Προσδιορισμός & χαρακτηρισμός μιας πρωτεΐνης ειδικής προειδοποίησης του φερμινίου: είναι ο μοναδικός της ρόλος η δραστηριότητα του υπεροξειδίου;

Reactive oxygen species (ROS) such as superoxide, peroxide, hydroxyl ion, and singlet oxygen radicals are by-products of specific metabolic reactions in plants. ROS damage cellular biomolecules and are thus actively scavenged. However, ROS also act as signal transducers during development and stress responses. Redox enzymes such as superoxide dismutase (SOD) contribute to achieving a balance between ROS being scavenged and being available as signal transducers. Spatio-temporally differential expression of SODs in various tissues plays a critical role in scavenging the superoxide radical. Using a nitroblue-tetrazolium-riboflavin-based in-gel SOD assay, up-regulation of two low mobility (~96 kDa and ~92 kDa) putative SODs was observed in rice spikelets. The two proteins were purified by gel electrophoresis and both were identified through mass spectrometry as homotetrameric versions of a single germin-like protein (GLP), encoded by the gene LOC_Os08g08960 (Rice Genome Annotation Project). Staining with a glycoprotein gel stain and enzymatic deglycosylation showed that the protein is post-translationally modified by N-glycosylation; differential modification probably accounts for the difference between the two forms. Biochemical characterization of this developmentally regulated spikelet-specific GLP (ssGLP) indicated that its SOD activity is Mn-SOD type and is highly stable in denaturing agents and at elevated temperatures. Tetrameric ssGLP lacks oxalate oxidase activity. The biological functions of GLPs have not been fully defined; among the different functions attributed to them their role in biotic stress tolerance is relatively well studied. Rice germins, especially some of those with genes on chromosome 8 in the same cluster as the LOC_Os08g08960 gene, although not this specific gene, have previously been found to contribute to the basal mechanism of resistance to pathogens causing rice blast and sheath blight disease. ssGLP was estimated to contribute nearly 11% of the total SOD activity in rice spikelets, indicating a crucial developmental role. Expression analysis with the in-gel SOD assay at the spatiotemporal level, as well as treatment with various hormones, chemicals, and abiotic variables such as wounding and light, indicated that the expression of ssGLP is affected by developmental rather than stress stimuli. Further, expression analysis at the spatio-temporal level through RT-PCR confirmed the developmental patterns observed previously at the protein level. In situ mRNA hybridization studies provided evidence for ssGLP gene expression in pollen and endosperm and in localized regions of leaves and for the production of antisense RNA. Consistent with the up-regulation in spikelets, in silico comparison of the rice GLP promoters revealed more seed and pollen-specific motifs in LOC_Os08g08960 than in other GLP genes. Additionally, the presence of a GAF protein domain typical of cyclic nucleotide phosphodiesterases is unique to ssGLP. Preliminary enzyme assays and in silico results suggested that ssGLP may act as a nucleotide-sugar pyrophosphatase/phosphodiesterase (NSPPase) similar to a previously described barley GLP. As a first step to a fuller definition of the enzyme activity of ssGLP and in particular to test the hypothesis that a different oligomeric configuration acts as NSPPase, a gene for recombinant ssGLP was constructed, cloned, and expressed in Pichia pastoris.Τα αντιδραστικά είδη οξυγόνου (ROS) όπως το υπεροξείδιο, το υπεροξείδιο, το ιόν υδροξυλίου και οι ρίζες οξυγόνου είναι υποπροϊόντα συγκεκριμένων μεταβολικών αντιδράσεων στα φυτά. Οι ROS βλάπτουν τα κυτταρικά βιομόρια και έτσι σαρώνονται ενεργά. Ωστόσο, οι ROS λειτουργούν επίσης ως μορφοτροπείς σήματος κατά τη διάρκεια της ανάπτυξης και των αντιδράσεων στο στρες. Τα οξειδοαναγωγικά ένζυμα όπως η δισμουτάση υπεροξειδίου (SOD) συμβάλλουν στην επίτευξη ισορροπίας μεταξύ των ROS που σαρώνονται και είναι διαθέσιμα ως μετατροπείς σήματος. Η χωροχρονικά διαφορική έκφραση των SOD σε διάφορους ιστούς παίζει κρίσιμο ρόλο στην απομάκρυνση της ρίζας υπεροξειδίου. Χρησιμοποιώντας μια δοκιμασία SOD με βάση το νιτρομπλε-τετραζόλιο-ριβοφλαβίνη, παρατηρήθηκε ρύθμιση δύο υποθετικών SOD χαμηλής κινητικότητας (~96 kDa και ~92 kDa) σε αιχμές ρυζιού. Οι δύο πρωτεΐνες καθαρίστηκαν με ηλεκτροφόρηση πηκτής και οι δύο αναγνωρίστηκαν μέσω φασματομετρίας μάζας ως ομοτετραμερείς εκδόσεις μιας μοναδικής πρωτεΐνης που μοιάζει με βλαστίνη (GLP), κωδικοποιημένη από το γονίδιο LOC_Os08g08960 (Rice Genome Annotation Project). Η χρώση με χρώση πηκτής γλυκοπρωτεΐνης και ενζυματική απογλυκοζυλίωση έδειξε ότι η πρωτεΐνη τροποποιείται μετα-μεταφραστικά με Ν-γλυκοζυλίωση. Η διαφορική τροποποίηση πιθανώς εξηγεί τη διαφορά μεταξύ των δύο μορφών. Ο βιοχημικός χαρακτηρισμός αυτού του αναπτυξιακά ρυθμιζόμενου ειδικού GLP spikelet (ssGLP) έδειξε ότι η δραστικότητά του SOD είναι τύπου Mn-SOD και είναι εξαιρετικά σταθερή σε παράγοντες μετουσίωσης και σε υψηλές θερμοκρασίες. Το τετραμερές ssGLP στερείται δραστικότητας οξειδάσης οξαλικού. Οι βιολογικές λειτουργίες των ΟΕΠ δεν έχουν καθοριστεί πλήρως. Μεταξύ των διαφόρων λειτουργιών που τους αποδίδονται, ο ρόλος τους στην ανοχή στο βιοτικό στρες είναι σχετικά καλά μελετημένος. Τα μικρόβια ρυζιού, ειδικά μερικά από εκείνα με γονίδια στο χρωμόσωμα 8 στο ίδιο σύμπλεγμα με το γονίδιο LOC_Os08g08960, αν και όχι αυτό το συγκεκριμένο γονίδιο, έχουν βρεθεί προηγουμένως ότι συμβάλλουν στον βασικό μηχανισμό αντοχής σε παθογόνα που προκαλούν έκρηξη ρυζιού και ασθένεια θήκης. Το ssGLP εκτιμάται ότι συνεισφέρει σχεδόν το 11% της συνολικής δραστηριότητας SOD στις αιχμές ρυζιού, υποδεικνύοντας έναν κρίσιμο αναπτυξιακό ρόλο. Η ανάλυση έκφρασης με τη δοκιμασία in-gel SOD σε χωροχρονικό επίπεδο, καθώς και η θεραπεία με διάφορες ορμόνες, χημικές ουσίες και αβιοτικές μεταβλητές όπως ο τραυματισμός και το φως, έδειξαν ότι η έκφραση του ssGLP επηρεάζεται από αναπτυξιακά και όχι από ερεθίσματα στρες. Περαιτέρω, η ανάλυση έκφρασης σε χωροχρονικό επίπεδο μέσω RT-PCR επιβεβαίωσε τα αναπτυξιακά πρότυπα που παρατηρήθηκαν προηγουμένως σε επίπεδο πρωτεΐνης. In situ μελέτες υβριδισμού mRNA παρείχαν στοιχεία για την έκφραση γονιδίων ssGLP στη γύρη και το ενδοσπέρμιο και σε εντοπισμένες περιοχές των φύλλων και για την παραγωγή αντινοηματικού RNA. Σύμφωνα με την αύξηση της ρύθμισης των ακίδων, η σύγκριση in silico των υποστηρικτών της ΟΕΠ ρυζιού αποκάλυψε περισσότερα μοτίβα σπόρων και γύρης σε LOC_Os08g08960 από ό, τι σε άλλα γονίδια ΟΕΠ. Επιπλέον, η παρουσία ενός τομέα πρωτεΐνης GAF χαρακτηριστικού των κυκλικών νουκλεοτιδικών φωσφοδιεστερασών είναι μοναδική για το ssGLP. Προκαταρκτικές δοκιμασίες ενζύμων και αποτελέσματα in silico έδειξαν ότι το ssGLP μπορεί να δράσει ως πυροφωσφατάση/φωσφοδιεστεράση νουκλεοτιδίου-σακχάρου (NSPPase) παρόμοιο με ένα GLP κριθαριού που περιγράφηκε προηγουμένως. Ως πρώτο βήμα για έναν πληρέστερο ορισμό της ενζυμικής δραστικότητας του ssGLP και ειδικότερα για να ελεγχθεί η υπόθεση ότι μια διαφορετική ολιγομερής διαμόρφωση δρα ως NSPPase, ένα γονίδιο για την ανασυνδυασμένη ssGLP κατασκευάστηκε, κλωνοποιήθηκε και εκφράστηκε στο Pichia pastoris

Proof of concept and early development stage of market-oriented high iron and zinc rice expressing dicot ferritin and rice nicotianamine synthase genes

Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 (OsNAS2) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 μg g−1 and up to 37 μg g−1, respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector.PRIFPRI3; ISI; 2 Promoting Healthy Diets and Nutrition for all; HarvestPlus; CRP4DGO; HarvestPlus; A4NHCGIAR Research Program on Agriculture for Nutrition and Health (A4NH

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement

Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds

CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype

Proof of concept and early development stage of market-oriented high iron and zinc rice expressing dicot ferritin and rice nicotianamine synthase genes

Abstract Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 (OsNAS2) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 μg g−1 and up to 37 μg g−1, respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector

The End of the Constitutional Beginning

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g−1 iron (Fe) and 16 μg g−1 zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g−1 Fe and 28 μg g−1 Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g−1 in glasshouse settings; in contrast, under field conditions, 4 μg g−1 was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g−1 Fe and 45.7 μg g−1 Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds