célia M.; Maroco, João; Oliveira, Maria MargaridaThe effects of antibiotics commonly used in Agrobacterium-mediated transformation were studied on Pinus pinaster tissues. Embryogenic tissue growth from three embryogenic lines and adventitious bud induction from cotyledons from three open-pollinated seed families were analysed. Cefotaxizme, carbenicillin and timentin commonly used for Agrobacterium elimination, at concentrations of 200–400 mg l –1 did not inhibit the embryogenic tissue growth on filter paper nor as clumps. Adventitious bud induction and bud number were significantly reduced for one of the tested families when using 400 mg l–1 cefotaxime or timentin. The selection agent kanamycin significantly inhibited growth of embryogenic tissue on filter paper in all the embryogenic lines and concentrations tested (20–50 mg l–1). Kanamycin also inhibited growth of embryogenic clumps after two subcultures at 5–50 mg l–1. In cotyledons, kanamycin inhibited adventitious bud formation in the three seed families used, regardless of the concentrations tested (5– 25 mg l–1). There was a significant effect of the seed family on the bud induction and the number of adventitious buds produced. From the results obtained, we propose the use of timentin to eliminate Agrobacterium in transformation experiments, at concentrations of 400 mg l–1 for embryogenic tissues and of 300 mg l–1 for cotyledons. For selection of transformed tissues carrying the kanamycin resistance gene, kanamycin should be used at 20 mg l–1 for embryogenic tissues on filter paper, at 5 mg l–1 when clumps are in direct contact with the selection medium, and bellow 5 mg l–1 for adventitious bud induction.