Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus.

KEY MESSAGE: Modification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callus Metabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

Changing biosynthesis of terpenoid percursors in rice through synthetic biology.

Many highly valued chemicals in the pharmaceutical, biotechnological, cosmetic, and biomedical industries belong to the terpenoid family. Biosynthesis of these chemicals relies on polymerization of Isopentenyl di-phosphate (IPP) and/or dimethylallyl diphosphate (DMAPP) monomers, which plants synthesize using two alternative pathways: a cytosolic mevalonic acid (MVA) pathway and a plastidic methyleritritol-4-phosphate (MEP) pathway. As such, developing plants for use as a platform to use IPP/DMAPP and produce high value terpenoids is an important biotechnological goal. Still, IPP/DMAPP are the precursors to many plant developmental hormones. This creates severe challenges in redirecting IPP/DMAPP towards production of non-cognate plant metabolites. A potential solution to this problem is increasing the IPP/DMAPP production flux in planta. Here, we aimed at discovering, understanding, and predicting the effects of increasing IPP/DMAPP production in plants through modelling. We used synthetic biology to create rice lines containing an additional ectopic MVA biosynthetic pathway for producing IPP/DMAPP. The rice lines express three alternative versions of the additional MVA pathway in the plastid, in addition to the normal endogenous pathways. We collected data for changes in macroscopic and molecular phenotypes, gene expression, isoprenoid content, and hormone abundance in those lines. To integrate the molecular and macroscopic data and develop a more in depth understanding of the effects of engineering the exogenous pathway in the mutant rice lines, we developed and analyzed data-centric, line-specific, multilevel mathematical models. These models connect the effects of variations in hormones and gene expression to changes in macroscopic plant phenotype and metabolite concentrations within the MVA and MEP pathways of WT and mutant rice lines. Our models allow us to predict how an exogenous IPP/DMAPP biosynthetic pathway affects the flux of terpenoid precursors. We also quantify the long-term effect of plant hormones on the dynamic behavior of IPP/DMAPP biosynthetic pathways in seeds, and predict plant characteristics, such as plant height, leaf size, and chlorophyll content from molecular data. In addition, our models are a tool that can be used in the future to help in prioritizing re-engineering strategies for the exogenous pathway in order to achieve specific metabolic goals.

Cyanovirin-N binds to select SARS-CoV-2 spike oligosaccharides outside of the receptor binding domain and blocks infection by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive stranded RNA virus which has caused the recent deadly pandemic called COVID-19. The SARS-CoV-2 virion is coated with a heavily glycosylated Spike glycoprotein which is responsible for attachment and entry into target cells. One, as yet unexploited strategy for preventing SARS-CoV-2 infections, is the targeting of the glycans on Spike. Lectins are carbohydrate-binding proteins produced by plants, algae, and cyanobacteria. Some lectins can neutralize enveloped viruses displaying external glycoproteins, offering an alternative therapeutic approach for the prevention of infection with virulent ß-coronaviruses, such as SARS-CoV-2. Here we show that the cyanobacterial lectin cyanovirin-N (CV-N) can selectively target SARS-CoV-2 Spike oligosaccharides and inhibit SARS-CoV-2 infection in vitro and in vivo. CV-N neutralizes Delta and Omicron variants in vitro better than earlier circulating viral variants. CV-N binds selectively to Spike with a Kd as low as 15 nM and a stoichiometry of 2 CV-N: 1 Spike but does not bind to the receptor binding domain (RBD). Further mapping of CV-N binding sites on Spike shows that select high-mannose oligosaccharides in the S1 domain of Spike are targeted by CV-N. CV-N also reduced viral loads in the nares and lungs in vivo to protect hamsters against a lethal viral challenge. In summary, we present an anti-coronavirus agent that works by an unexploited mechanism and prevents infection by a broad range of SARS-CoV-2 strains.

Functional expression of the nitrogenase Fe protein in transgenic rice.

Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N2. Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis-trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co), two fundamental roles for NifH in N2 fixation. NifH functionality was, however, limited by poor [4Fe-4S] cluster occupancy, highlighting the importance of in vivo [Fe-S] cluster insertion and stability to achieve biological N2 fixation in planta. Nevertheless, the expression and activity of a nitrogenase component in rice plants represents the first major step to engineer functional nitrogenase in cereal crops.

Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe-9S-C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe-S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe-4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.

The Biosynthesis of Non-Endogenous Apocarotenoids in Transgenic Nicotiana glauca.

Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified in saffron, Buddleja, and gardenia. In this study, the enzyme from Buddleja, BdCCD4.1, was constitutively expressed in Nicotiana glauca, a tobacco species with carotenoid-pigmented petals. The transgenic lines produced significant levels of crocins in their leaves and petals. However, the accumulation of crocins was, in general, higher in the leaves than in the petals, reaching almost 302 µg/g DW. The production of crocins was associated with decreased levels of endogenous carotenoids, mainly ß-carotene. The stability of crocins in leaf and petal tissues was evaluated after three years of storage, showing an average reduction of 58.06 ± 2.20% in the petals, and 78.37 ± 5.08% in the leaves. This study illustrates the use of BdCCD4.1 as an effective tool for crocin production in N. glauca and how the tissue has an important impact on the stability of produced high-value metabolites during storage.

Rice callus as a high-throughput platform for synthetic biology and metabolic engineering of carotenoids.

Carotenoids are a large class of important lipid-soluble phytonutrients that are widely used as nutritional supplements due to their health-promoting activities. For example, ß-carotene is the precursor for vitamin A synthesis, and astaxanthin is a powerful antioxidant. However, these carotenoids cannot be synthesized de novo by humans. These properties of ß-carotene and astaxanthin make them attractive targets for metabolic engineering in rice (Oryza sativa) endosperm because rice is an important staple food in developing countries, and rice endosperm is devoid of carotenoids. In this chapter, we introduce an assay based on rice embryogenic callus for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation. The system is also an ideal platform to characterize cereal endosperm specific promoters. Four diverse cereal endosperm specific promoters were demonstrated to be active in rice callus despite their restricted activity in mature plants. The use of endosperm specific promoters that are expressed in rice callus, but remain silent in regenerated vegetative tissue, directs accumulation of carotenoids in the endosperm without interfering with plant growth. Rice callus is a useful platform for improving gene editing methods and for further optimizing pathway engineering. Thus, the rice callus platform provides a unique opportunity to test strategies for metabolic engineering of synthetic carotenoid pathways, leading to novel carotenoid-biofortified crops.

Development of a facile genetic transformation system for the Spanish elite rice paella genotype Bomba.

We report the development of an efficient and reproducible genetic transformation system for the recalcitrant Spanish elite rice paella genotype, Bomba. Preconditioned embryos derived from dry seeds were bombarded with gold particles carrying a plasmid containing a screenable and a selectable marker. We confirmed integration and expression of hpt and gusA in the rice genome. Transformation frequency was ca: 10% in several independent experiments. We show Mendelian inheritance of the input transgenes and zygosity determination of the transgenic lines in the T1 generation. A unique and critical step for the regeneration of plants from transformed tissue was shading during the early stages of regeneration, combined with a specific cytokinin:auxin ration at the onset of shifting callus to regeneration media.

Rapid production of SaCas9 in plant-based cell-free lysate for activity testing.

Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with 10 different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.

Metabolic Engineering of Crocin Biosynthesis in Nicotiana Species.

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. ß-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 µg/g DW in leaves, while in N. tabacum 36 µg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications.

Multilevel interactions between native and ectopic isoprenoid pathways affect global metabolism in rice.

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.

Physicochemical characterization of the recombinant lectin scytovirin and microbicidal activity of the SD1 domain produced in rice against HIV-1.

KEY MESSAGE: Rice-produced SD1 retains its physicochemical properties and provides efficient pre-exposure HIV-1 prophylaxis against infection in vitro. Scytovirin (SVN) is an HIV-neutralizing lectin that features two structural domains (SD1 and SD2) that bind to HIV-1 envelope glycoproteins. We expressed SD1 in rice seeds as a potential large-scale production platform and confirmed that rice-derived SD1 binds the HIV-1 envelope glycoprotein gp120 in vitro. We analyzed the thermodynamic properties of SD1 compared to full-size SVN (produced in E. coli) by isothermal titration and differential scanning calorimetry to characterize the specific interactions between SVN/SD1 and gp120 as well as to high-mannose oligosaccharides. SVN bound with moderate affinity (Kd = 1.5 µM) to recombinant gp120, with 2.5-fold weaker affinity to nonamannoside (Kd of 3.9 µM), and with tenfold weaker affinity to tetramannoside (13.8 µM). The melting temperature (Tm) of full-size SVN was 59.1 °C and the enthalpy of unfolding (ΔHunf) was 16.4 kcal/mol, but the Tm fell when SVN bound to nonamannoside (56.5 °C) and twice as much energy was required for unfolding (ΔHunf = 33.5 kcal/mol). Interestingly, binding to tetramannoside destabilized the structure of SD1 (ΔTm ~ 11.5 °C) and doubled the enthalpy of unfolding, suggesting a dimerization event. The similar melting phenomenon shared by SVN and SD1 in the presence of oligomannose confirmed their conserved oligosaccharide-binding mechanisms. SD1 expressed in transgenic rice was able to neutralize HIV-1 in vitro. SD1 expressed in rice, therefore, is suitable as a microbicide component.

The Coordinated Upregulated Expression of Genes Involved in MEP, Chlorophyll, Carotenoid and Tocopherol Pathways, Mirrored the Corresponding Metabolite Contents in Rice Leaves during De-Etiolation.

Light is an essential regulator of many developmental processes in higher plants. We investigated the effect of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1/2 genes (OsHDR1/2) and isopentenyl diphosphate isomerase 1/2 genes (OsIPPI1/2) on the biosynthesis of chlorophylls, carotenoids, and phytosterols in 14-day-old etiolated rice (Oyza sativa L.) leaves during de-etiolation. However, little is known about the effect of isoprenoid biosynthesis genes on the corresponding metabolites during the de-etiolation of etiolated rice leaves. The results showed that the levels of α-tocopherol were significantly increased in de-etiolated rice leaves. Similar to 1-deoxy-D-xylulose-5-phosphate synthase 3 gene (OsDXS3), both OsDXS1 and OsDXS2 genes encode functional 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activities. Their expression patterns and the synthesis of chlorophyll, carotenoid, and tocopherol metabolites suggested that OsDXS1 is responsible for the biosynthesis of plastidial isoprenoids in de-etiolated rice leaves. The expression analysis of isoprenoid biosynthesis genes revealed that the coordinated expression of the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, chlorophyll, carotenoid, and tocopherol pathway genes mirrored the changes in the levels of the corresponding metabolites during de-etiolation. The underpinning mechanistic basis of coordinated light-upregulated gene expression was elucidated during the de-etiolation process, specifically the role of light-responsive cis-regulatory motifs in the promoter region of these genes. In silico promoter analysis showed that the light-responsive cis-regulatory elements presented in all the promoter regions of each light-upregulated gene, providing an important link between observed phenotype during de-etiolation and the molecular machinery controlling expression of these genes.

Genome editing in cereal crops: an overview.

Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations.

Transgenic and genome-edited fruits: background, constraints, benefits, and commercial opportunities.

Breeding has been used successfully for many years in the fruit industry, giving rise to most of today's commercial fruit cultivars. More recently, new molecular breeding techniques have addressed some of the constraints of conventional breeding. However, the development and commercial introduction of such novel fruits has been slow and limited with only five genetically engineered fruits currently produced as commercial varieties-virus-resistant papaya and squash were commercialized 25 years ago, whereas insect-resistant eggplant, non-browning apple, and pink-fleshed pineapple have been approved for commercialization within the last 6 years and production continues to increase every year. Advances in molecular genetics, particularly the new wave of genome editing technologies, provide opportunities to develop new fruit cultivars more rapidly. Our review, emphasizes the socioeconomic impact of current commercial fruit cultivars developed by genetic engineering and the potential impact of genome editing on the development of improved cultivars at an accelerated rate.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

Genome editing in fruit, ornamental, and industrial crops.

The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies.

Nitrogen inputs influence vegetative metabolism in maize engineered with a seed-specific carotenoid pathway.

KEY MESSAGE: Metabolomic profiling of a maize line engineered with an endosperm-specific carotenogenic pathway revealed unexpected metabolic readjustments of primary metabolism in leaves and roots. High-carotenoid (HC) maize was engineered to accumulate high levels of carotenoids in the endosperm. The metabolic interventions influenced the flux through non-target pathways in tissues that were not affected by the targeted intervention. HC maize at the vegetative stage also showed a reduced susceptibility to insect feeding. It is unknown, however, whether the metabolic history of the embryo has any impact on the metabolite composition in vegetative tissues. We, therefore, compared HC maize and its isogenic counterpart (M37W) to test the hypothesis that boosting the carotenoid content in the endosperm triggers compensatory effects in core metabolism in vegetative tissues. Specifically, we investigated whether the metabolite composition of leaves and roots at the V6 stage differs between HC and M37W, and whether N inputs further alter the core metabolism of HC compared to M37W. We found an increase in the abundance of organic acids from the tricarboxylic acid (TCA) cycle in HC even under restricted N conditions. In contrast, low levels of carotenoids and chlorophyll were measured regardless of N levels. Sugars were also significantly depleted in HC under low N. We propose a model explaining the observed genotype-dependent and input-dependent effects, in which organic acids derived from the TCA cycle accumulate during vegetative growth and contribute to the increased demand for pyruvate and/or acetyl-CoA in the endosperm and embryo. This response may in part reflect the transgenerational priming of vegetative tissues in the embryo induced by the increased demand for metabolic precursors during seed development in the previous generation.