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.

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.

Stimulation of photosynthesis and enhancement of growth and yield in Arabidopsis thaliana treated with amine-functionalized mesoporous silica nanoparticles.

Mesoporous silica nanoparticles (MSNs) of 50 nm diameter particle size with a pore size of approximately 14.7 nm were functionalized with amino groups (Am-MSNs) and the effects of exposure to these positively charged Am-MSNs on each of the life cycle stages of Arabidopsis thaliana were investigated. After growth in half strength MS medium amended with Am-MSNs (0-100 µg/mL) for 7 and 14 days, seed germination rate and seedling growth were significantly increased compared with untreated controls. The seedlings were then transferred to soil and irrigated with Am-MSNs solutions every 3 days until seed harvesting. After four weeks growth in soil, Am-MSNs treated plants showed up-regulation of chlorophyll and carotenoid synthesis-related genes, an increase in the content of photosynthetic pigments and an amplification of plant photosynthetic capacity. All these changes in plants were closely correlated with greater vegetative growth and higher seed yield. In all the experiments, 20 and 50 µg/mL of Am-MSNs were found to be more effective with respect to other treatments, while Am-MSNs at the highest level of 100 µg/mL did not result in oxidative stress or cell membrane damage in the exposed plants. To the best of our knowledge, this is the first report evaluating both physiological and molecular responses following exposure to plants of these specific Am-MSNs throughout their whole life cycle. Overall, these findings indicate that following exposure Am-MSNs play a major role in the increase in seed germination, biomass, photosynthetic pigments, photosynthetic capacity and seed yield in A. thaliana.

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.

Nanoapplication of a Resistance Inducer to Reduce Phytophthora Disease in Pineapple (Ananas comosus L.).

Treatment of plants with a variety of abiotic and biotic inducers causes induced resistance to pathogen attack. In this study, the effect of four resistance inducers on plant diseases caused by Phytophthora cinnamomi was screened in vivo initially by using lupin, a susceptible model plant. Lupin pretreated with 0.5 mM salicylic acid (SA) showed effective resistance against P. cinnamomi with restricted lesions. Then, mesoporous silica nanoparticles (MSNs) with particle size around 20 nm and approximate pore size of 3.0 nm were synthesized and functionalized for loading and importing SA to pineapple plantlets. Decanethiol gatekeepers were introduced to the surface of MSNs via glutathione (GSH)-cleavable disulfide linkages to cover the pore entrance, which was confirmed through using Raman spectroscopy. Through free diffusion, the loading efficiency of SA in MSNs gated with gatekeepers was 11.7%, but was lower in MSNs without gatekeepers (8.0%). In addition, in vitro release profile of SA from gatekeeper-capped MSNs indicated that higher concentrations of GSH resulted in more cargo release. Moreover, the experiments in planta showed that the application of MSNs as a resistance inducer delivery system significantly improved pineapple resistance to P. cinnamomi in terms of inhibiting lesion development and improving root growth of infected plants, compared to the use of free SA and MSNs without gatekeepers. The analysis of SA, GSH, and defense-related genes, of PR1 and PR5, further confirmed that the slow and prolonged release of SA from MSNs inside the roots of pineapple plants was achieved through a redox-stimuli release mechanism. Therefore, the application of MSNs with redox-responsive gatekeepers has shown great potential as an efficient tool for delivering chemicals into plants in a controllable way.

Delivery of Abscisic Acid to Plants Using Glutathione Responsive Mesoporous Silica Nanoparticles.

An intracellular glutathione (GSH) responsive phytochemical delivery system based on thiol gated mesoporous silica nanoparticles (MSNs) was developed and tested on the model plant Arabidopsis thaliana. In the present study, monodispersed MSNs with particle diameters of ~20 nm and pore sizes of ~2.87 nm were synthesized and modified. Abscisic acid (ABA), a key phytohormone, was entrapped in the mesopores of MSNs and then the pore entrances of MSNs were covered with decanethiol gatekeepers through GSH-cleavable disulfide linkages. An in vitro release test of ABA from decanethiol gated MSNs proved that there was efficient loading and entrapment of phytochemicals in the absence of a GSH redox trigger. Most importantly, in planta experiments demonstrated that GSH-mediated release of ABA from the pores of MSNs significantly reduced the leaf stomatal aperture and inhibited water loss of treated plants. Moreover, compared with the usage of free ABA, the controlled release of the encapsulated phytohormone from MSNs markedly prolonged the expression of the ABA inducible marker gene (AtGALK2) and finally, improved the drought resistance ability of Arabidopsis seedlings under drought stress. Therefore, the concept of using short-chain molecules as gatekeepers to encapsulate biomolecules in MSNs was demonstrated. The application of MSNs with redox-responsive gatekeepers has been shown in this study to be a potential and efficient technique to deliver phytochemicals into plants and release them in a controllable fashion.

Active defence by an Australian native host, Lomandra longifolia, provides resistance against Phytophthora cinnamomi.

Resistance is rare against the oomycete plant pathogen Phytophthora cinnamomi Rands. Only a limited number of species have been recorded as field-resistant species in Australia. However, understanding the nature of resistance of those species when grown under controlled conditions is challenging because of their slow growth and the inherent difficulties of working with a root pathogen. We assessed the Australian native species, Lomandra longifolia Labill., as a resistant species by analysing in detail the response of roots to infection by P. cinnamomi in a series of comparative tests with Lupinus angustifolius L., a highly susceptible species. Following inoculation of L. longifolia roots, lesion length and colonisation percentage were significantly less than in roots of the susceptible species. Moreover, there was no statistical difference in root growth rate, whole-plant FW and leaf relative chlorophyll content between controls and inoculated L. longifolia. We then examined three key cellular responses that are related to resistance: the production of the reactive oxygen species, H2O2, callose formation and lignin deposition in L. longifolia roots following inoculation with P. cinnamomi. The upregulation of these resistance-related components in the early hours after inoculation suggested their involvement in resistance and that this is controlled by the coordinated response of multiple components. Resistance assessment and a detailed investigation of cellular resistance components along with gene expression analysis provides a platform for further understanding of the mechanisms of resistance against this generalist pathogen and presents opportunities for manipulating susceptible species for disease resistance.

Mesoporous silica nanoparticles enhance seedling growth and photosynthesis in wheat and lupin.

The application of mesoporous silica nanoparticles (MSNs) as a smart delivery system to agricultural crops is gaining attention but the release of nanoparticles into the environment may pose a potential threat to biological systems. We investigated the effects of MSNs on the growth and development of wheat and lupin plants grown under controlled conditions. We report a dramatic increase in the growth of wheat and lupin plants exposed to MSNs. We also found that, in leaves, MSNs localised to chloroplasts and that photosynthetic activity was significantly increased. In addition, absorption and cellular distribution of MSNs by the two plant species following root uptake were observed using scanning electron microscopy equipped with energy dispersive spectroscopy (SEM-EDS). Following uptake of MSNs at 500 and 1000 mg L(-1), there was enhancement of seed germination, increased plant biomass, total protein and chlorophyll content. Treatment of both species with MSNs at the highest concentration (2000 mg L(-1)) did not result in oxidative stress or cell membrane damage. These findings show that MSNs can be used as novel delivery systems in plants and that over the range of concentrations tested, MSNs do not have any negative impacts on plant growth or development.

A New Insight into Growth Mechanism and Kinetics of Mesoporous Silica Nanoparticles by in Situ Small Angle X-ray Scattering.

The growth mechanism and kinetics of mesoporous silica nanoparticles (MSNs) were investigated for the first time by using a synchrotron time-resolved small-angle X-ray scattering (SAXS) analysis. The synchrotron SAXS offers unsurpassed time resolution and the ability to detect structural changes of nanometer sized objects, which are beneficial for the understanding of the growth mechanism of small MSNs (∼20 nm). The Porod invariant was used to quantify the conversion of tetraethyl orthosilicate (TEOS) in silica during MSN formation, and the growth kinetics were investigated at different solution pH and temperature through calculating the scattering invariant as a function of reaction time. The growth of MSNs was found to be accelerated at high temperature and high pH, resulting in a higher rate of silica formation. Modeling SAXS data of micelles, where a well-defined electrostatic interaction is assumed, determines the size and shape of hexadecyltrimethylammonium bromide (CTAB) micelles before and after the addition of TEOS. The results suggested that the micelle size increases and the micelle shape changes from ellipsoid to spherical, which might be attributed to the solubilization of TEOS in the hydrophobic core of CTAB micelles. A new "swelling-shrinking" mechanism is proposed. The mechanism provides new insights into understanding MSN growth for the formation of functional mesoporous materials exhibiting controlled morphologies. The SAXS analyses were correlated to the structure of CTAB micelles and chemical reaction of TEOS. This study has provided critical information to an understanding of the growth kinetics and mechanism of MSNs.

Functionalized mesoporous silica nanoparticles with redox-responsive short-chain gatekeepers for agrochemical delivery.

The controlled release of salicylic acid (SA), a key phytohormone, was mediated by using a novel decanethiol gatekeeper system grafted onto mesoporous silica nanoparticles (MSNs). The decanethiol was conjugated only to the external surfaces of the MSNs through glutathione (GSH)-cleavable disulfide linkages and the introduction of a process to assemble gatekeepers only on the outer surface so that the mesopore area can be maintained for high cargo loading. Raman and nitrogen sorption isotherm analyses confirmed the successful linkage of decanethiol to the surface of MSNs. The in vitro release of SA from decanethiol gated MSNs indicated that the release rate of SA in an environment with a certain amount of GSH was significantly higher than that without GSH. More importantly, in planta experiments showed the release of SA from decanethiol gated MSNs by GSH induced sustained expression of the plant defense gene PR-1 up to 7 days after introduction, while free SA caused an early peak in PR-1 expression which steadily decreased after 3 days. This study demonstrates the redox-responsive release of a phytohormone in vitro and also indicates the potential use of MSNs in planta as a controlled agrochemical delivery system.

Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition.

Electron microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been invaluable tools for the study of the micromorphology of plant cuticles. However, for electron microscopy, the preparation techniques required may invariably introduce artefacts in cuticle preservation. Further, there are a limited number of methods available for quantifying the image data obtained through electron microscopy. Therefore, in this study, optical microscopy techniques were coupled with staining procedures and, along with SEM were used to qualitatively and quantitatively assess the ultrastructure of plant leaf cuticles. Leaf cryosections of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) were stained with either fat-soluble azo stain Sudan IV or fluorescent, diarylmethane Auramine O and were observed under confocal laser scanning microscope (CLSM). For all the plant species tested, the cuticle on the leaf surfaces could be clearly resolved in many cases into cuticular proper (CP), external cuticular layer (ECL), and internal cuticular layer (ICL). Novel image data analysis procedures for quantifying the epicuticular wax micromorphology were developed, and epicuticular waxes of L. angustifolius were described here for the first time. Together, application of a multifaceted approach involving the use of a range of techniques to study the plant cuticle has led to a better understanding of cuticular structure and provides new insights into leaf surface architecture.

Nanostructured liquid crystalline particles as an alternative delivery vehicle for plant agrochemicals.

Agrochemical spray formulations applied to plants are often mixed with surfactants that facilitate delivery of the active ingredient. However, surfactants cause phytotoxicity and off-target effects in the environment. We propose the use of nanostructured liquid crystalline particles (NLCP) as an alternative to surfactant-based agrochemical delivery. For this, we have compared the application of commercial surfactants, di (2-ethylhexyl) sulfosuccinate and alkyl dimethyl betaine, with NLCP made from phytantriol, at concentrations of 0.1%, 1% and 5% on the adaxial surface of leaves of four plant species Ttriticum aestivum (wheat), Zea mays (maize), Lupinus angustifolius (lupin), and Arabidopsis thaliana. In comparison with the application of surfactants there was less phytotoxicity on leaves of each species following treatment with NLCP. Following treatment of leaves with NLCP analysis of cuticular wax micromorphology revealed less wax solubilization in the monocot species. The results clearly show that there are advantages in the use of NLCP rather than surfactants for agrochemical delivery.

Analysis of global host gene expression during the primary phase of the Arabidopsis thaliana-Plasmodiophora brassicae interaction.

Microarray analysis was used to investigate changes in host gene expression during the primary stages of the interaction between the susceptible plant Arabidopsis thaliana (L.) Heynh ecotype Col-0 and the biotrophic pathogen Plasmodiophora brassicae Woronin. Analyses were conducted at 4, 7 and 10 days after inoculation (DAI) and revealed significant induction or suppression of a relatively low number of genes in a range of functional categories. At 4 DAI, there was induced expression of several genes known to be critical for pathogen recognition and signal transduction in other resistant host-pathogen interactions. As the pathogen further colonised root tissue and progressed through the primary plasmodium stage to production of zoosporangia at 7 and 10 DAI, respectively, fewer genes showed changes in expression. The microarray results were validated by examining a subset of induced genes at 4 DAI by quantitative real-time reverse transcriptase PCR (RT-qPCR) analysis all of which correlated positively with the microarray data. The two A. thaliana mutants jar1 and coiI tested were found to be susceptible to P. brassicae. The involvement of defence-related hormones in the interaction was further investigated and the findings indicate that addition of salicylic acid can suppress clubroot disease in A. thaliana plants.