Specialized Metabolites From the Husks of Rice Oryza sativa L. and Their Biological Activities.

Rice (Oryza sativa L.) husk harbors a substantial proportion of biological metabolites, as one of the most plentiful agriculture by-products in rice milling process, rice husk remains poorly utilized. As a continuing search for potential bioactive molecules from the husk of rice, a totally of twelve conponents (1-12), including six sterol ferulates (1-6), one flavonoid (7), one dipeptide (8), and four phenylpropanoid derivatives (9-12) were obtained. All the chemical structures were elucidated based on comprehensive spectroscopic data. Wherein, compounds 1 and 2 were yield as previous undescribed metabolites, and the comprehensive NMR data for compounds 3 and 4 were first presented in its entirety. Motivated by the similarity of the structural motifs of components 1-6 to that of reported sterol ferulates, the antioxidant and anti-inflammatory effects for compounds 1-6 were evaluated in vitro. Among them, compounds 5/6 had a significant antioxidant activity compare to that of vitamin E in both DPPH and reducing power assay up to the concentration 40 µg/ml; while compounds 1 and 2 exhibited weak suppressive effect on the production of nitric oxide, with the IC50 values of 53.27 ± 1.37 µM.

Adaptation of Ustilago maydis to phenolic and alkaloid responsive metabolites in maize B73.

The adaptation of pathogenic fungi to plant-specialized metabolites is necessary for their survival and reproduction. The biotrophic fungus Ustilago maydis can cause maize smut and produce tumors in maize (Zea mays), resulting in reduced maize yield and significant economic losses. Qualitative analysis using UPLC-MS/MS revealed that the infection of maize variety B73 with U. maydis resulted in increased levels of phytohormones, phenolics, and alkaloids in maize seedling tissues. However, correlation analysis showed that nearly all compounds in the mechanical damage group were significantly negatively correlated with the shoot growth indexes of maize B73. The correlation coefficients of 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and maize B73 shoot length and shoot weight were r = -0.56 (p < 0.01) and r = -0.75 (p < 0.001), respectively. In the inoculation group, these correlations weakened, with the correlation coefficients between HMBOA and maize B73 shoot length and shoot weight being r = 0.02 and r = -0.1, respectively. The correlation coefficients between 6-methoxy-2-benzoxazolinone (MBOA) and the shoot weight were r = -0.73 (p < 0.001) and r = -0.15 in the mechanical damage group and inoculation group, respectively. These findings suggest that increased concentrations of these compounds are more positively associated with mechanical damage than with U. maydis infection. At high concentrations, most of these compounds had an inhibitory effect on U. maydis. This study investigated the ability of U. maydis to regulate various compounds, including phytohormones, phenolic acids, and alkaloids in maize B73, providing evidence that U. maydis has adapted to the specialized metabolites produced by maize B73.

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

Large-Scale Production of Specialized Metabolites In Vitro Cultures.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

The role of red and white light in optimizing growth and accumulation of plant specialized metabolites at two light intensities in medical cannabis (Cannabis sativa L.).

The cultivation of medical cannabis (Cannabis sativa L.) is expanding in controlled environments, driven by evolving governmental regulations for healthcare supply. Increasing inflorescence weight and plant specialized metabolite (PSM) concentrations is critical, alongside maintaining product consistency. Medical cannabis is grown under different spectra and photosynthetic photon flux densities (PPFD), the interaction between spectrum and PPFD on inflorescence weight and PSM attracts attention by both industrialists and scientists. Plants were grown in climate-controlled rooms without solar light, where four spectra were applied: two low-white spectra (7B-20G-73R/Narrow and 6B-19G-75R/2Peaks), and two high-white (15B-42G-43R/Narrow and 17B-40G-43R/Broad) spectra. The low-white spectra differed in red wavelength peaks (100% 660 nm, versus 50:50% of 640:660 nm), the high-white spectra differed in spectrum broadness. All four spectra were applied at 600 and 1200 µmol m-2 s-1. Irrespective of PPFD, white light with a dual red peak of 640 and 660 nm (6B-19G-75R/2Peaks) increased inflorescence weight, compared to white light with a single red peak of 660 nm (7B-20G-73R/Narrow) (tested at P = 0.1); this was associated with higher total plant dry matter production and a more open plant architecture, which likely enhanced light capture. At high PPFD, increasing white fraction and spectrum broadness (17B-40G-43R/Broad) produced similar inflorescence weights compared to white light with a dual red peak of 640 and 660 nm (6B-19G-75R/2Peaks). This was caused by an increase of both plant dry matter production and dry matter partitioning to the inflorescences. No spectrum or PPFD effects on cannabinoid concentrations were observed, although at high PPFD white light with a dual red peak of 640 and 660 nm (6B-19G-75R/2Peaks) increased terpenoid concentrations compared to the other spectra. At low PPFD, the combination of white light with 640 and 660 nm increased photosynthetic efficiency compared with white light with a single red peak of 660nm, indicating potential benefits in light use efficiency and promoting plant dry matter production. These results indicate that the interaction between spectrum and PPFD influences plant dry matter production. Dividing the light energy in the red waveband over both 640 and 660 nm equally shows potential in enhancing photosynthesis and plant dry matter production.

Enhanced accumulation of biologically active compounds in lichens with potential functional food applications.

Lichens have great potential as food, functional food additives or flavourings. The presence of specific substances with multiple biological activities is one of the characteristics of lichens. However, research on lichens as a food source or functional food additive is limited. The present study simulated, for the first time, the potential bioaccessibility of active compounds from 6 lichen species in simulated gastric and intestinal conditions. An in vitro digestion showed that the lichen substances had different bioaccessibility and stability during digestion. It was found that the application of some metabolic modulators significantly altered the accumulation of metabolites in most species. In addition, the study demonstrated the antimicrobial activity of the tested extracts as well as of 14 isolated lichen metabolites. These multi-directional studies demonstrate the potential of lichens in terms of their use as antimicrobial functional food additives.

Local adaptation to the native environment affects pyrethrin variability in Dalmatian pyrethrum populations.

The insecticidal compound pyrethrin is synthesized in Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch.Bip.; Asteraceae), a plant species endemic to the eastern Mediterranean. Pyrethrin is a mixture of six compounds, pyrethrin I and II, cinerin I and II, and jasmolin I and II. For this study we sampled 15 natural Dalmatian pyrethrum populations covering the entire natural distribution range of the species; Croatian coastal regions and the islands, inland Bosnia and Herzegovina and Montenegro. The plants were grown in a field experiment under uniform growing conditions to exclude a short-term response to environmental factors and instead observe variation in pyrethrin content and composition among and within populations due to genetic adaptation to the native environment. The drivers of local adaptation were explored by examining the role of bioclimatic factors as a cause of population differentiation. Pyrethrins were extracted by ultrasound-assisted extraction, and the extracts were analyzed by HPLC-UV-DAD. The populations differed significantly in the content and composition of pyrethrins. The highest levels of total pyrethrins (1.27% flower DW), were found in population P14 Budva and the significantly highest levels of pyrethrin I in population P14 Vranjske Njive, Podgorica (66.47% of total pyrethrin). Based on bioclimatic conditions of the sampling sites, populations were grouped into five bioclimatic groups (A, B, C, D, and E), which showed qualitative and quantitative variability in pyrethrin content. The most abundant bioclimatic group was bioclimatic group E, which was characterized by the highest average values for pyrethrin I (53.87% of total pyrethrin), total pyrethrin content (1.06% flower DW) and the ratio of pyrethrin I and II (1.85). The correlation analysis between the pyrethrin compounds and some of the bioclimatic variables (e. g., BIO03 Isothermality and BIO04 Temperature seasonality) showed their significant contribution in explaining the variation of pyrethrins in T. cinerariifolium. The differences in pyrethrin content and composition may be partly due to genetic adaptation to the ecological conditions of the native environment. The obtained data would enable the selection of source populations for breeding programs aimed at producing cultivars with desirable biochemical properties and adaptation to different bioclimatic conditions.

Comparative metabolomics reveals how the severity of predation by the invasive insect Cydalima perspectalis modulates the metabolism re-orchestration of native Buxus sempervirens.

The recent biological invasion of box tree moth Cydalima perspectalis on Buxus trees has a major impact on European boxwood stands through severe defoliation. This can hinder further regrowth and threaten survival of populations. In a mesocosm approach and controlled larval density over a 2-month period, responses of B. sempervirens essential and specialized metabolites were characterized using metabolomics, combining 1H-NMR and LC-MS/MS approaches. This is the first metabolome depiction of major Buxus responses to boxwood moth invasion. Under severe predation, remaining green leaves accumulate free amino acids (with the noticeable exception of proline). The leaf trans-4-hydroxystachydrine and stachydrine reached 10-13% and 2-3% (DW), while root content was lower but also modulated by predation level. Larval predation promoted triterpenoid and (steroidal) alkaloid synthesis and diversification, while flavonoids did not seem to have a relevant role in Buxus resistance. Our results reveal the concomitant responses of central and specialized metabolism, in relation to severity of predation. They also confirm the potential of metabolic profiling using 1H-NMR and LC-MS to detect re-orchestration of metabolism of native boxwood after severe herbivorous predation by the invasive box-tree moth, and thus their relevance for plant-insect relationships and ecometabolomics.

Metabolites-induced co-evolutionary warfare between plants, viruses, and their associated vectors: So close yet so far away.

Agriculture and global food security encounter significant challenges due to viral threats. In the following decades, several molecular studies have focused on discovering biosynthetic pathways of numerous defensive and signaling compounds, as key regulators of plant interactions, either with viruses or their associated vectors. Nevertheless, the complexities of specialized metabolites mediated plant-virus-vector tripartite viewpoint and the identification of their co-evolutionary crossroads toward antiviral defense system, remain elusive. The current study reviews the various roles of plant-specialized metabolites (PSMs) and how plants use these metabolites to defend against viruses. It discusses recent examples of specialized metabolites that have broad-spectrum antiviral properties. Additionally, the study presents the co-evolutionary basis of metabolite-mediated plant-virus-insect interactions as a potential bioinspired approach to combat viral threats. The prospects also show promising metabolic engineering strategies aimed at discovering a wide range of PSMs that are effective in fending off viruses and their related vectors. These advances in understanding the potential role of PSMs in plant-virus interactions not only serve as a cornerstone for developing plant antiviral systems, but also highlight essential principles of biological control.

Steroidal scaffold decorations in Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato, and eggplant. Although it has been known that SGAs play important roles in defense in plants and "anti-nutritional" effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral, and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum) are reported to have potent anti-tumor, anti-proliferative, and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine, are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome-guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana, and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the starting cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine, and malonyl-solamargine SGA products. We further identified six enzymes from cultivated eggplant that catalyze the production of α-solasonine, α-solamargine, and malonyl-solamargine SGAs from solasodine aglycone via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high-value, steroidal bioactive molecules in heterologous hosts using synthetic biology.

Disarming the defenses: Insect detoxification of plant defense-related specialized metabolites.

The ability of certain insects to feed on plants containing toxic specialized metabolites may be attributed to detoxification enzymes. Representatives of a few large families of detoxification enzymes are widespread in insect herbivores acting to functionalize toxins and conjugate them with polar substituents to decrease toxicity, increase water solubility and enhance excretion. Insects have also developed specific enzymes for coping with toxins that are activated upon plant damage. Another source of detoxification potential in insects lies in their microbiomes, which are being increasingly recognized for their role in processing plant toxins. The evolution of insect detoxification systems to resist toxic specialized metabolites in plants may in turn have selected for the great diversity of such metabolites found in nature.

Effects of individual traits vs. trait syndromes on assemblages of various herbivore guilds associated with central European Salix.

Plants employ diverse anti-herbivore defences that can covary to form syndromes consisting of multiple traits. Such syndromes are hypothesized to impact herbivores more than individual defences. We studied 16 species of lowland willows occurring in central Europe and explored if their chemical and physical traits form detectable syndromes. We tested for phylogenetic trends in the syndromes and explored whether three herbivore guilds (i.e., generalist leaf-chewers, specialist leaf-chewers, and gallers) are affected more by the detected syndromes or individual traits. The recovered syndromes showed low phylogenetic signal and were mainly defined by investment in concentration, richness, or uniqueness of structurally related phenolic metabolites. Resource acquisition traits or inducible volatile organic compounds exhibited a limited correlation with the syndromes. Individual traits composing the syndromes showed various correlations to the assemblages of herbivores from the three studied guilds. In turn, we found some support for the hypothesis that defence syndromes are composed of traits that provide defence against various herbivores. However, individual traits rather than trait syndromes explained more variation for all studied herbivore assemblages. The detected negative correlations between various phenolics suggest that investment trade-offs may occur primarily among plant metabolites with shared metabolic pathways that may compete for their precursors. Moreover, several traits characterizing the recovered syndromes play additional roles in willows other than defence from herbivory. Taken together, our findings suggest that the detected syndromes did not solely evolve as an anti-herbivore defence.

Introducing the glycyrrhizic acid and glabridin rich genotypes from the cultivated Iranian licorice (Glycyrrhiza glabra L.) populations to exploit in production systems.

Currently, the stable, uniform, and highly efficient production of raw materials for pharmaceutical companies has received special attention. To meet these criteria and reduce harvesting pressure on the natural habitats of licorice (Glycyrrhiza glabra L.), cultivation of this valuable plant is inevitable. In the present study, to introduce the glycyrrhizic acid (GA)- and glabridin-rich genotypes from cultivated Iranian licorice, forty genotypes from eight high-potential wild populations were cultivated and evaluated under the same environmental conditions. The GA content varied from 5.00 ± 0.04 mg/g DW (TF2 genotype) to 23.13 ± 0.02 mg/g DW (I5 genotype). The highest and lowest glabridin content were found in the K2 (0.72 ± 0.021 mg/g DW) and M5 (0.02 ± 0.002 mg/g DW) genotypes, respectively. The rutin content in the leaves of the studied genotypes varied from 1.27 ± 0.02 mg/g DW in E4 to 3.24 ± 0.02 mg/g DW in BO5 genotypes. The genotypes from the Ilam population were characterized by higher vegetative growth and yield traits in the aerial parts and roots. The average root dry yield was 2.44 tons per hectare (t/ha) among the studied genotypes and a genotype from Ilam (I5) yielded the maximum value (3.08 ± 0.034 t/ha). The highest coefficient of variation among the genotypes was observed for leaf width (CV = 34.9%). The GA and glabridin-rich genotypes introduced in this study can be used in the future breeding programs to release new bred licorice cultivars.

Harnessing controlled environment systems for enhanced production of medicinal plants.

Medicinal plants (MPs) are valued for their contributions to human health. However, the growing demand for MPs and the concerns regarding their quality and sustainability have prompted the reassessment of conventional production practices. Controlled environment cropping systems, such as vertical farms, offer a transformative approach to MP production. By enabling precise control over environment factors, such as light, carbon dioxide, temperature, humidity, nutrients, and airflow, controlled environments can improve the consistency, concentration, and yield of bioactive phytochemicals in MPs. This review explores the potential of controlled environment systems for enhancing MP production. First, we describe how controlled environments can overcome the limitations of conventional production in improving the quality of MP. Next, we propose strategies based on plant physiology to manipulate environment conditions for enhancing the levels of bioactive compounds in plants. These strategies include improving photosynthetic carbon assimilation, light spectrum signalling, purposeful stress elicitation, and chronoculture. We describe the underlying mechanisms and practical applications of these strategies. Finally, we highlight the major knowledge gaps and challenges that limit the application of controlled environments, and discuss future research directions.

Spatial and Temporal Resolution of Cyanobacterial Bloom Chemistry Reveals an Open-Ocean Trichodesmium thiebautii as a Talented Producer of Specialized Metabolites.

While the ecological role that Trichodesmium sp. play in nitrogen fixation has been widely studied, little information is available on potential specialized metabolites that are associated with blooms and standing stock Trichodesmium colonies. While a collection of biological material from a T. thiebautii bloom event from North Padre Island, Texas, in 2014 indicated that this species was a prolific producer of chlorinated specialized metabolites, additional spatial and temporal resolution was needed. We have completed these metabolite comparison studies, detailed in the current report, utilizing LC-MS/MS-based molecular networking to visualize and annotate the specialized metabolite composition of these Trichodesmium blooms and colonies in the Gulf of Mexico (GoM) and other waters. Our results showed that T. thiebautii blooms and colonies found in the GoM have a remarkably consistent specialized metabolome. Additionally, we isolated and characterized one new macrocyclic compound from T. thiebautii, trichothilone A (1), which was also detected in three independent cultures of T. erythraeum. Genome mining identified genes predicted to synthesize certain functional groups in the T. thiebautii metabolites. These results provoke intriguing questions of how these specialized metabolites affect Trichodesmium ecophysiology, symbioses with marine invertebrates, and niche development in the global oligotrophic ocean.

The root of plant-plant interactions: Belowground special cocktails.

Plants interact with each other via a multitude of processes among which belowground communication facilitated by specialized metabolites plays an important but overlooked role. Until now, the exact targets, modes of action, and resulting phenotypes that these metabolites induce in neighboring plants have remained largely unknown. Moreover, positive interactions driven by the release of root exudates are prevalent in both natural field conditions and controlled laboratory environments. In particular, intraspecific positive interactions suggest a genotypic recognition mechanism in addition to non-self perception in plant roots. This review concentrates on recent discoveries regarding how plants interact with one another through belowground signals in intra- and interspecific mixtures. Furthermore, we elaborate on how an enhanced understanding of these interactions can propel the field of agroecology forward.

Carbon-based nanomaterials as inducers of biocompounds in plants: Potential risks and perspectives.

Biocompounds are metabolites synthesized by plants, with clinically proven capacity in preventing and treating degenerative diseases in humans. Carbon-based nanomaterials (CNMs) are atomic structures that assume different hybridization and shape. Due to the reactive property, CNMs can induce the synthesis of metabolites, such as biocompounds in cells and various plant species, by generating reactive oxygen species (ROS). In response, plants positively or negatively regulate the expression of various families of genes and enzymes involved in physiological and metabolomic pathways of plants, such as carbon and nitrogen metabolism, which are directly involved in plant development and growth. Likewise, ROS can modulate the expression of enzymes and genes related to the adaptation of plants to stress, such as the glutathione ascorbate cycle, the shikimic acid, and phenylpropanoid pathways, from which the largest amount of biocompounds in plants are derived. This document exposes the ability of three CNMs (fullerene, graphene, and carbon nanotubes) to positively or negatively regulate the activity of enzymes and genes involved in various plant species' primary and secondary metabolism. The mechanism of action of CNMs on the production of biocompounds and the effect of the translocation of CNMs on the growth and content of primary metabolites in plants are described. Adverse effects of CNMs on plants, prospects, and possible risks involved are also discussed. The use of CNMs as inducers of biocompounds in plants could have implications and relevance for human health, crop quality, and plant adaptation and resistance to biotic and abiotic stress.

Machine learning assists prediction of genes responsible for plant specialized metabolite biosynthesis by integrating multi-omics data.

BACKGROUND: Plant specialized (or secondary) metabolites (PSM), also known as phytochemicals, natural products, or plant constituents, play essential roles in interactions between plants and environment. Although many research efforts have focused on discovering novel metabolites and their biosynthetic genes, the resolution of metabolic pathways and identified biosynthetic genes was limited by rudimentary analysis approaches and enormous number of candidate genes. RESULTS: Here we integrated state-of-the-art automated machine learning (ML) frame AutoGluon-Tabular and multi-omics data from Arabidopsis to predict genes encoding enzymes involved in biosynthesis of plant specialized metabolite (PSM), focusing on the three main PSM categories: terpenoids, alkaloids, and phenolics. We found that the related features of genomics and proteomics were the top two crucial categories of features contributing to the model performance. Using only these key features, we built a new model in Arabidopsis, which performed better than models built with more features including those related with transcriptomics and epigenomics. Finally, the built models were validated in maize and tomato, and models tested for maize and trained with data from two other species exhibited either equivalent or superior performance to intraspecies predictions. CONCLUSIONS: Our external validation results in grape and poppy on the one hand implied the applicability of our model to the other species, and on the other hand showed enormous potential to improve the prediction of enzymes synthesizing PSM with the inclusion of valid data from a wider range of species.

Hidden Treasure: Halophilic Fungi as a Repository of Bioactive Lead Compounds.

The pressing demand for novel compounds to address contemporary health challenges has prompted researchers to venture into uncharted territory, including extreme ecosystems, in search of new natural pharmaceuticals. Fungi capable of tolerating extreme conditions, known as extremophilic fungi, have garnered attention for their ability to produce unique secondary metabolites crucial for defense and communication, some of which exhibit promising clinical significance. Among these, halophilic fungi thriving in high-salinity environments have particularly piqued interest for their production of bioactive molecules. This review highlights the recent discoveries regarding novel compounds from halotolerant fungal strains isolated from various saline habitats. From diverse fungal species including Aspergillus, Penicillium, Alternaria, Myrothecium, and Cladosporium, a plethora of intriguing molecules have been elucidated, showcasing diverse chemical structures and bioactivity. These compounds exhibit cytotoxicity against cancer cell lines such as A549, HL60, and K-562, antimicrobial activity against pathogens like Escherichia coli, Bacillus subtilis, and Candida albicans, as well as radical-scavenging properties. Notable examples include variecolorins, sclerotides, alternarosides, and chrysogesides, among others. Additionally, several compounds display unique structural motifs, such as spiro-anthronopyranoid diketopiperazines and pentacyclic triterpenoids. The results emphasize the significant promise of halotolerant fungi in providing bioactive compounds for pharmaceutical, agricultural, and biotechnological uses. However, despite their potential, halophilic fungi are still largely unexplored as sources of valuable compounds.

The Putative GATA Transcription Factor SbGATA22 as a Novel Regulator of Dhurrin Biosynthesis.

Cyanogenic glucosides are specialized metabolites produced by over 3000 species of higher plants from more than 130 families. The deployment of cyanogenic glucosides is influenced by biotic and abiotic factors in addition to being developmentally regulated, consistent with their roles in plant defense and stress mitigation. Despite their ubiquity, very little is known regarding the molecular mechanisms that regulate their biosynthesis. The biosynthetic pathway of dhurrin, the cyanogenic glucoside found in the important cereal crop sorghum (Sorghum bicolor (L.) Moench), was described over 20 years ago, and yet no direct regulator of the biosynthetic genes has been identified. To isolate regulatory proteins that bind to the promoter region of the key dhurrin biosynthetic gene of sorghum, SbCYP79A1, yeast one-hybrid screens were performed. A bait fragment containing 1204 base pairs of the SbCYP79A1 5' regulatory region was cloned upstream of a reporter gene and introduced into Saccharomyces cerevisiae. Subsequently, the yeast was transformed with library cDNA representing RNA from two different sorghum developmental stages. From these screens, we identified SbGATA22, an LLM domain B-GATA transcription factor that binds to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region. Transient assays in Nicotiana benthamiana show that SbGATA22 localizes to the nucleus. The expression of SbGATA22, in comparison with SbCYP79A1 expression and dhurrin concentration, was analyzed over 14 days of sorghum development and in response to nitrogen application, as these conditions are known to affect dhurrin levels. Collectively, these findings suggest that SbGATA22 may act as a negative regulator of SbCYP79A1 expression and provide a preliminary insight into the molecular regulation of dhurrin biosynthesis in sorghum.