Boosting nutritional quality of Urtica dioica L. to resist climate change.

Introduction: More than ever, traditional agricultural practices need a shift towards more resilient, sustainable, modern and adaptable practices that benefit the health of the planet and people. Today's consumers are constantly on the lookout for novel, highly nutritious foods that have a positive impact on their overall health and well-being. Nettle (Urtica dioica L.) is gaining recognition not only as a popular medicinal plant, but also as a desirable green leafy vegetable rich in phytonutrients. As it is difficult and even expensive to control the quality standards of wild-collected plants, the implementation of sustainable cultivation methods, especially hydroponics, with effective greenhouse management could be a possible solution to obtain a standardized product with high nutritional value. Therefore, the aim of this study was to investigate the effects of four nutrient solutions differing in the content of macro- and micronutrients (especially nitrogen, potassium, calcium, magnesium and iron) and two consecutive cuts on the number of leaves, yield, nitrate and mineral content and the content of specialized metabolites of stinging nettle from a floating hydroponic system. Methods: Nettle plants were cultivated in a hydroponic system using the floating hydroponics technique. The two-factorial experiment was performed with nutrient solution and consecutive cuts as factors. Results: The highest yield (2.49 kg/m2) was achieved after the 1st cut with plants cultivated in the nutrient solution with higher nutrient concentration. All tested nutrient solutions resulted in high levels of minerals and bioactive compounds in the plant material (ascorbic acid content of 102.30 mg/100 g fw and total phenolics content of 465.92 mg GAE/100 g fw), confirming floating hydroponics as a sustainable approach for cultivating nettle with enhanced nutritional value and antioxidant potential. Conclusion: It is important to highlight that the nutrient solution with the lowest nutrient composition yielded the highest concentrations of calcium (5.54%) and iron (180.67 mg/kg dw). Furthermore, it exhibited elevated levels of specific phenolic compounds, including caffeoylmaleic acid, ellagic acid, ferulic acid, naringin, and rutin trihydrate. Notably, this solution demonstrated the lowest nitrate content (4225.33 mg/kg fw) in the plant material. Therefore, it can be recommended as a preferable formulation for hydroponic nettle cultivation.

How to Increase the Nutritional Quality of Stinging Nettle Through Controlled Plant Nutrition§.

Research background: As food production faces major challenges, modern agricultural practices are increasingly focused on conserving resources, reducing negative environmental impacts and sustainably producing food with a high content of health-promoting phytochemicals. During production, many factors can affect the quality and chemical composition of a final food product. Proper selection of cultivating conditions, especially a balanced nutrition, can significantly increase nutritional value and result in foods with strong biological and functional properties. Stinging nettle is a rich source of minerals, vitamins, pigments, phenols and other bioactive compounds and can be consumed as a green leafy vegetable with beneficial effects on human health. Therefore, the aim of this study is to determine the nutritional quality and antioxidant capacity of stinging nettle leaves under the influence of different nutrient solution (NS) treatments and three harvest cycles. Experimental approach: The experiment was conducted in a floating hydroponic system in which treatments with different nutrient solutions were applied and three harvest cycles were carried out. After each harvest, the following treatments were applied: treatment 1 - depletion of nutrient solution by adding water, treatment 2 - supplementation of nutrient solution by adding initial nutrient solution and treatment 3 - correction of nutrient solution by adding nutrients. Among the bioactive compounds, minerals, ascorbic acid, phenols and photosynthetic pigments content, as well as antioxidant capacity were analysed spectrophotometrically, while individual phenols were determined by liquid chromatography. Results and conclusions: Different nutrition solution treatments and the number of harvest cycles had a significant effect on the content of the analysed bioactive compounds. The highest mass fraction (on fresh mass basis) of total phenols expressed as gallic acid equivalents (377.04 mg/100 g), total flavonoids expressed as catechol equivalents (279.54 mg/100 g), ascorbic acid (112.37 mg/100 g) and pigments (total chlorophylls 1.84, and total carotenoids 0.36 mg/g) as well as the highest antioxidant capacity expressed as Trolox equivalents (35.47 µmol/g) were recorded in the samples supplemented with nutrient solution (treatment NS2) and analysed after the third harvest. Novelty and scientific contribution: This is the first time that stinging nettle leaves have been produced in a floating hydroponic system by controlled plant nutrition. We have set this type of nutritional manipulation with multiple harvest cycles as an innovative technique for the production of novel food with improved nutritional value that can be consumed as green leafy vegetables.

RNA Sequencing Analyses for Deciphering Potato Molecular Responses.

Understanding the molecular mechanisms of potato development and responses to environmental stressors is of utmost importance for achieving stable crop yields. RNA sequencing (RNA-Seq) provides an insight into responses of all of the organism genes to the environmental and developmental cues and thus provides insights into underlying modes of action. In this chapter, we guide a researcher through some of the most important steps in the analysis of transcriptomics data. The initial topic of experimental design is followed by a more wet-lab-oriented section on RNA-Seq sample preparation. Next, we present intermediate steps of data retrieval, quality control, mapping, and differential expression of the dataset and a section on how to expose your data to the public (i.e., public repositories) and make it findable, accessible, interoperable, and reusable (FAIR). In the last four sections, we describe specific tools or Web applications, which ease the exploration of generated results in the context of their gene function and network-based visualizations, specifically GoMapMan, GSEA, DiNAR, and Biomine Explorer. All sections are accompanied by potato dataset examples and include general hints and tricks, as well as potato specificities that one should be aware of.

A Comprehensive Guide to Potato Transcriptome Assembly.

We have witnessed a rapid advancement in high-throughput genome sequencing and the maturation of long-read technologies. However, an accurate assembly of polyploid potato genomes still remains challenging. Sequencing the double-monoploid genome of Solanum tuberosum Group Phureja (Xu et al., Nature 475:189-195, 2011) has enabled functional studies of polyploid potato cultivars using RNA sequencing (RNA-Seq) technologies, although with the limitation of not covering cultivar-specific gene expression. The accumulated RNA-Seq datasets from these cultivars can be leveraged to assemble tetraploid potato transcriptomes that enable the analysis of genes that are not limited to reference genome annotations. To increase transcriptomes' quality, short-read assemblies are nowadays complemented with full-length transcriptome sequencing using Pacific Biosciences or Oxford Nanopore platforms. In this chapter we give a detailed guide on a pipeline for de novo transcriptome assembly of polyploid potato genotypes and their integration into a pan-transcriptome.

Cultivar-specific transcriptome and pan-transcriptome reconstruction of tetraploid potato.

Although the reference genome of Solanum tuberosum Group Phureja double-monoploid (DM) clone is available, knowledge on the genetic diversity of the highly heterozygous tetraploid Group Tuberosum, representing most cultivated varieties, remains largely unexplored. This lack of knowledge hinders further progress in potato research. In conducted investigation, we first merged and manually curated the two existing partially-overlapping DM genome-based gene models, creating a union of genes in Phureja scaffold. Next, we compiled available and newly generated RNA-Seq datasets (cca. 1.5 billion reads) for three tetraploid potato genotypes (cultivar Désirée, cultivar Rywal, and breeding clone PW363) with diverse breeding pedigrees. Short-read transcriptomes were assembled using several de novo assemblers under different settings to test for optimal outcome. For cultivar Rywal, PacBio Iso-Seq full-length transcriptome sequencing was also performed. EvidentialGene redundancy-reducing pipeline complemented with in-house developed scripts was employed to produce accurate and complete cultivar-specific transcriptomes, as well as to attain the pan-transcriptome. The generated transcriptomes and pan-transcriptome represent a valuable resource for potato gene variability exploration, high-throughput omics analyses, and breeding programmes.

Beetroot mineral composition affected by mineral and organic fertilization.

In modern agriculture, besides providing high and stable yields, it is imperative to produce products with a high nutritive quality. The goal of this study was to determine the effect of different fertilization regimes on the macro- and micronutrients in beetroot. A 3-year field trial was set up according to a Latin square method with four types of fertilization (unfertilized control, 50 t stable manure ha-1, and 500 and 1,000 kg NPK 5-20-30 ha-1). The mineral content was determined as follows (mg 100 g-1 in fresh weight of beetroot): 14-29 P, 189-354 K, 18-34 Ca, 17-44 Mg, 0.67-1.83 Fe, 0.41-0.65 Mn and 0.28-0.44 Zn. The highest beetroot P content was determined for the treatment with stable manure, especially in a year with dry climatic conditions. The highest beetroot K content was determined for the treatment with 1,000 kg NPK 5-20-30 ha-1, but at the same time for the same treatment, a general decreasing trend of micronutrient content was determined, due to the possible antagonistic effect of added potassium. For better mineral status of beetroot, application of combined mineral and organic fertilizers supplemented with additional foliar application of micronutrients can be suggested.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Primary Metabolism, Phenylpropanoids and Antioxidant Pathways Are Regulated in Potato as a Response to Potato virus Y Infection.

Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus-plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed.

Potato virus Y infection hinders potato defence response and renders plants more vulnerable to Colorado potato beetle attack.

In the field, plants are challenged by more than one biotic stressor at the same time. In this study, the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa decemlineata Say; CPB) and Potato virus Y(NTN) (PVY(NTN) ) were investigated through analyses of gene expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile compounds. CPB larval growth was enhanced when reared on secondary PVY(NTN) -infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVY(NTN) -infected plants, ethylene signalling pathway induction and induction of auxin response transcription factors were attenuated, while no differences were observed in jasmonic acid (JA) signalling pathway. Similarly to rearing on virus-infected plants, CPB larvae gained more weight when reared on plants silenced in JA receptor gene (coi1). Although herbivore-induced defence mechanism is regulated predominantly by JA, response in coi1-silenced plants only partially corresponded to the one observed in PVY(NTN) -infected plants, confirming the role of other plant hormones in modulating this response. The release of ß-barbatene and benzyl alcohol was different in healthy and PVY(NTN) -infected plants before CPB larvae infestation, implicating the importance of PVY(NTN) infection in plant communication with its environment. This was reflected in gene expression profiles of neighbouring plants showing different degree of defence response. This study thus contributes to our understanding of plant responses in agro-ecosystems.

Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.

A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense.

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.