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Chickpea (Cicer arietinum) is a major player in the FAO Zero Hunger program “toolkit”. It is the second most cultivated food legume, a source of sustainable protein (and other nutrients) and contributes to improved soil health and lower fertilization input. Grain yield, seed protein content and nutritional quality were found to be largely affected by Genotype x Environment. In the Mediterranean regions, higher frequency, intensity and duration of droughts, as well as hotter droughts are being registered, thus interfering with agroecosystems’ structure, composition, and functions. A controlled conditions assay (non-invasive phenotyping@PhenoLab) was undertaken in two genotypes from the Portuguese chickpea breeding program (Elvas, INIAV). Results revealed that high temperature (32º C during the reproductive phase) and water regime (40% vs 10% soil water content) play a significant role in chickpea development, seed production and composition, and protein content. As seed biochemical signatures allow to discriminate between genotypes, the power protein-based methodologies in genotype assessment are highlighted. Because some proteins can resist gastrointestinal digestion and influence human health, the processed seed proteome (i.e. following soaking, boiling and in-vitro digestion) in three consecutive years was further analysed in field-grown chickpeas (Elvas, INIAV). The seed proteome was found to be highly conserved, with minor changes being attributed to the seed development conditions. Furthermore, in vitro digestion efficiently removed many anti-nutritional proteins. Combined phenotyping and omic´s approaches contribute to the mechanistic knowledge of how severe terminal drought and high temperature modulate sink capacity and productivity (yield and quality). The integrated use of phenomics and omics methodologies has significant potential to increase our understanding of plant growth and development and, thus, an efficient, knowledge-based management of crops and resources. Work supported by EMPHASIS-GO (HORIZON-INFRA-2021-DEV-02 contract n. 101079772), by the European Plant Phenotyping Network 2020 (EXCELLENT SCIENCE - Research Infrastructures contract n. 731013) and by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P. namelly: UCIBIO – Research Unit on Applied Molecular Biosciences (FCT UIDP/04378/2020; FCT UIDB/04378/ 2020); 4HB - Associate Laboratory Institute for Health and Bioeconomy (FCT LA/P/0140/2020); LEAF – Linking Landscape, Environment, Agriculture and Food (FCT UID/AGR/04129/2020); PhD fellowship SFRH/BD/70345/2010. info:eu-repo/semantics/publishedVersion

Plants have evolved sophisticated mechanisms to coordinate carbon metabolism during growth and development both under optimal and stress conditions. In coffee-rust biotrophic interactions, plants try to limit pathogen access to nutrients (e.g., sugars and sugar derivatives) and trigger immune responses, while Hemileia vastatrix (Hv) attempts to circumvent plant defences and control the host's primary metabolism for its own benefit. Previous proteomics data highlighted the up-regulation of proteins from photosynthesis, primary metabolism, and redox-related enzymes along the coffee resistance response. Coffee Kawisari hybrid - Hv interactions (resistant and susceptible reactions) were evaluated using a single sample fractionation method for metabolite and protein extraction. The microscopic evaluation of the Hv infection process revealed that coffee resistance was associated with early hypersensitive response and accumulation of phenolic-like compounds in host cell walls. GC-TOF-MS untargeted metabolomics allowed the identification of metabolic components, such as sugars, sugar derivatives, amino acids, phenylpropanoids, chlorogenic acids, alkaloids, and fatty acids (using the Golm Metabolome database). The overrepresentation of several caffeoylquinic acids in the resistance reaction may be linked to the accumulation of the phenolic-like compounds that were cytologically observed at the infection sites. Furthermore, sugar-related features also played a role in distinguishing between resistant and susceptible reactions, such as glucose and galactose. The cellular availability of mono and disaccharides is the result of the activity of several enzymes, e.g., sucrose synthase and invertases, that can be targeted by Hv in its strategy to manipulate plant carbon metabolism. The activity profile of these enzymes along the infection will be discussed. Proteomic analysis of the same samples (using the single sample fractionation method previously mentioned) is foreseen. The ultimate goal is to establish a connection between the metabolite and protein signatures. Financial support by the Access to Research Infrastructures, Horizon2020 Programme of the EU (EPPN2020 Grant Agreement 731013) and Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES (PTDC/ASP- PLA/29779/2017), UNIT LEAF (UID/AGR/04129/2020), UCIBIO (UIDP/04378/2020; UIDB/04378/ 2020) and i4HB (LA/P/0140/2020). N/A

The five Nordic countries span the most northern region for field cultivation in the world. This presents challenges per se with short growing seasons, long days and a need for frost tolerance. Climate change has additionally increased risks for micro-droughts and water logging as well as pathogens and pests expanding northwards. Thus, Nordic agriculture demands crops that are adapted to the special Nordic growth conditions and future climate scenarios. A focus on crop varieties and traits important to Nordic agriculture, including the unique resource of nutritious wild crops, can meet these needs. In fact, with a future longer growing season due to climate change the region could contribute proportionally more to the global agricultural production. This also applies to other northern regions, including the Arctic. To address current growth conditions, mitigate impacts of climate change and meet market demands, the adaptive capacity of crops that both perform well in northern latitudes and are more climate resilient has to be increased, and better crop management systems be built. This requires functional phenomics approaches that integrate versatile high-throughput phenotyping, physiology and bioinformatics. This review stresses key target traits, the opportunities of latitudinal studies and infrastructure needs for phenotyping to support Nordic agriculture.

Chickpeas (Cicer arietinum L.) are included in the Zero Hunger program, are the world’s second most cultivated food legume, and have one of the best nutritional compositions among the grain legumes. Drought typically reduces grain yield, namely when occurring during pod set and seed filling, which is further reduced in combination with elevated temperatures. With this EPPN2020 project, we focused the phenotyping during the critical flowering and seed filling stages. Phenological responses to terminal drought and heat were evaluated by non-invasive thermo-, multireflectance- and multifluorescence imaging in the Phenolab at the University of Copenhagen (DK) to relate physiological responses at plant level with the activity signatures of key enzymes of carbohydrate and antioxidant metabolism and its final impact on seed yield and quality. Two chickpea genotypes with high production potential were evaluated: Elvar, adapted to dry conditions; Electra, selected due to large seed size. The impact of high temperature was combined with a 40% or 10% watering regime and compared with plants kept at 40% watering regime and ambient temperature. The sensor based phenotyping in the Phenolab was complemented with seed yield and quality analysis. Both seed weight and seed number were negatively affected by high temperatures irrespectively of the watering regime. Seeds from both genotypes obtained under high temperature conditions showed increased protein content and lower starch. Protein content was further affected by the watering regime. Alterations in protein-to-starch indicates modifications in sink-source relationships. The differential effects were mirrored in distinct sensor based and biochemical biosignatures. This study contributes to the mechanistic knowledge on how combined effects of severe terminal drought and high temperature modulate sink capacity and chickpea productivity (yield and quality) and identifies predictive biomarkers for the breeding. info:eu-repo/semantics/publishedVersion

In sugar beet (Beta vulgaris subsp. vulgaris, Chenopodiaceae), sucrose is stored in a taproot developed by a genus-specific type of anomalous secondary thickening from supernumerary cambial rings. In a previous study (Jammer & al. 2020), we showed that the transition from primary root development to the formation of the sucrose-storing taproot is accompanied by a clear shift in physiological signatures: characteristic changes in the activities of several key enzymes of carbohydrate metabolism were associated with the onset of taproot development and sucrose storage, resulting in three distinct physiological stages (prestorage, transition, secondary growth and sucrose storage). In order to investigate the regulatory mechanisms behind these changes in enzyme activities, we performed a microarray expression analysis for the genes encoding the respective carbohydrate-metabolic enzymes. The data obtained from the microarray were validated for selected transcripts by semi-quantitative reverse transcription PCR. A high level of similarity between the developmental changes in transcript levels and enzyme activities at the transition stage suggests that the majority of the respective enzymes are predominantly regulated on the transcriptional level during the early stages of taproot development.

Additional file 1: Table S1. Experimental setup of the chemicals used in each enzymatic assay. In cases where the extract volume added was smaller than 25μL, buffer was added to reach a total extraction volume of 25μL in each well. In every well, the total volume of the reaction was 160 μL.

Additional file 2: Table S2. Activity profile of the nine antioxidant enzymatic signature in different plants and tissue. The values are normalized per mg protein (Mean v [nkat/mg prot]) ± standard deviation; N.D. not detectable. The activities for Arabidopsis leaves, maize leaves and maize roots are average of 3 independent plant replicates whereas for sugar beet roots and barley leaves 3 distinct extractions were performed on the same plant sample, consisting of at least 15 individual plants for barley leaves and 3 plants for sugar beet roots.

Additional file 4: Figure S2. Antioxidant enzymatic activities during salt stress. Nicotiana tabacum plants were grown for 60 days in the greenhouse, thereafter, detached leaves were fed with 0,2M NaCl or 0,5M NaCl via their petioles. Leaves were sampled every two days for a total of 14 days after NaCl application and the enzymatic activities of the 9 antioxidant scavenging enzymes were tested and normalized by protein content. Bars indicate standard deviations of three independent biological replicates.

Additional file 3: Figure S1. Antioxidant enzymatic activities during drought. Nicotiana tabacum plants were grown for 60 days in the greenhouse, thereafter, water was withheld for half of the plants whereas the other half was kept watered. Leaves were harvested every two days for a total of 14 days after watering was stopped and the enzymatic activities of the 9 antioxidant scavenging enzymes were tested and normalized by protein content. Bars indicate standard deviations of three independent biological replicates.

Additional file 3. Case study on the effect of palmitate treatment on Aldolase activity. Additional methods used in the case study. The file describes experimental procedures of in vitro palmitate treatment and analysis of enzyme gene expression. Obtained data were aligned with results from enzyme activity assays. https://doi.org/10.6084/m9.figshare.7859777 .

Additional file 1. Additional line graphs, spider graphs, PCA biplots and table of PCA loadings for all tested concentrations and time points, visual effects on representative samples, flowchart of experimental design and scheme of data organization and inputs used for analyses.

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.