Spatio-temporal expression of polyphenol oxidase unveils the dynamics of L-DOPA accumulation in faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is a winter season grain legume and a rich source of the anti-parkinson drug, L-3,4-dihydroxyphenylalanine (L-DOPA). The biosynthesis of L-DOPA in plants is not uniform and remains largely unexplored. While the hydroxylase activities of Tyrosine Hydroxylase (TH), the Cytochrome P450 (CYP450) class of enzymes, and Polyphenol Oxidases (PPOs) on tyrosine substrate have been reported in plants, only the roles of PPOs in L-DOPA biosynthesis have been recently established in velvet bean (Mucuna pruriens). To understand the differential accumulation of L-DOPA in different tissues of faba bean, profiling of L-Tyrosine, L-DOPA, Tyramine, and Dopamine in different tissues was performed. Differential accumulation of L-DOPA depended on tissue type and maturity. Furthermore, dopamine biosynthesis through L-DOPA from L-Tyr was confirmed in faba bean. The expression analysis of PPOs in leaf and flower tissues revealed the selective induction of only four (HePPO-2, HePPO-7, HePPO-8b, and HePPO-10) out of ten genes encoding different PPOs mined from the faba bean genome. Higher accumulation of L-DOPA in young leaves and flower buds than in mature leaves and flowers was accompanied by significantly higher expression of HePPO-10 and HePPO-7, respectively. The role of various transcription factors contributing to such metabolite dynamics was also predicted. Further exploration of this mechanism using a multi-omics approach can provide meaningful insight and pave the way for enhancing L-DOPA content in crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01449-2.

Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender ( https://www.biorender.com ).

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Chitosan nanoparticles and their combination with methyl jasmonate for the elicitation of phenolics and flavonoids in plant cell suspension cultures.

Productivity enhancement approaches, such as elicitation can overcome the limitations of low metabolite(s) yield in in vitro plant cell culture platforms. Application of biotic/abiotic elicitors triggers molecular responses that lead to a concomitant enhancement in the production of metabolites. Nanoparticles have been tested as alternatives to commonly studied biotic/abiotic elicitors. However, most nanoparticles explored are of metallic origin, which raises concerns about their cytotoxicity, disposal post-elicitation, and may limit downstream applications of metabolites. Here, we report the synthesis and application of biopolymeric methyl jasmonate-loaded chitosan nanoparticles (MJ-CNPs) and empty CNPs (size <100 nm) as nano-elicitors, which were simple to synthesize, cost-effective and safe. Enzymatic and metabolic investigations revealed that MJ-CNPs and empty CNPs improve and prolong phenylalanine ammonia-lyase enzyme activity and production of phenolics and flavonoids. The data provides the first evidence of MJ-CNPs and empty CNPs as nano-elicitors that prolong the production of metabolites in plant cell suspension cultures.

Reduced Genotoxicity of Gold Nanoparticles With Protein Corona in Allium cepa.

Increased usage of gold nanoparticles (AuNPs) in biomedicine, biosensing, diagnostics and cosmetics has undoubtedly facilitated accidental and unintentional release of AuNPs into specific microenvironments. This is raising serious questions concerning adverse effects of AuNPs on off-target cells, tissues and/or organisms. Applications utilizing AuNPs will typically expose the nanoparticles to biological fluids such as cell serum and/or culture media, resulting in the formation of protein corona (PC) on the AuNPs. Evidence for PC altering the toxicological signatures of AuNPs is well studied in animal systems. In this report, we observed significant genotoxicity in Allium cepa root meristematic cells (an off-target bioindicator) treated with high concentrations (≥100 µg/ml) of green-synthesized vanillin capped gold nanoparticles (VAuNPs). In contrast, protein-coated VAuNPs (PC-VAuNPs) of similar concentrations had negligible genotoxic effects. This could be attributed to the change in physicochemical characteristics due to surface functionalization of proteins on VAuNPs and/or differential bioaccumulation of gold ions in root cells. High elemental gold accumulation was evident from µ-XRF mapping in VAuNPs-treated roots compared to treatment with PC-VAuNPs. These data infer that the toxicological signatures of AuNPs are influenced by the biological route that they follow to reach off-target organisms such as plants. Hence, the current findings highlight the genotoxic risk associated with AuNPs, which, due to the enhanced utility, are emerging as new pollutants. As conflicting observations on the toxicity of green-synthesized AuNPs are increasingly reported, we recommend that detailed studies are required to investigate the changes in the toxicological signatures of AuNPs, particularly before and after their interaction with biological media and systems.

Review of the structures and functions of algal photoreceptors to optimize bioproduct production with novel bioreactor designs for strain improvement.

Microalgae are important renewable feedstock to produce biodiesel and high-value chemicals. Different wavelengths of light influence the growth and metabolic activities of algae. Recent research has identified the light-sensing proteins called photoreceptors that respond to blue or red light. Structural elucidations of algal photoreceptors have gained momentum over recent years. These include channelrhodopsins, PHOT proteins, animal-like cryptochromes, and blue-light sensors utilizing flavin-adenine dinucleotide proteins. Pulsing light has also been investigated as a means to optimize energy inputs into bioreactors. This study summarizes the current structural and functional basis of photoreceptor modulation to optimize the growth, production of carotenoids and other high-value metabolites from microalgae. The review also encompasses novel photobioreactor designs that implement different light regimes including light wavelengths and time to optimize algal growth and desired metabolite profiles for high-value products.

Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin.

Vanillin production by metabolic engineering of proprietary microbial strains has gained impetus due to increasing consumer demand for naturally derived products. Here, we demonstrate the use of rice cell cultures metabolically engineered with vanillin synthase gene (VpVAN) as a plant-based alternative to microbial vanillin production systems. VpVAN catalyzes the signature step to convert ferulic acid into vanillin in Vanilla planifolia. As ferulic acid is a phenylpropanoid pathway intermediate in plant cells, rice calli cells are ideal platform for in vivo vanillin synthesis due to the availability of its precursor. In this study, rice calli derived from embryonic rice cells were metabolically engineered with a codon-optimized VpVAN gene using Agrobacterium-mediated transformation. The putative transformants were selected based on their proliferation on herbicide-supplemented N6D medium. Expression of the transgenes were confirmed through a ß-glucuronidase (GUS) reporter assay and polymerase chain reaction (PCR) analysis provided evidence of genetic transformation. The semiquantitative RT-PCR and real-time (RT)-qPCR revealed expression of VpVAN in six transgenic calli lines. High-performance liquid chromatography identified the biosynthesis of vanillin in transgenic calli lines, with the highest yielding line producing 544.72 (± 102.50) µg of vanillin-g fresh calli. This work serves as a proof-of-concept to produce vanillin using metabolically engineered rice cell cultures.

Designer nanoparticles for plant cell culture systems: Mechanisms of elicitation and harnessing of specialized metabolites.

Plant cell culture systems have become an attractive and sustainable approach to produce high-value and commercially significant metabolites under controlled conditions. Strategies involving elicitor supplementation into plant cell culture media are employed to mimic natural conditions for increasing the metabolite yield. Studies on nanoparticles (NPs) that have investigated elicitation of specialized metabolism have shown the potential of NPs to be a substitute for biotic elicitors such as phytohormones and microbial extracts. Customizable physicochemical characteristics allow the design of monodispersed-, stimulus-responsive-, and hormone-carrying-NPs of precise geometries to enhance their elicitation capabilities based on target metabolite/plant cell culture type. We contextualize advances in NP-mediated elicitation, especially stimulation of specialized metabolic pathways, the underlying mechanisms, impacts on gene regulation, and NP-associated cytotoxicity. The novelty of the concept lies in unleashing the potential of designer NPs to enhance yield, harness metabolites, and transform nanoelicitation from exploratory investigations to a commercially viable strategy.

Next-generation metabolic engineering approaches towards development of plant cell suspension cultures as specialized metabolite producing biofactories.

Plant cell suspension culture (PCSC) has emerged as a viable technology to produce plant specialized metabolites (PSM). While Taxol® and ginsenoside are two examples of successfully commercialized PCSC-derived PSM, widespread utilization of the PCSC platform has yet to be realized primarily due to a lack of understanding of the molecular genetics of PSM biosynthesis. Recent advances in computational, molecular and synthetic biology tools provide the opportunity to rapidly characterize and harness the specialized metabolic potential of plants. Here, we discuss the prospects of integrating computational modeling, artificial intelligence, and precision genome editing (CRISPR/Cas and its variants) toolboxes to discover the genetic regulators of PSM. We also explore how synthetic biology can be applied to develop metabolically optimized PSM-producing native and heterologous PCSC systems. Taken together, this review provides an interdisciplinary approach to realize and link the potential of next-generation computational and molecular tools to convert PCSC into commercially viable PSM-producing biofactories.

A Short History and Perspectives on Plant Genetic Transformation.

Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30 years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understanding their role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.

Agonist, antagonist and signaling modulators of ABA receptor for agronomic and post-harvest management.

Abscisic acid (ABA) is a ubiquitous phytohormone, plays important roles in several physiological processes, including stress adaptation, flowering, seed germination, fruit ripening, and leaf senescence etc. ABA binds with START domain proteins called Pyrabactin Resistance1 (PYR1)/PYR1-like (PYL)/Regulatory Components of ABA Receptors (RCARs) and controls the activity of PP2C phosphatase proteins and in turn the ABA-dependent signaling pathway. Fourteen ABA receptors have been identified in the model plant Arabidopsis thaliana and have shown to be involved in various biological functions. Under field conditions, exogenous application of ABA produces inadequate physiological response due to its rapid conversion into the biologically inactive metabolites. ABA shows selective binding preferences to PYL receptor subtypes and hence produces pleiotropic physiological and phenotypic effects which limit the usage of ABA in agriculture. An agrochemical meant for ameliorating the undesirable physiological effect of the plant should ideally have positive biological attributes without affecting the normal growth, development, and yield. Therefore, to overcome the limitations of ABA for its usage in various agricultural applications, several types of ABA-mimicking agents have been developed. Many compounds have been identified as having significant ABA-agonist/antagonist activity and can be employed to reverse the excessive/moderate ABA action. The present review highlights the potential usage of ABA signaling modulators for managing agronomic and postharvest traits. Besides, designing, development and versatile usage of ABA-mimicking compounds displaying ABA agonists and antagonist activities are discussed in detail.

In silico study revealed major conserve architectures and novel features of pyrabactin binding to Oryza sativa ABA receptors compare to the Arabidopsis thaliana.

Enhancing water use efficiency (WUE) of crops in irrigated agriculture and drought tolerance in rain-fed agriculture is the major goal for sustaining and enhancing agricultural productivity in the future. The phytohormone abscisic acid (ABA) signaling pathway is a major target for the agronomic management of WUE and genetic improvement of drought tolerance in crops. The START domain proteins PYRABACTIN RESISTANCE1 (PYR1)/PYR1-like (PYL)/Regulatory Components of ABA Receptors (RCARs) of the model plant Arabidopsis thaliana have been characterized as bona fide ABA receptors (ABARs). ABA signaling pathway can be activated or repressed by using specific agonist and antagonist against ABAR and therefore, can be used to control ABA-mediated physiological changes in plants. In the present work, we have reported the 3 D structure models of three ABARs (OsPYL1-3) from drought-tolerant Indica rice N22 (Oryza sativa L. sp. Indica cv N22) in apo- and ligand-bound conformations developed using comparative modeling techniques. Subsequently, these models were used in docking study to investigate the binding mode of known ABAR agonists and antagonists. Further, molecular dynamics studies on the selected systems verified the residues involved in protein-ligand interactions. The study identified the important ligand-binding features for the future development of specific agonists/antagonists to modulate the ABA activity in O. sativa and provides in silico models for designing and virtual screening to identify potent ABA receptor ligands.Communicated by Ramaswamy H. Sarma.

Genetic variability and inter species relationship between wild and cultivated yams (Dioscorea spp.) from Koraput, India based on molecular and morphological markers.

Wild yams (Dioscorea spp.) are important tuber crops used both as vegetable and medicine by the tribal people of Koraput, India. There is deficiency of documented information on genetic structure and diversity of wild yams and its genetic assessment is necessary for crop improvement program. The present study assessed the level of genetic diversity of eight wild and one cultivated yam species of Koraput by using different morphological and molecular markers. Significant variation in different yield and morphological traits was observed among the studied yam species. The major morphological traits such as branch number, stem thickness, tuber depth, tuber length, number of tubers per plant and yield showed high genetic heritability accompanied with high genetic advance and major determinants of phenotypic diversity. Molecular profiling was carried out by taking five simple sequence repeat markers. A total of 10 polymorphic bands with an average of two were detected at the loci of the five markers across the nine yam species. Genetic similarity analysis revealed that some wild yam species such as D. oppositifolia, D. hamiltonii and D. pubera showed higher genetic similarity with cultivated (D. alata) species. The knowledge of the extent of genetic variations of wild yam species is important for planning of the genetic conservation and the utilization of this resource especially for genetic improvement.

Data assessing genotypic variations in selected traditional rice landraces of Jeypore tract of Odisha, India based on photosynthetic traits.

Variations in photosynthetic characteristics and dry matter accumulation were investigated in thirty selected rice (Oryza sativa L.) landraces from Jeypore tract of Odisha, India to find the possibility of their use in crop improvement programs. Leaf gas exchange measurements, photosystem (PS) II activity and leaf pigment estimates were conducted at the flowering stage. Significant differences were noticed in the CO2 photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E), internal CO2 concentration (Ci), water use efficiency (WUE) and carboxylation efficiency (CE) among the landraces. In addition, significant variation was observed in leaf chlorophyll content, PS II activity and dry matter accumulation (DMA). Further, multiple correlations between photosynthetic characteristics and other physiological traits revealed that leaf photosynthesis was not significantly influenced by PS II photochemical activity, leaf area and pigment contents but it was regulated by stomatal conductance, water use efficiency and carboxylation efficiency. Taken together, data presented here shows that some of the landraces had superior photosynthetic traits along with better DMA under prevailing environmental condition and can be used for future crop improvement programs aimed for an increase of leaf photosynthesis in rice.

Data on genetic potentiality of folk rice (Oryza sativa L.) genotypes from Koraput, India in reference to drought tolerance traits.

Precise physiological and molecular marker-based assessment provides information about the extent of genetic diversity, which helps for effective breeding programmes. We have conducted detailed physiological and molecular marker-based assessment of selected eight indigenous rice landraces from Koraput, India along with tolerant (N22) and susceptible (IR64) check varieties under control and simulated drought stress using polyethylene glycol (PEG) 6000. After exposure to different levels of drought stress, relative germination performance (RGP), seedling vigour index (SVI) and relative growth index (RGI) were significantly declined in all the rice landraces compared to the control plants and significant varietal differences were observed. Genetic relationship among the studied rice landraces was assessed with 24 previously reported drought tolerance linked Simple Sequence Repeat (SSR) markers. A total of 53 alleles were detected at the loci of the 24 markers across the 10 rice accessions. The Nei's gene diversity (He) and the polymorphism information content (PIC) ranged from 0 to 0.665 and 0 to 0.687, respectively. Six SSR loci, RM276, RM411, RM3, RM263, RM216 and RM28199, provided the highest PIC values and are potential for exploring the genetic diversity of studied rice lines for drought tolerance. Four rice genotypes (Butkichudi, Haldichudi, Machakanta and Kalajeera) showed the highest genetic distance with tolerant check variety (N22) and can be considered as valuable genetic resources for drought breeding program.

Leaf photosynthesis and antioxidant response in selected traditional rice landraces of Jeypore tract of Odisha, India to submergence.

Submergence tolerance in rice is important for improving yield under rain-fed lowland rice ecosystem. In this study, five traditional rice landraces having submergence tolerance phenotype were selected. These five rice landraces were chosen based on the submergence-tolerance screening of 88 rice landraces from various lowland areas of Jeypore tract of Odisha in our previous study. These five rice landraces were further used for detailed physiological assessment under control, submergence and subsequent re-aeration to judge their performance under different duration of submergence. Seedling survival was significantly decreased with the increase of plant height and significant varietal difference was observed after 14 days of complete submergence. Results showed that submergence progressively declined the leaf photosynthetic rate, stomatal conductance, instantaneous water use efficiency, carboxylation efficiency, photosystem II (PSII) activity and chlorophyll, with greater effect observed in susceptible check variety (IR 42). Notably, higher activities of antioxidative enzymes and ascorbate level were observed in traditional rice landraces and were found comparable with the tolerant check variety (FR 13A). Taken together, three landraces such as Samudrabali, Basnamundi and Gadaba showed better photosynthetic activity than that of tolerant check variety (FR 13A) and showed superior antioxidant response to submergence and subsequent re-aeration. These landraces can be considered as potential donors for the future submergence tolerance breeding program.