Sara Amâncio

RAPD Assessment for Identification of Clonal Identity and Genetic Stability of in vitro Propagated Chestnut Hybrids

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the clonal identity of four in vitro propagated chestnut rootstock hybrids (Castanea sativa × C. crenata) described as originally isolated from the same mother tree. To confirm genetic stability after in vitro multiplication for more than 4 years, RAPD patterns of in vitro and donor plants were compared. From 40 arbitrary 10-mer primers used to amplify DNA, 21 provided patterns and were chosen for comparisons. Although significant differences were found in growth parameters between in vitro material of the putative clones, RAPD profiling showed polymorphism in none but one. This accession may then be withdrawn from the same clonal origin as the other three. As expected, no polymorphism was detected between the material propagated in vitro and the donor plants they originated from.

Antioxidant defence system in plantlets transferred from in vitro to ex vitro: effects of increasing light intensity and CO2 concentration

Grapevine and chestnut plantlets multiplied in vitro were acclimatised under four treatments combining two irradiances, 150 and 300 mol m −2 s − 1 , respectively, three and six times the irradiance in vitro, and two [CO2], ambient and the double (700 ppm). During acclimatisation the plants were monitored for the variation of glutathione pools, glutathione reductase activity and chlorophyll fluorescence to identify any of these parameters as a specific indicator of autotrophic behaviour and to assess the effect of high [CO2] in preventing photoinhibition and oxidative stress symptoms due to high irradiance. Grapevine under high light and chestnut under high light and high [CO2] displayed a faster growth and in new leaves GSH approached the values measured in normal field leaves. Concerning chlorophyll fluorescence parameters, the quantum yield of electron transport in PSII given by e allowed to identify photoinhibition symptoms in chestnut under high light, and the alleviation of these symptoms by increasing [CO2]. In grapevine a net benefit from high [CO2] was reflected in e values very close to in vivo leaves. The results suggest that GSH and e are reliable indicators of autotrophic functioning, discriminating between the environmental conditions that accelerate the acquisition of full autotrophy.

Improvement of acclimatization of micropropagated grapevine: Photosynthetic competence and carbon allocation

Grapevine plantlets multiplied in vitro were acclimatized at 40 or 90 μmol m−2 s−1 photon flux density for 12 or 16 h per day, respectively. In the high-light regime a decrease in total chlorophyll and an increase in chlorophyll a/chlorophyll b ratio occurred. However, at high-light intensity lower photosynthetic capacities and higher apparent photosynthesis were measured than at the low-light regime. In leaves expanded during acclimatization, the light compensation point was higher in plantlets under high-light while quantum yield was higher in low-light conditions. High-light also gave rise to an increase in carbohydrate concentration. As a whole, the results suggest that high-light increases carbon assimilation and growth although with a low investment in the photosynthetic apparatus.

In vitro propagation of chestnut (Castanea sativa×C. crenata): Effects of rooting treatments on plant survival, peroxidase activity and anatomical changes during adventitious root formation

Abstract In order to improve plant survival and to achieve a better understanding of the rooting process of chestnut ( Castanea sativa × C. crenata ) shoot cultures of mature origin, different rooting treatments were compared. For root induction, the basal ends of the shoots were dipped into 1 g l −1 IBA solution for 1 min or planted for 5 days in 3 mg l −1 IBA agar medium. For root development, the induced shoots were transferred either to auxin-free agar medium (in vitro rooting) or to a peat:perlite substrate (ex vitro rooting). Rooted shoots were subsequently acclimatized. After dipping induction, the rooting percentage was higher when root development was performed in vitro (97%) than ex vitro (77%). After induction with IBA in agar medium, the root development conditions did not affect the rooting percentage (93% and 87%, respectively, for in vitro and ex vitro rooting). In the acclimatization stage, 100% survival was obtained with microplants with ex vitro-developed roots, compared to only 50% for microplants with in vitro-developed roots. After the root inductive treatments, peroxidase activity in the shoots was characterized by an initial reduction during the first 12 h, followed by a transient peak at day 1. From day 4 to day 6, peroxidase activity increased. This increase was faster in the dipped shoots, but on day 8, no difference in activity could be observed between the treatments. The sequential anatomical changes during the rooting process were similar in both root induction treatments. The first cellular divisions were observed in some of the cambial derivative cells 24 h after auxin induction and a meristemoid became individualised by days 3–4. Identifiable root primordia with a conical shape were present after 6–8 days. Roots with organized tissue systems emerged from the stem 10–12 days after the root induction treatment.

Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress

BackgroundQuantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes.ResultsUsing the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both.ConclusionsEach method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis.

Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine

Grapevine is an extremely important crop worldwide. In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress (WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, a cross-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors.

Involvement of free and conjugated polyamines and free amino acids in the adventitious rooting of micropropagated cork oak and grapevine shoots

Abstract The recent advances in biotechnology have boosted interest in the differentiation processes, including adventitious rooting. Differentiation processes depend on endogenous factors, among which auxins and polyamines are believed to play a major role. A positive correlation between polyamine accumulation and the induction of adventitious rooting by auxin has been observed in numerous woody species, suggesting that polyamines could be used as markers of the rooting process. The aim of the present work is to investigate whether primary and secondary nitrogen metabolism is involved in adventitious root induction by auxin treatments in cork oak ( Quercus suber L.) and grapevine ( Vitis vinifera L.) shoots cultured in vitro. For this purpose, we followed the profile of free and conjugated polyamines, free amino acid pools and 15 N-labelling profiles during root induction and expression. We have also observed the effects of cyclohexylamine (CHA), an inhibitor of spermidine synthase. Taking the results together, it is possible to conclude that: (a) glutamine is the most abundant free amino acid in grapevine, while in cork oak, asparagine and arginine are the major amino acids; (b) in grapevine, auxin did not significantly affect the glutamine levels, but changed the 15 N-enrichment and labelling pattern of arginine; (c) auxin affected asparagine levels and 15 N-labelling pattern of glutamine in cork oak; (d) in both cork oak and grapevine, free putrescine (Put) can be considered as a marker of in vitro root induction; (e) in both species, the presence of CHA resulted in the accumulation of free Put; (f) no Put catabolism was detected in the form of 15 N-NMR products, namely 15 N-γ-aminobutyric acid; (g) the CHA-induced accumulation of Put only increased grapevine rooting rate.

Mineral stress affects the cell wall composition of grapevine (Vitis vinifera L.) callus

Grapevine (Vitis vinifera L.) is one of the most economically important fruit crops in the world. Deficit in nitrogen, phosphorus and sulfur nutrition impairs essential metabolic pathways. The influence of mineral stress in the composition of the plant cell wall (CW) has received residual attention. Using grapevine callus as a model system, 6 weeks deficiency of those elements caused a significant decrease in growth. Callus CWs were analyzed by Fourier transform infrared spectroscopy (FT-IR), by quantification of CW components and by immunolocalization of CW epitopes with monoclonal antibodies. PCA analysis of FT-IR data suggested changes in the main components of the CW in response to individual mineral stress. Decreased cellulose, modifications in pectin methyl esterification and increase of structural proteins were among the events disclosed by FT-IR analysis. Chemical analyses supported some of the assumptions and further disclosed an increase in lignin content under nitrogen deficiency, suggesting a compensation of cellulose by lignin. Moreover, polysaccharides of callus under mineral deficiency showed to be more tightly bonded to the CW, probably due to a more extensive cross-linking of the cellulose-hemicellulose network. Our work showed that mineral stress impacts the CW at different extents according to the withdrawn mineral element, and that the modifications in a given CW component are compensated by the synthesis and/or alternative linking between polymers. The overall results here described for the first time pinpoint the CW of Vitis callus different strategies to overcome mineral stress, depending on how essential they are to cell growth and plant development.

Activation of the Ascorbate‐Glutathione Cycle Is an Early Response of Micropropagated Vitis vinifera L. Explants Transferred to ex Vitro

Micropropagated grapevine (Vitis vinifera L. var. Touriga Nacional) was transferred from heterotrophic growth conditions and PPFD of 50 μmol m−2 s−1 to ex vitro under PPFD fourfold higher, which causes photooxidative stress. We have analyzed the first 7 d of growth, focusing on the protective role of the ascorbate‐glutathione cycle and relating its activity to the functioning of photosynthetic electron transport. Immediately after transfer, reversible photoinhibition was observed. Both the photochemical efficiency of PSII and PSII excitation capture efficiency were transiently affected. Concomitantly, a transient rise in H2O2 content was observed on day 1, followed by increased activity of the ascorbate‐glutathione cycle as determined by changes to ascorbate and glutathione levels and redox states, enzyme activities, and expression of associated genes. The upregulation of ascorbate peroxidase (APX1 and APX3), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GOR2) was observed from day 1, followed by catalase (CAT) on day 2. Accompanying changes to ascorbate‐glutathione cycle activity, the expression of superoxide dismutase isoforms increased. A slight H2O2 peak, not accompanied by photoinhibition, was observed on day 6. A decrease in the ascorbate and glutathione pools also pointed to the development of new leaves, with the concurring translocation of metabolites.

Oxidative stress homeostasis in grapevine (Vitis vinifera L.)

Plants can maintain growth and reproductive success by sensing changes in the environment and reacting through mechanisms at molecular, cellular, physiological and developmental levels. Each stress condition prompts a unique response although some overlap between the reactions to abiotic stress (drought, heat, cold, salt or high light) and to biotic stress (pathogens) does occur. A common feature in the response to all stresses is the onset of oxidative stress, through the production of reactive oxygen species (ROS). As hydrogen peroxide and superoxide are involved in stress signaling, a tight control in ROS homeostasis requires a delicate balance of systems involved in their generation and degradation. If the plant lacks the capacity to generate scavenging potential, this can ultimately lead to death. In grapevine, antioxidant homeostasis can be considered at whole plant levels and during the development cycle. The most striking example lies in berries and their derivatives, such as wine, with nutraceutical properties associated with their antioxidant capacity. Antioxidant homeostasis is tightly regulated in leaves, assuring a positive balance between photosynthesis and respiration, explaining the tolerance of many grapevine varieties to extreme environments. In this review we will focus on antioxidant metabolites, antioxidant enzymes, transcriptional regulation and cross-talk with hormones prompted by abiotic stress conditions. We will also discuss three situations that require specific homeostasis balance: biotic stress, the oxidative burst in berries at veraison and in vitro systems. The genetic plasticity of the antioxidant homeostasis response put in evidence by the different levels of tolerance to stress presented by grapevine varieties will be addressed. The gathered information is relevant to foster varietal adaptation to impending climate changes, to assist breeders in choosing the more adapted varieties and to suitable viticulture practices.