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According to the climate projections, drought will increase in frequency and severity. Since water stress (WS) impacts a grapevine's physiology and yield negatively, the evaluation and selection of tolerant genotypes are needed. To analyse the WS effects on the morphology and cell division of three grapevines (Vitis vinifera L.) varieties, "Touriga Franca" (TF), "Touriga Nacional" (TN) and "Viosinho" (VS), in vitro-grown plants were exposed to 10% polyethylene glycol 6000 (PEG) (−0.4 MPa) or 20% PEG (−0.8 MPa), incorporated in the culture medium, for four weeks. Control plants were kept in culture media without PEG. The VS and TN plants showed the highest mean numbers of nodes, shoots and leaves and average mitotic indexes under 20% PEG. The TF and TN plants showed the lowest frequencies of mitotic anomalies under 10% PEG. The VS plant growth was less affected by WS, but TF and TN presented more regular mitosis under moderate WS. Globally, in vitro culture constitutes a cost-effective experimental system for studying grapevine responses to WS and the preliminary selection of resilient genotypes. These approaches could be applied to study plant responses to other abiotic stresses based on additional evaluation techniques (e.g., transcriptional analyses or genome-wide association studies). [ABSTRACT FROM AUTHOR] more...

The main role of the nucleolus is ribosomal biogenesis, but it also responds to stress with changes in number, area, and/or morphology. Nucleoli with transcriptionally active ribosomal RNA genes are selectively stained by silver nitrate. Hypothesising that fire recurrence and/or polyethylene glycol (PEG)-induced water stress would cause nucleolar changes in Pinus pinaster roots, nucleolar parameters were analysed upon the germination of seeds harvested in post-fire naturally regenerated stands with different fire regimes. An unburned stand was used as a control. The nucleoli number varied from 1 to 15 among stands and PEG treatments, but a mode of five or six nucleoli was found. The number of nucleoli per interphase (N) increased (p < 0.001) with fire recurrence (stand effect). Increasing PEG concentration (treatment effect) decreased the nucleoli number, notably in the stand with the highest fire recurrence. The nucleolar area decreased (p < 0.001) with increased nucleoli number, fire recurrence, and/or PEG concentration. Fire recurrence and water scarcity are forecasted for future climate scenarios. As demonstrated earlier, these factors could influence protein synthesis or the cell cycle's regularity, which are crucial processes for root growth and pine seedling establishment. Furthermore, this work revealed that nucleolar parameters could be suitable biomarkers for pine stress studies. [ABSTRACT FROM AUTHOR] more...

Background: “Touriga Franca” (TF) and “Touriga Nacional” (TN) are grapevine varieties cultivated in the ‘Douro Superior’ subregion (Northern Portugal) that experience stressful environmental conditions during the summer. Objectives: Aiming to profile the expression of stressresponsive genes by quantitative real-time PCR (qPCR) in TF and TN plants growing naturally, three candidate reference genes were first tested under controlled conditions. Methods: To simulate a summer’s day, TF and TN in vitro plants were exposed to 32 ◦C–3 h (heat acclimation) and 42 ◦C–1 h (severe heat stress, HS) followed by two recovery periods (32 ◦C–3 h and 24 ◦C–24 h). Leaf samples were collected at the end of each phase. Control plants were kept at 24 ◦C. Results: Among the candidate reference genes, the UBC and VAG pair showed the highest stability. The suitability of these genes for qPCR was validated by heat shock protein 17.9A (HSP17.9A) gene profiling. The HSP17.9A expression was up-regulated in both varieties and all experimental phases except in TF control plants. TN showed the highest HSP17.9A relative expression ratio after severe HS. Conclusions: TN responded faster than TF to the induced heat shocks. The UBC, VAG, and HSP17.9A genes revealed to be suitable for further qPCR assays in TF and TN grapevine varieties. [ABSTRACT FROM AUTHOR] more...

The degree of tolerance to heat stress (HS) differs among grapevine varieties. HS affects the duration and the regularity of the cell cycle in plants. The cytogenetic studies in grapevine are scarce, and the consequences of HS in the mitosis are barely known. This work intends to evaluate the consequences of induced HS in the mitotic cell cycle and chromosomes of four wine-producing varieties: Touriga Franca (TF), Touriga Nacional (TN), Rabigato and Viosinho using a cytogenetic approach. HS (1h at 42 ºC) was induced in plants of the four grapevine varieties that grew in vitro for 11 months. Plants of the same varieties and with equal age were used as control (maintained in vitro at 25 ºC). Three plants per variety and treatment (control and HS) were analysed. After HS, root-tips were collected in all plants and immediately fixed to be used for the preparation of mitotic chromosome spreads. In total, 6,116 root-tip cells were scored. Among the 5,973 dividing cells, 24.33% showed different types of irregularities in all mitotic phases (prophase, metaphase, anaphase, and telophase). After HS, the mitotic index (MI) decreased in the varieties TF and Viosinho, and increased in TN and Rabigato, relatively to the control. However, the average values of MI did not show statistically significant differences (p ˃ 0.05) among varieties, treatments and for the variety x treatment interaction. The percentage of dividing cells with anomalies (%DCA) increased after HS in all varieties relatively to the control. The average values of %DCA presented statistically significant differences (p < 0.05) only between treatments. As far as we know, this work constitutes the first cytogenetic evaluation of the HS effects in the mitotic cell cycle and chromosomes of grapevine using meristematic cells of root-tips. TN has been considered tolerant to various abiotic stresses (drought and excessive heat and light) based on other methodologies. TF and Viosinho have been referred as tolerant to abiotic stress without deeper studies available, and till the development of our investigation, the sensibility of Rabigato was unknown. Among the varieties analysed in this work, Rabigato revealed to be the less tolerant to HS. This research can be useful for selection of grapevine clones more tolerant to HS for commercialization and for the improvement of the economic sustainability. more...

Grapevine varieties from "Douro Superior" (NE Portugal) experience high temperatures, solar radiation, and water deficit during the summer. This summer's stressful growing conditions induce nucleic acids, lipids, and protein oxidation, which cause cellular, physiological, molecular, and biochemical changes. Cell cycle anomalies, mitosis delay, or cell death may occur at the cellular level, leading to reduced plant productivity. However, the foliar application of kaolin (KL) can mitigate the impact of abiotic stress by decreasing leaf temperature and enhancing antioxidant defence. Hence, this study hypothesised that KL-treated grapevine plants growing in NE Portugal would reveal, under summer stressful growing conditions, higher progression and stability of the leaf mitotic cell cycle than the untreated (control) plants. KL was applied after veraison for two years. Leaves, sampled 3 and 5 weeks later, were cytogenetically, molecularly, and biochemically analysed. Globally, integrating these multidisciplinary data confirmed the decreased leaf temperature and enhanced antioxidant defence of the KL-treated plants, accompanied by an improved regularity and completion of the leaf cell cycle relative to the control plants. Nevertheless, the KL efficacy was significantly influenced by the sampling date and/or variety. In sum, the achieved results confirmed the hypothesis initially proposed. [ABSTRACT FROM AUTHOR] more...

The cultivation of walnuts (Juglans regia L.) has become increasingly popular worldwide due to the nutritional value of the nuts. Plant biostimulants (PBs) and nutrient-rich products have been increasingly used in agriculture to improve yield, quality, and abiotic stress tolerance. However, farmers need fast laboratory studies to determine the most suitable treatment per crop or ecosystem to take full advantage of these products. Evaluating nucleolar activity and protein content can provide clues about the most appropriate treatment. This study aimed to determine how five commercial products, four PBs based on seaweed extract and/or free amino acids and one boron-enriched fertiliser used as foliar sprays, affect walnut cv's nucleolar activity and protein content. "Franquette" from an orchard located in NE Portugal was compared to untreated (control) plants. All treatments brought a low leaf mitotic index. The control showed the smallest nucleolar area, highest protein content, and highest frequency of nucleolar irregularities. Fitoalgas Green®, Sprint Plus®, and Tradebor® showed the highest nucleolar area and lowest frequencies of nucleolar irregularities. The recruitment of proteins/enzymes for response against abiotic stresses may explain the high protein content in the control. Hence, the enhanced abiotic stress tolerance of the treated trees explains their lower protein content and frequency of nucleolar anomalies. Globally, the Fitoalgas Green®, Sprint Plus®, and Tradebor® seem better suited for "Franquette" walnut trees under the edaphoclimatic conditions where trials were conducted. [ABSTRACT FROM AUTHOR] more...

Climate change will increase the frequency of drought, heat waves, and wildfires. We intended to analyse how fire recurrence and/or induced water stress can affect seed germination and root cell division in Pinus pinaster Aiton. Seeds from stands with no prior fire history and from post-fire regeneration (in areas burnt once, twice, and thrice) in northern Portugal were germinated in distilled water (control) and polyethylene glycol (PEG) to simulate water stress for four weeks, followed by a recovery period. Roots were analysed cytogenetically. The germination index of the Pinus pinaster seeds was not statistically influenced by the induction of osmotic stress, nor by the fire recurrence of the stands. The mean germination time (MGT) was 10–29 days and 1–36 days for the stress and recovery periods, respectively, and increased with PEG concentration. The 20% PEG treatment inhibited root growth after germination. The 10% PEG treatment induced a high frequency of cytogenetic anomalies, mostly in the sites which experienced fire exposure. While fire recurrence did not affect the germination rate, it seemed to reduce the water stress response, negatively impacting cell division and impair root growth. [ABSTRACT FROM AUTHOR] more...