Centre for Plant Biotechnology and Genomics

Yield reliability under diverse environments is important to address climate stress consequences in wheat production systems. Breeding for reliability under a changing climate remains a challenge in wheat. We assessed the performance of 18 hexaploid (Triticum aestivum L.) genotypes in three field environments at a location within the semi-arid Canadian Prairies over four years with a primary aim to establish knowledge of the phenotypic plasticity and yield reliability in the parental lines as it relates to heat and drought stress tolerance. We collected data on various physiological traits along with some agronomic and morphological attributes, uncovering significant variation across early seeded rainfed, early seeded irrigated, and late seeded rainfed (hot and dry) environments. Eight high yielding hexaploid genotypes ‘01S0263-28’, ‘AC Foremost’, AC Karma’, ‘Cutler’, ‘MN03358-4’, ‘Reeder’, ‘Stettler’, and ‘Superb’ showed higher grain Δ¹³C. Six of these genotypes ‘01S0263-28’, ‘AC Foremost’, ‘MN03358-4’, ‘Reeder’, ‘Stettler’, and ‘Superb’ showed higher water use efficiency under irrigated as well as hot and dry environment compared to the low yielding lines ‘Red Fife’,’8021-V2’ and ‘BW278’. Only four genotypes ‘01S0263-28’, ‘MN03358-4’, ‘Reeder’, and ‘Stettler’ were found with higher yield reliability index. The grain yield relationship with leaf rolling, glaucousness, and canopy temperature was found to be weak. The flag leaf stomatal numbers increased with water stress in high yielding lines which were otherwise low in stomatal numbers. Contrastingly, water stress significantly reduced the stomatal numbers in low yielding lines that were otherwise high in stomatal numbers. The results highlight the stomatal adaptability of different genotypes in response to drought. Taken together, these results provide baseline information that the genotypes with high grain Δ¹³C and WUE, and low stomata numbers are more yield reliable under variable field environments, and this information can guide the breeding of climate-resilient germplasm that expresses consistent and reliable grain yield production in the semi-arid Prairies.

Key message Enhanced antioxidant enzymes activity, particularly superoxide dismutase and catalase, along with autophagy process in reproductive organs, can improve the resilience of rapeseed to heat stress, thereby securing crop yield in the face of global warming. Abstract Climate change and global warming have increasingly influenced yield and quality of rapeseed (Brassica napus) almost all across the world. The response of reproductive organs to high-temperature stress was studied in two rapeseed varieties, SAFI5 and DH13 with contrasting levels of heat stress tolerance. Pollen germination, viability, and seed set showed a significant reduction in the heat-sensitive variety (DH13). Superoxide quantification revealed higher accumulation in heat-sensitive variety, leading to decreased seed formation and floret fertility most probably due to declined pollen viability and stigma receptivity. Further microscopic analysis of the anther and pistil demonstrated a significant overlay between the damaged areas and the location of O2⁻ accumulation. The sensitive variety showed higher O2⁻ accumulation and a wider damage area than the tolerant one, suggesting that superoxide could incapacitate anther and pistil due to structural injury. Moreover, the activity levels and expression of superoxide dismutase and catalase antioxidant enzymes were significantly higher in the anther and pistil of the tolerant variety. Histochemical analysis also indicated markedly higher autophagosome formation in tolerant variety’s anther and pistil. Consistently, the expression levels of autophagy and ubiquitin-proteasome system (UPS)-related genes including BnATG8d, BnEXO70B, BnATl1 4A, and BnNBR1, as well as ubiquitin-activating enzyme E1, were higher in both reproductive organs of the tolerant variety. Interestingly, the areas of autophagosome formation overlapped with the areas in which higher superoxide accumulation and structural changes happened, suggesting a specific role of autophagy in oxidative stress response.

Climate change and global warming have increasingly influenced yield and quality of rapeseed ( Brassica napus ) almost all across the world. The response of reproductive organs to high-temperature stress was studied in two rapeseed varieties, SAFI5 and DH13 with contrasting levels of heat stress tolerance. Pollen germination, viability, and seed set showed a significant reduction in the heat-sensitive variety (DH13). Superoxide quantification revealed higher accumulation in heat-sensitive variety, leading to decreased seed formation and floret fertility most probably due to declined pollen viability and stigma receptivity. Further microscopic analysis of the anther and pistil demonstrated a significant overlay between the damaged areas and the location of O 2 ⁻ accumulation. The sensitive variety showed higher O 2 -accumulation and a wider damage area than the tolerant one, suggesting that superoxide could incapacitate anther and pistil due to structural injury. Moreover, the activity levels and expression of superoxide dismutase and catalase antioxidant enzymes were significantly higher in the anther and pistil of the tolerant variety. Histochemical analysis also indicated markedly higher autophagosome formation in tolerant variety’s anther and pistil. Consistently, the expression levels of autophagy and ubiquitin-proteasome system (UPS)-related genes including BnATG8d , BnEXO70B , BnATl1 4A , and BnNBR1 , as well as ubiquitin-activating enzyme E1 , were higher in both reproductive organs of the tolerant variety. Interestingly, the areas of autophagosome formation overlapped with the areas in which higher superoxide accumulation and structural changes happened, suggesting a specific role of autophagy in oxidative stress response.

Crop genomes accumulate deleterious mutations—a phenomenon known as the cost of domestication. Precision genome editing has been proposed to eliminate such potentially harmful mutations; however, experimental demonstration is lacking. Here we identified a deleterious mutation in the tomato transcription factor SUPPRESSOR OF SP2 (SSP2), which became prevalent in the domesticated germplasm and diminished DNA binding to genome-wide targets. We found that the action of SSP2 is partially redundant with that of its paralog SSP in regulating shoot and inflorescence architecture. However, redundancy was compromised during tomato domestication and lost completely in the closely related species Physalis grisea, in which a single ortholog regulates shoot branching. We applied base editing to directly repair the deleterious mutation in cultivated tomato and obtained plants with compact growth that provide an early fruit yield. Our work shows how deleterious variants have sensitized modern genotypes for phenotypic tuning and illustrates how repairing deleterious mutations with genome editing may allow predictable crop improvement.

Global climate change exacerbates abiotic stresses, as drought, heat, and salt stresses are anticipated to increase significantly in the coming years. Plants coexist with a diverse range of microorganisms. Multiple inter-organismic relationships are known to confer benefits to plants, including growth promotion and enhanced tolerance to abiotic stresses. In this study, we investigated the mutualistic interactions between 3 fungal endophytes originally isolated from distinct arid environments and an agronomically relevant crop, Solanum lycopersicum . We demonstrated a significant increase in shoot biomass under drought conditions in co-cultivation with Penicillium chrysogenum isolated from Antarctica, Penicillium minioluteum isolated from the Atacama Desert, Chile, and Serendipita indica isolated from the Thar Desert, India. To elucidate plant gene modules commonly induced by the different endophytes that could explain the observed drought tolerance effect in tomato, a comprehensive transcriptomics analysis was conducted. This analysis led to the identification of a shared gene module in the fungus-infected tomato plants. Within this module, gene network analysis enabled us to identify genes related to abscisic acid (ABA) signaling, ABA transport, auxin signaling, ion homeostasis, proline biosynthesis, and jasmonic acid signaling, providing insights into the molecular basis of drought tolerance commonly mediated by fungal endophytes. Our findings highlight a conserved response in the mutualistic interactions between endophytic fungi isolated from unrelated environments and tomato roots, resulting in improved shoot biomass production under drought stress.

Solvothermal polymerization (STP) of diaminomaleonitrile (DAMN) was evaluated using a wide variety of solvents and in the temperature range from 80 to 170 °C. The highest yields, almost quantitative, were achieved with protic n-alcohols such as n-pentanol or n-hexanol at 130 and 150 °C, respectively. The kinetic behavior was studied by gravimetry and the DAMN consumption was monitored by UV–vis spectroscopy and HPLC. GC–MS identified byproducts of the DAMN hydrolysis and oxidation reactions, which were significantly reduced when n-pentanol or n-hexanol were used with respect to hydrothermal conditions. This led to an exploration of compositional changes and microstructural variations by FTIR and NMR spectroscopy and simultaneous thermal analysis. n-Hexanol appears to be an ideal eco-friendly solvent for the DAMN self-STP. The results presented here are not only of interest for the design of polymeric materials based on C=N structures but also show remarkable implications for prebiotic chemistry.

Background: Cross-reactivity between nonspecific lipid transfer proteins could cause anaphylaxis, further influencing food avoidance and nutrient deficiencies. The one affecting olive pollen (Ole e 7) and peach (Pru p 3) may underlie a variety of pollen-food syndromes, though a deep molecular analysis is necessary. Methods: Three Ole e 7-monosensitised patients (MON_OLE), three Pru p 3-monosensitised patients (MON_PRU) and three bisensitised patients (BI) were selected. For epitope mapping, both digested proteins were incubated with patient sera, and the captured IgE-bound peptides were characterised by LC-MS. Results: The analysis revealed two Ole e 7 epitopes and the three Pru p 3 epitopes previously described. Interestingly, the “KSALALVGNKV” Ole e 7 peptide was recognised by MON_OLE, BI and MON_PRU patients. Conversely, all patients recognised the “ISASTNCATVK” Pru p 3 peptide. Although complete sequence alignment between both proteins revealed 32.6% identity, local alignment considering seven residue fragments showed 50 and 57% identity when comparing “ISASTNCATVK” with Ole e 7 and “KSALALVGNKV” with Pru p 3. Conclusions: This study mapped sIgE-Ole e 7-binding epitopes, paving the way for more precise diagnostic tools. Assuming non-significant sequence similarity, structural homology and shared key residues may underlie the potential cross-reactivity between Ole e 7 and Pru p 3 nsLTPs.

The domestication process in grapevines has facilitated the fixation of desired traits. Nowadays, vegetative propagation through cuttings enables easier preservation of these genotypes compared to sexual reproduction. Nonetheless, even with vegetative propagation, various phenotypes are often present within the same vineyard due to the accumulation of somatic mutations. These mutations are not the sole factors influencing phenotype. Alongside somatic variations, epigenetic variation has been proposed as a pivotal player in regulating phenotypic variability acquired during domestication. The emergence of these epialleles might have significantly influenced grapevine domestication over time. This study aims to investigate the impact of domestication on methylation patterns in cultivated grapevines. Reduced-representation bisulfite sequencing was conducted on 18 cultivated and wild accessions. Results revealed that cultivated grapevines exhibited higher methylation levels than their wild counterparts. Differential Methylation Analysis between wild and cultivated grapevines identified a total of 9955 differentially methylated cytosines, of which 78% were hypermethylated in cultivated grapevines. Functional analysis shows that core methylated genes (consistently methylated in both wild and cultivated accessions) are associated with stress response and terpenoid/isoprenoid metabolic processes. Meanwhile, genes with differential methylation are linked to protein targeting to the peroxisome, ethylene regulation, histone modifications, and defense response. Collectively, our results highlight the significant roles that epialleles may have played throughout the domestication history of grapevines.

Recombinational repair is an important mechanism that allows DNA replication to overcome damaged templates, so the DNA is duplicated timely and correctly. The RecFOR pathway is one of the common ways to load RecA, while the RuvABC complex operates in the resolution of DNA intermediates. We have generated deletions of recO, recR and ruvB genes in Thermus thermophilus, while a recF null mutant could not be obtained. The recO deletion was in all cases accompanied by spontaneous loss of function mutations in addA or addB genes, which encode a helicase‐exonuclease also key for recombination. The mutants were moderately affected in viability and chromosome segregation. When we generated these mutations in a Δppol/addAB strain, we observed that the transformation efficiency was maintained at the typical level of Δppol/addAB, which is 100‐fold higher than that of the wild type. Most mutants showed increased filamentation phenotypes, especially ruvB, which also had DNA repair defects. These results suggest that in T. thermophilus (i) the components of the RecFOR pathway have differential roles, (ii) there is an epistatic relationship of the AddAB complex over the RecFOR pathway and (iii) that neither of the two pathways or their combination is strictly required for viability although they are necessary for normal DNA repair and chromosome segregation.

Plants share their habitats with a multitude of different microbes. This close vicinity promoted the evolution of inter-organismic interactions between plants and many different microorganisms that provide mutual growth benefits both to the plant and the microbial partner. The symbiosis of Arabidopsis thaliana with the beneficial root colonizing endophyte Serendipita indica represents a well-studied system. Co-colonization of Arabidopsis roots with S. indica significantly promotes plant growth. Due to the notable phenotypic alterations of fungus-infected root systems, the involvement of a reprogramming of plant hormone levels, especially that of indole-3-acetic acid, has been suggested earlier. However, until now, the molecular mechanism by which S. indica promotes plant growth remains largely unknown. This study used comprehensive transcriptomics, metabolomics, reverse genetics, and life cell imaging to reveal the intricacies of auxin-related processes that affect root growth in the symbiosis between A. thaliana and S. indica . Our experiments revealed the essential role of tightly controlled auxin conjugation in the plant–fungus interaction. It particularly highlighted the importance of two GRETCHEN HAGEN 3 ( GH3 ) genes, GH3.5 and GH3.17 , for the fungus infection-triggered stimulation of biomass production, thus broadening our knowledge about the function of GH3s in plants. Furthermore, we provide evidence for the transcriptional alteration of the PIN2 auxin transporter gene in roots of Arabidopsis seedlings infected with S. indica and demonstrate that this transcriptional adjustment affects auxin signaling in roots, which results in increased plant growth.

Probiotics are valuable microorganisms effective in reducing malnutrition-related infections in children. In this work, a collection of lactobacilli strains representative of traditional Andean fermented beverages was in vitro screened for their capability to survive the gastrointestinal transit, to adhere to the intestinal epithelium and to compete under simulated conditions of the child gut microbiota. The results allowed the selection of the riboflavin overproducing strain Lactiplantibacillus plantarum CECT 9435 based on its good rate of survival under in vitro gastrointestinal conditions when included in a food matrix representing the fortified food supplement Incaparina. The strain also showed good adhesion to HT29 cells producing mucus and outstanding performance in E. coli competition for the adhesion to this epithelial cell line. L. plantarum CECT 9435 gut performance was also evaluated in the child intestinal microbiota simulated in a dynamic gut model (BFBL simulator). The viability of the probiotic candidate in the gut conditions was high during the 7-day intervention period, reaching over 1 × 10⁷ counts in each of the reactors simulating the three colonic regions. The transient viability of L. plantarum CECT 9435 within the child gut microbiota and its adhesion capacity to intestinal cells could facilitate the strain potential benefits as probiotic added to fortified supplementary foods destined to malnourished children.

Food security is threatened by climate change, with heat and drought being the main stresses affecting crop physiology and ecosystem services, such as plant–pollinator interactions. We hypothesize that tracking and ranking pollinators' preferences for flowers under environmental pressure could be used as a marker of plant quality for agricultural breeding to increase crop stress tolerance. Despite increasing relevance of flowers as the most stress sensitive organs, phenotyping platforms aim at identifying traits of resilience by assessing the plant physiological status through remote sensing‐assisted vegetative indexes, but find strong bottlenecks in quantifying flower traits and in accurate genotype‐to‐phenotype prediction. However, as the transport of photoassimilates from leaves (sources) to flowers (sinks) is reduced in low‐resilient plants, flowers are better indicators than leaves of plant well‐being. Indeed, the chemical composition and amount of pollen and nectar that flowers produce, which ultimately serve as food resources for pollinators, change in response to environmental cues. Therefore, pollinators' preferences could be used as a measure of functional source‐to‐sink relationships for breeding decisions. To achieve this challenging goal, we propose to develop a pollinator‐assisted phenotyping and selection platform for automated quantification of Genotype × Environment × Pollinator interactions through an insect geo‐positioning system. Pollinator‐assisted selection can be validated by metabolic, transcriptomic, and ionomic traits, and mapping of candidate genes, linking floral and leaf traits, pollinator preferences, plant resilience, and crop productivity. This radical new approach can change the current paradigm of plant phenotyping and find new paths for crop redomestication and breeding assisted by ecological decisions.

Grafting in tomato (Solanum lycopersicum L.) has mainly been used to prevent damage by soil-borne pathogens and the negative effects of abiotic stresses, although productivity and fruit quality can also be enhanced using high vigor rootstocks. In the context of a low nutrients input agriculture, the grafting of elite cultivars onto rootstocks displaying higher Nitrogen Use Efficiency (NUE) supports a direct strategy for yield maximization. In this study we assessed the use of plants overexpressing the Arabidopsis (AtCDF3) or tomato (SlCDF3) CDF3 genes, previously reported to increase NUE in tomato, as rootstocks to improve yield in the grafted scion under low N inputs. We found that the AtCDF3 gene induced greater production of sugars and amino acids, which allowed for greater biomass and fruit yield under both sufficient and limiting N supplies. Conversely, no positive impact was found with the SlCDF3 gene. Hormone analyses suggest that gibberellins (GA4), auxin and cytokinins (tZ) might be involved in the AtCDF3 responses to N. The differential responses triggered by the two genes could be related, at least in part, to the mobility of the AtCDF3 transcript through the phloem to the shoot. Consistently, a higher expression of the target genes of the transcription factor, such as glutamine synthase 2 (SlGS2) and GA oxidase 3 (SlGA3ox), involved in amino acid and gibberellin biosynthesis, respectively, was observed in the leaves of this graft combination. Altogether, our results provided further insights into the mode of action of CDF3 genes and their biotechnology potential for transgrafting approaches.

A mutant deficient in polynucleotide phosphorylase (PNPase) activity was previously constructed in Enterococcus faecalis 14; a strain producing a leaderless two-peptide enterocin DD14 (EntDD14). Here, we examined the impact of the absence of PNPase on the expression and synthesis of EntDD14, at the transcriptional and functional levels. As result, EntDD14 synthesis augmented in line with the growth curve, reaching a two- to fourfold increase in the ΔpnpA mutant compared to the E. faecalis 14 wild-type strain (WT). EntDD14 synthesis has reached its highest level after 9 h of growth in both strains. Notably, high expression level of the ddABCDEFGHIJ cluster was registered in ΔpnpA mutant. Transcriptional and in silico analyses support the existence of ddAB and ddCDEFGHIJ independent transcripts, and analysis of the fate of ddAB and ddCDEFGHIJ mRNAs indicated that the differences in mRNA levels and the high EntDD14 activity are likely due to a better stability of the two transcripts in the ΔpnpA mutant, which should result in a higher translation efficiency of the ddAB EntDD14 structural genes and their other protein determinants. Consequently, this study shows a potential link between the mRNA stability and EntDD14 synthesis, secretion and immunity in a genetic background lacking PNPase.

Research on microalgae has surged due to its diverse biotechnological applications and capacity for accumulating bioactive compounds. Despite considerable advancements, microalgal cultivation remains costly, prompting efforts to reduce expenses while enhancing productivity. This study proposes a cost-effective approach through the coculture of microalgae and bacteria, exploiting mutualistic interactions. An engineered consortium of Chlorella vulgaris and Stutzerimonas stutzeri strain J3BG demonstrated biofilm-like arrangements, indicative of direct cell-to-cell interactions and metabolite exchange. Strain J3BG's enzymatic characterization revealed amylase, lipase, and protease production, sustaining mutual growth. Employing Response Surface Methodology (RSM), Artificial Neural Network (ANN), and Genetic Algorithm (GA) in a hybrid modeling approach resulted in a 2.1-fold increase in chlorophyll production. Optimized conditions included a NaNO3 concentration of 128.52 mg/l, a 1:2 (Algae:Bacteria) ratio, a 6-day cultivation period, and a pH of 5.4, yielding 10.92 ± 0.88 mg/l chlorophyll concentration.

Simple Summary Azotobacter vinelandii is a model organism used to study biological nitrogen fixation, a process by which nitrogen gas is transformed into ammonia, a form of nitrogen that can be assimilated by most organisms. This requires the synthesis and transfer of specific iron-containing cofactors to enzymes involved in nitrogen fixation. There are large gaps in our knowledge of iron uptake and utilization by A. vinelandii. In this review, our goal is to summarize current knowledge, propose novel elements based on our current understanding of bacterial iron homeostasis, and highlight those areas requiring more detailed research. Abstract Iron is an essential nutrient for all life forms. Specialized mechanisms exist in bacteria to ensure iron uptake and its delivery to key enzymes within the cell, while preventing toxicity. Iron uptake and exchange networks must adapt to the different environmental conditions, particularly those that require the biosynthesis of multiple iron proteins, such as nitrogen fixation. In this review, we outline the mechanisms that the model diazotrophic bacterium Azotobacter vinelandii uses to ensure iron nutrition and how it adapts Fe metabolism to diazotrophic growth.

Engineering bacterial properties requires precision and fine-tuning for optimal control of the desired application. In consequence, it is essential to accurately turn the function of interest from OFF to ON state and vice versa, avoiding any type of residual activation. For this type of purpose, light switches have revealed a clean and powerful tool in which control does not depend on the addition of chemical compounds that may remain in the media. To reach this degree of directed regulation through light, the switch based on the cyanobacterial two-component system CcaSR system was previously adapted to manipulate Pseudomonas putida for transcription of a gene of interest. In this chapter, we describe how to induce biofilm formation by placing the expression of the c-di-GMP-producing diguanylate cyclase PleD from Caulobacter sp. under the control of the CcaSR system. The regulation through optogenetics accomplished with this protocol promotes higher exploitation of biofilm beneficial features in a cheaper and cleaner way compared to chemical induction.

Light quality influence on barley development is poorly understood. We exposed three barley genotypes with either sensitive or insensitive response to two light sources producing different light spectra, fluorescent bulbs, and metal halide lamps, keeping constant light intensity, duration, and temperature. Through RNA-seq, we identified the main genes and pathways involved in the genotypic responses. A first analysis identified genotypic differences in gene expression of development-related genes, including photoreceptors and flowering time genes. Genes from the vernalization pathway of light quality-sensitive genotypes were affected by fluorescent light. In particular, vernalization-related repressors reacted differently: HvVRN2 did not experience relevant changes, whereas HvOS2 expression increased under fluorescent light. To identify the genes primarily related to light quality responses, and avoid the confounding effect of plant developmental stage, genes influenced by development were masked in a second analysis. Quantitative expression levels of PPD-H1, which influenced HvVRN1 and HvFT1, explained genotypic differences in development. Upstream mechanisms (light signaling and circadian clock) were also altered, but no specific genes linking photoreceptors and the photoperiod pathway were identified. The variety of light-quality sensitivities reveals the presence of possible mechanisms of adaptation of winter and facultative barley to latitudinal variation in light quality, which deserves further research.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, β-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.