This study aims to develop a technical itinerary for Ca biofortification of three potato varieties (Agria, Picasso and Rossi) during the 2018 production cycle. As such, a parcel was selected about 20 x 20 m (in Campos Moledo and Casal Galharda) and 20 x 24 m (in Campo Boas Águas), located in the western region of Portugal. In this context, plant biofortification was promoted throughout the respective production cycle, after planting. There were 4 leaf applications with four different concentrations with calcium chloride or, alternatively, calcium nitrate. To study the interactions between Ca and other chemical elements in the tuber and assess the index of biofortification were used different techniques, such as μ-EDXRF M4 Tornado ™ and scanning electron microscopy with X-ray energy dispersive spectroscopy (SEM-EDS). At harvest, it was found that the tubers produced in the Moledo, Boas Águas and Casal Galharda fields, showed an increase in Ca content of about 2.6, between 2 - 2.5 and between 1.5 - 2.5 times, respectively (relative to the application of Ca(NO₃)₂ and CaCl₂) with a predominant location in the tuber. Calcium is the most abundant mineral element in the human body and one of the most important to its functioning. It’s required in relatively large quantities, essential for bone synthesis and metabolism, tooth mineralization and for the regulation of intracellular processes in various tissues. It is known, that Ca deficiency may promote low bone density and fragility, and may also determine the evolution of pathologies, namely osteoporosis and rickets. Calcium biofortification is a strategy that promotes nutrient enhancement in food crops and can in the long run provide increased nutrient uptake and accumulation in the human body. Thus, the biofortification in Ca allows the development of a product for human consumption that presents functional characteristics at the physiological level. It was concluded that it is possible to develop products such as chips or purees enriched in this macroelement, enabling the creation of a value-added product.

Grain sorghum (Sorghum bicolor (L.) Moench) is one of the major crops used for various purposes, including animal and human nutrition. The most relevant strategy for create new sorghum hybrids is the use of lines with cytoplasmic male sterility (CMS). However, the process of creating sterile lines and lines that restore fertility is very laborious and time-consuming. The breeders need to know the genotype of the original parental forms of sorghum by the presence of the main genes that control fertility to speed up breeding work. One of these is the Rf1 gene. Our study aimed to identify alleles of the Rf1 gene in collection samples of grain sorghum (Sorghum bicolor (L.) Moench) adapted to the arid conditions of southern Russia. The studies carried out on southern Russia (FSBSI “ARC “Donskoy”, Zernograd, Russia) in 2018-2019. The presence of alleles of the Rf1 fertility gene using the Xtxp18 SSR marker by PCR analysis in collection samples of grain sorghum (313 pcs.) was studied. A crossed some samples with two sterile lines - "Demetra S" and "Dzhetta S" (developed in FSBSI "ARC "Donskoy") were doing in 2019. The assessment of the fertility of self-pollinated lines was carried out in the field using a 3-point scale. The polymorphism of the Xtxp18 marker, a wide allelic diversity of the Rf1 gene in collection samples of sorghum and the association of the identified Rf1 alleles with the fertility and sterility of self-pollinated hybrids of grain sorghum as a result of the study was performed. This result will make it possible to deepen understanding of the influence of the Rf1 gene alleles on the level of fertility of sorghum plants in the future, as well as to accelerate the breeding process to create sterile lines and their fertile analogues for further obtaining commercial hybrids.

Zinc deficiency leads to loss of brain function, changes in growth and weakening of the immune system. This micronutrient has a fundamental role at the regulatory, functional and structural levels. Biofortification, which is a process in which there is an enrichment of both content and the bioavailability of micronutrients in edible tissues of staple foods, may be used to overcome these deficiencies. Two wheat crops (fields 1 and 2) located in Beja, Portugal, with two varieties (Paiva and Roxo) of Triticum aestivum, were selected to be part of a zinc biofortification itinerary. Both varieties were sprayed three times with a zinc fertilizer in two different concentrations and were compared to the control samples. To quantify and locate Zn in the wheat flour and within the grains, XRF analyser and μ-EDWRF analyser were used, respectively, at harvest. Applying XRF analyser to wheat flour, the biofortification index of Zn increased between 1,2 – 1,7 times for Paiva and Roxo increased between 1,3 – 1,4 times in field 1. In field 2, the results varied between 2,3 – 2,5 times for Paiva and between 2,1 – 2,4 times for Roxo. The μ-EDWRF analyses revealed that Zn was preferentially located in the embryo and aleurone in both varieties. To sum up, Zn biofortification resulted in a higher accumulation of the mineral in the embryo and aleurone, particularly in the samples with higher levels of Zn applications. Thus, the biofortification of wheat will allow the development of functional foods with added value and differentiators in the market.

The micronutrient deficiency affects more than two million individuals, there for it is a public
health problem that can lead to various diseases, so we should pay more attention to it
nowadays. In order to combat this problem, several alternatives have appeared, namely
biofortification, which consists on increasing the amount of nutrients in food crops. Zinc is one of
the most relevant micronutrients for the body and has three main roles: catalytic, structural and
regulatory. The deficiency of this micronutrient leads to several health issues. In order to put zinc
biofortification into practice, a technical itinerary was outlined which took place in the fields
located in Palmela (Portugal). This work aims to optimize a response to zinc biofortification of
two grape varieties Fernão Pires and Syrah. Biofortification was performed with leaf applications
of zinc oxide (ZnO) and zinc sulfate (ZnSO4) throughout the production cycle. In order to assess
the efficiency of biofortification, atomic absorption spectrophotometry and μ-EDXRF M4
TornadoTM were used in this process. Both techniques demonstrated an increase in zinc content.
Considering both grape varieties measured by atomic absorption spectrophotometry, there was
an increase of 1.2 times and 2.2 times for Fernão Pires and Syrah respectively. Through tissue
analysis, Fernão Pires and Syrah showed a substantial rise in zinc levels in the grape’s skin, with
the application of zinc oxide or zinc sulfate.

The shrub Cistus incanus L. is well adapted to Mediterranean conditions thanks to its morpho-anatomical, physiological and biochemical traits. However, its distribution and survival in coastal dunes will be likely threatened by ongoing runaway climate change. We investigated how the harsh climatic conditions generated by the 2015 summer heat wave triggered specific anatomical, physiological and biochemical responses of this species in its natural environment. These adjustments were compared to those measured in summer 2014. The drier and hotter conditions of summer 2015 determined an increment in leaf lamina thickness, due to a greater palisade parenchyma, thus leading to an increase in the whole leaf mass per area. These morphoanatomical adjustments enhanced leaf resistance against dehydration, optimized carbon assimilation, and delayed leaf senescence.
In addition, the activation of secondary metabolic pathways, in particular the biosynthesis of tannins and monoterpenes, helped prevent oxidative stress through the consumption of excess reducing power, thus contributing to the maintenance of physiological performances even under hotter and drier conditions. In conclusion, our study offers new evidence on the integration of morphophysiological and metabolic adjustments of this species growing in its natural habitat to cope with ongoing climate change.

Microcystins are cyanobacterial toxins, effectively inhibiting protein phosphatases 1 and 2A, enzymes that are involved in plant cytoskeleton (i.e. microtubules and F-actin) organization and cell cycle progression. Therefore, studies on the toxicity of cyanobacterial products on plant cytoskeleton have so far focused mainly on the effects of microcystin variants on microtubules. In this study, we investigated the effects of the extracts of two non-microcystin-producing (NMP) cyanobacterial strains, Microcystis viridis TAU-MAC 1810 and Planktothrix agardhii TAU-MAC 0514, on the cytoskeleton and cell cycle of Oryza sativa (rice) root cells. Rice seedling roots were exposed for various time periods (1, 12 and 24h) to aqueous extracts of the aforementioned strains. Treated root tips were excised, fixed and underwent either immunostaining for α-tubulin or fluorescent phalloidin staining for F-actin, and DAPI staining for DNA. Fluorescent specimens were observed by confocal laser scanning microscopy (CLSM). Corrected total cell fluorescence (CTCF) was calculated to quantify cytoskeletal disorders. Cell cycle stages were recognized according to cytoskeletal arrangement and/or chromatin state, in order to assess cell cycle progression alterations, and the data were statistically analyzed. Also, a number of treated seedlings were stained with Evans Blue to determine dead cells. Treatment with the extracts affected both microtubules and F-actin, while cell cycle stage frequency was also altered. Although belonging to well-known toxic, microcystin-producing species, the above cyanobacterial strains have been found (by LC-MS/MS analysis) not to produce microcystins. These findings suggest that bioactive cyanobacterial compounds, other than microcystins, could be able to disrupt the cytoskeleton and cell cycle progression in plant cells.

Crassulacean acid metabolism (CAM) is an adaptation of certain plants, to arid and water-stressed environments. The expression of the CAM cycle may be strongly modulated by developmental and environmental factors. Mesembryanthemum crystallinum is a well-known facultative halophyte, that can shift its photosynthetic carbon fixation pathway from C₃ to CAM under salinity and other abiotic stress factors. However, until now there has been no study about the volatile organic compounds (VOCs) that are emitted by M. crystallinum in its various life cycles, C₃, and CAM. Plants emit a part of the photosynthetically assimilated carbon into the atmosphere in the form of VOCs. Under normal conditions, isoprenoids (isoprene and monoterpenes) are the most abundant VOCs though methanol, acetaldehyde and C-6 compounds are also emitted in great quantities. Under stress conditions, the emission of these compounds generally is altered. The study of how emissions change depending of stress conditions has become a useful “in vivo” indicator of plant vitality and of the plant response to abiotic stresses. Within this work, we aimed to analyse the VOCs emitted from C₃ or CAM-induced M. crystallinum in order to evaluate the possible role that VOCs may have in the C₃/CAM transition and consequently in the adaptation of this plant to salinity. Results showed that M. crystallinum emits different kind of VOCs: aldehydes, hydrocarbons, ketones, alcohols and terpenoids. VOC emissions were generally higher in plants representing C₃, with only few exceptions as butanone, octanal and ethyl-hexanol that were similar in the III phase of CAM and C₃ plants. Regarding the emission of terpenoids, we could observe that whereas plants in the C₃ mode of photosynthesis emitted three types of monoterpenes: a-pinene, carene and limonene, plants in CAM state did not emit any terpenoid compound.

This study aims to develop a technical itinerary for Mg biofortification of two tomato varieties (H1534 and H9205) during the 2018 production cycle. In this framework, plant biofortification was promoted after planting and during the respective production cycle throughout six leaf applications with four different treatments of magnesium sulfate. At harvest, the accumulation of Mg and the interactions with others chemical elements in tomato tissues were studied, as well as the biofortification index. The content of Mg increased 90% and 78.8%, in H1534 and H9205 varieties, respectively, comparatively with controls, as seen by μ-EDXRF M4 Tornado ™ system. Using scanning electron microscopy, with X-ray energy dispersive spectroscopy (SEM-EDS), it was possible to identify in which area of the tomato tissue the Mg predominates. Mg is a micronutrient involved in protein synthesis, muscle and nerve functions, blood glucose control and blood pressure regulation. In human body almost 53% of magnesium (Mg) is involved in the development and maintenance of bone and other calcified tissues. In addition, Mg deficiency is uncommon in humans because of the kidney urinary excretion limits. However, there are some groups at risk of Mg inadequacy, people with gastrointestinal diseases, type 2 diabetes, chronic alcoholism. Magnesium biofortification is a strategy that boosts nutrient enhancement in food crops and can increase nutrient uptake and accumulation in the human body. Considering that biofortification with Mg consists in the development of a functional product, it is concluded that it’s possible to develop a new product such as biofortified tomato pulp.

Some microbes are important players in plant’s fitness, contributing to their nutrients acquisition and protection against diverse biotic and abiotic stresses¹. Despite the vast knowledge acquired during the last decades about the effects in plants of plant growth promoting (PGP) bacteria², apart from those of the legume-rhizobial interactions³, not much is known about the response of bacteria to the interaction with plant.

With the aim to decipher the transcription profile of a non-rhizobial strain in its interaction with the plant, a PGP Pseudomonas strain isolated from Brassica napus roots and capable to protect the plant against biotic and abiotic stresses was inoculated onto rapeseed seedlings. Eleven days post-inoculation, we obtained the RNA-Seq profile of bacterial cells colonizing the seedlings’ roots. RNA from free living cells was used as control. Our analyses allowed us to identify 1378 bacterial genes differentially expressed (log2 fold change > 2; adjusted p value < 0.05). Most overexpressed genes in the interaction are related to biofilm formation, bacterial immunity and infection and bacterial survival to antimicrobial compounds -likely excreted by the plant-. However, genes implicated in PGP traits which had been previously demonstrated in vitro for this strain, appeared to be not significantly overexpressed, suggesting a latter PGP action in the interaction. Based on this RNA-Seq experiment, our results shed light into bacterial mechanisms to effectively colonize plant roots, to survive to plant defense mechanisms as well as to promote plant immunity.

Funding: This work was funded from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 750795.

¹Menéndez, E., & Garcia-Fraile, P. (2017). AIMS microbiology, 3(3), 502.

²Hayat, R., et al., (2010). Annals of microbiology, 60(4), 579-598.

³Oldroyd, G. E., et al., (2011). Annual review of genetics, 45, 119-144.

The response of five mustard cultivars; Pusa Tarak, RH-0742, Pusa Agrini, Giriraj and RH-406 to 0, 100 and 200 mg Cd/kg soil was evaluated in terms of photosynthetic and growth characteristics, antioxidant metabolism, oxidative stress and S-assimilation. 200 Cd soil was found to show more detrimental effects on photosynthetic and growth characteristics than 100 Cd. Among these cultivars, cv. Giriraj showed maximum resistant against 200 Cd stress and showed least reduction in photosynthetic and growth parameters with maximum increase in antioxidant metabolism. Further the influence of optimum-S (100 mg S kg^-1 soil) and excess S (200 mg S kg^-1 soil) in the form of different sources (gypsum, magnesium sulfate, elemental sulfur, and ammonium sulfate) was studied and their involvement in countering Cd induced toxicity was evaluated. Both optimum-S and excess S has positive impact on photosynthesis and growth of plants under control condition while excess S more conspicuously alleviated the detrimental effects of Cd. Among different S sources, elemental S proved to be more beneficial in alleviating Cd stress as compared to other sources by modulating activities of antioxidant enzymes and sustained lower level of lipid peroxidation by reducing contents of H₂O_2, and TBARS. Sulfur induced aforesaid results were due to production of S containing amino acids like cysteine which is a constituent of reduced glutathione and Cys rich heavy metal chelators like metallothionines and phytochelatins. These results suggest that S application in elemental form can more potentially induce antioxidant potential, S-assimilation, photosynthetic attributes and most efficiently help Cd sequestration playing crucial role in plant tolerance to Cd stress.