Identification of Stress-Responsive Metabolites in Plants Using an Untargeted Metabolomics Approach.

Stress can affect different groups of plant metabolites and multiple signaling pathways. Untargeted metabolomics enables the collection of whole-spectrum data for the entire metabolite content present in plant tissues at that point in time. We present a thorough approach for large-scale, untargeted metabolomics of plant tissues using reverse-phase liquid chromatography connected to high-resolution mass spectrometry (LC-MS) of dilute methanolic extract. MZmine is a specialized computer software that automates the alignment and baseline modification of all derived mass peaks across all samples, resulting in precise information on the relative abundance of hundreds of metabolites reflected by thousands of mass signals. Further processing with statistic and bioinformatic techniques will provide a comprehensive perspective of the variations and connections among groups of samples.

Towards realizing nano-enabled precision delivery in plants.

Nanocarriers (NCs) that can precisely deliver active agents, nutrients and genetic materials into plants will make crop agriculture more resilient to climate change and sustainable. As a research field, nano-agriculture is still developing, with significant scientific and societal barriers to overcome. In this Review, we argue that lessons can be learned from mammalian nanomedicine. In particular, it may be possible to enhance efficiency and efficacy by improving our understanding of how NC properties affect their interactions with plant surfaces and biomolecules, and their ability to carry and deliver cargo to specific locations. New tools are required to rapidly assess NC-plant interactions and to explore and verify the range of viable targeting approaches in plants. Elucidating these interactions can lead to the creation of computer-generated in silico models (digital twins) to predict the impact of different NC and plant properties, biological responses, and environmental conditions on the efficiency and efficacy of nanotechnology approaches. Finally, we highlight the need for nano-agriculture researchers and social scientists to converge in order to develop sustainable, safe and socially acceptable NCs.

Targeted mutagenesis of the vacuolar H+ translocating pyrophosphatase gene reduces grain chalkiness in rice.

Grain chalkiness is a major concern in rice production because it impacts milling yield and cooking quality, eventually reducing market value of the rice. A gene encoding vacuolar H+ translocating pyrophosphatase (V-PPase) is a major quantitative trait locus in indica rice, controlling grain chalkiness. Higher transcriptional activity of this gene is associated with increased chalk content. However, whether the suppression of V-PPase could reduce chalkiness is not clear. Furthermore, natural variation in the chalkiness of japonica rice has not been linked with V-PPase. Here, we describe promoter targeting of the japonica V-PPase allele that led to reduced grain chalkiness and the development of more translucent grains. Disruption of a putative GATA element by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 suppressed V-PPase activity, reduced grain chalkiness and impacted post-germination growth that could be rescued by the exogenous supply of sucrose. The mature grains of the targeted lines showed a much lower percentage of large or medium chalk. Interestingly, the targeted lines developed a significantly lower chalk under heat stress, a major inducer of grain chalk. Metabolomic analysis showed that pathways related to starch and sugar metabolism were affected in the developing grains of the targeted lines that correlated with higher inorganic pyrophosphate and starch contents and upregulation of starch biosynthesis genes. In summary, we show a biotechnology approach of reducing grain chalkiness in rice by downregulating the transcriptional activity of V-PPase that presumably leads to altered metabolic rates, including starch biosynthesis, resulting in more compact packing of starch granules and formation of translucent rice grains.

Comprehensive Risk Assessment of Carbon Nanotubes Used for Agricultural Applications.

Carbon-based nanomaterials (CBNs) are often used for potential agricultural applications. Since CBNs applied to plants can easily enter plant organs and reach the human diet, the consequences of the introduction of CBNs into the food chain need to be investigated. We created a platform for a comprehensive investigation of the possible health risks of multiwalled carbon nanotubes (CNTs) accumulated in the organs of exposed tomato plants. Quantification and visualization of CNTs absorbed by plant organs were determined by microwave-induced heating (MIH) and radio frequency (RF) heating methods. Feeding mice with CNT-contaminated tomatoes showed an absence of toxicity for all assessed animal organs. The amount of CNTs accumulated inside the organs of mice fed with CNT-containing fruits was assessed by an RF heating technique and was found to be negligible. Our work provides the experimental evidence that the amount of CNTs accumulated in plant organs as a result of nanofertilization is not sufficient to induce toxicity in mice.

Advanced applications of sustainable and biological nano-polymers in agricultural production.

Though still in its infancy, the use of nanotechnology has shown promise for improving and enhancing agriculture: nanoparticles (NP) offer the potential solution to depleted and dry soils, a method for the controlled release of agrochemicals, and offer an easier means of gene editing in plants. Due to the continued growth of the global population, it is undeniable that our agricultural systems and practices will need to become more efficient in the very near future. However, this new technology comes with significant worry regarding environmental contamination. NP applied to soils could wash into aquifers and contaminate drinking water, or NP applied to food crops may carry into the end product and contaminate our food supply. These are valid concerns that are not likely to be fully answered in the immediate future due to the complexity of soil-NP interactions and other confounding variables. Therefore, it is obviously preferred that NP used outdoors at this early stage be biodegradable, non-toxic, cost-effective, and sustainably manufactured. Fortunately, there are many different biologically derived, cost-efficient, and biocompatible polymers that are suitable for agricultural applications. In this mini-review, we discuss some promising organic nanomaterials and their potential use for the optimization and enhancement of agricultural practices.

Enhancement of drought tolerance in rice by silencing of the OsSYT-5 gene.

Improvement of drought tolerance of crops is a great challenge in conditions of increasing climate change. This report describes that the silencing of the synaptotagmin-5 (OsSYT-5) gene encoding the rice Ca2+ sensing protein with a C2 domain led to a significant improvement of rice tolerance to water deficit stress. Transgenic lines with suppressed expression of the OsSYT-5 gene exhibited an enhanced photosynthetic rate but reduced stomatal conductance and transpiration during water deficit stress. The abscisic acid (ABA) content under both normal and drought conditions was elevated in the leaves of the transgenic rice as compared to the wild type. The silencing of the OsSYT-5 gene affected the expression of several genes associated with ABA-related stress signaling in the transgenic rice plants. In the water deficit experiment, the transgenic lines with a silenced OsSYT-5 gene exhibited symptoms of drought stress seven days later than the wild type. Transgenic lines with suppressed OsSYT-5 gene expression exhibited higher pollen viability and produced more grains compared to the wild type at both normal and drought stress conditions.

Whole-Transcriptome Responses to Environmental Stresses in Agricultural Crops Treated with Carbon-Based Nanomaterials.

Carbon-based nanomaterials (CBNs) such as carbon nanotubes (CNTs) and graphene can be beneficial to crops exposed to abiotic stresses such as drought and high salinity. Our findings suggest that the improvement observed in stressed crops treated with CBNs can be associated with CBN-induced restoration of gene expression. When subjected to salt stress, sorghum seedlings showed modified expression in 51 stress-related genes. The introduction of CNTs or graphene into the salty growth medium resulted in the restoration of the expression of 29 affected genes, resembling that of untreated sorghum seedlings. RNA-Seq approach allowed us to analyze the total gene expression of CBN-treated rice exposed to water-deficit stress and gene expression of CBN-treated tomato plants exposed to salt stress. The application of CNTs or graphene resulted in full or partial restoration of expression of 458 and 1620 genes, respectively, affected by water-deficit stress in rice. Similarly, CBN treatment of NaCl-exposed tomato seedlings led to full or partial restoration of 1639 and 1391 salt-affected transcripts, respectively. Of the genes with restored expression, many of them were identified as major stress-response genes and major transcriptional factors (aquaporins, dehydrins, and heat shock proteins/co-chaperons, NAC, WRKY) and were associated with key stress-signaling pathways (ABA-signaling, InsP3 signaling, and MAPK signaling) in all three tested plant species. These findings provide evidence that CBNs can provide halotolerance and drought tolerance by normalizing the expression of affected stress genes.

Modification of soybean growth and abiotic stress tolerance by expression of truncated ERECTA protein from Arabidopsis thaliana.

ERECTA gene family encodes leucine-rich repeat receptor-like kinases that control major aspects of plant development such as elongation of aboveground organs, leaf initiation, development of flowers, and epidermis differentiation. To clarify the importance of ERECTA signaling for the development of soybean (Glycine max), we expressed the dominant-negative ERECTA gene from Arabidopsis thaliana that is truncated in the kinase domain (AtΔKinase). Expression of AtΔKinase in soybean resulted in the short stature, reduced number of leaves, reduced leaf surface area and enhanced branching in the transgenic plants. The transgenic AtΔKinase soybean plants exhibited increased tolerance to water deficit stress due to the reduction of total leaf area and reduced transpiration compared to the wild-type plants. Production of seeds in AtΔKinase lines was higher compared to wild type at regular conditions of cultivation and after exposure to drought stress. Transgenic seedlings expressing AtΔKinase were also able to withstand salt stress better than the wild-type. Established results demonstrated the significance of native soybean genes (GmER and GmERL) in development and stress response of soybean, and suggested that the truncated ERECTA gene of Arabidopsis thaliana can be used to manipulate the growth and stress response of different crop species.

Improvement of Commercially Valuable Traits of Industrial Crops by Application of Carbon-based Nanomaterials.

Carbon-based nanomaterials (CBNs) have great potential as a powerful tool to improve plant productivity. Here, we investigated the biological effects of graphene and carbon nanotubes (CNTs) on fiber-producing species (cotton, Gossypium hirsutum) and ornamental species (vinca, Catharanthus roseus). The exposure of seeds to CNTs or graphene led to the activation of early seed germination in Catharanthus and overall higher germination in cotton and Catharanthus seeds. The application of CBNs resulted in higher root and shoot growth of young seedlings of both tested species. Cultivation of Catharanthus plants in soil supplemented with CBNs resulted in the stimulation of plant reproductive system by inducing early flower development along with higher flower production. Catharanthus plants cultivated in CNTs or graphene supplemented soil accelerated total flower production by 37 and 58%, respectively. Additionally, CBNs reduced the toxic effects caused by NaCl. Long-term application of CBNs to crops cultivated under salt stress conditions improved the desired phenotypical traits of Catharanthus (higher flower number and leaf number) and cotton (increased fiber biomass) compared to untreated plants of both species cultivated at the same stress condition. The drought stress experiments revealed that introduction of CBNs to matured Catharanthus plant increased the plant survival with no symptoms of leaf wilting as compared to untreated Catharanthus growing in water deficit conditions.

Carbon-based nanomaterials as stimulators of production of pharmaceutically active alkaloids in cell culture of Catharanthus roseus.

Carbon-based nanomaterials (CBNs) were previously described as regulators of plant cell division. Here, we demonstrated the ability of multi-walled carbon nanotubes (MWCNT) and graphene to enhance biomass production in callus culture of the medicinal plant Catharanthus roseus cultivated in dark conditions. Furthermore, both tested CBNs were able to stimulate biosynthesis of total produced alkaloids in CBN-exposed callus culture of Catharanthus. In one case, total alkaloids in CBN-exposed Catharanthus were double that of unexposed Catharanthus. Analysis of metabolites by HPLC revealed that production of the pharmaceutically active alkaloids vinblastine and vincristine was dramatically enhanced in callus exposed to MWCNT or graphene in both dark and light conditions of callus cultivation. In vitro assays (MTT, flow cytometry) demonstrated that total alkaloid extracts derived from Catharanthus callus treated with CBNs significantly reduced cell proliferation of breast cancer (MCF-7) and lung cancer (A549) cell lines compared to the application of extracts derived from untreated Catharanthus callus.

The impact of tomato fruits containing multi-walled carbon nanotube residues on human intestinal epithelial cell barrier function and intestinal microbiome composition.

Carbon nanomaterials (CNMs) can positively regulate seed germination and enhance plant growth. However, clarification of the impact of plant organs containing absorbed CNMs on animal and human health is a critical step of risk assessment for new nano-agro-technology. In this study, we have taken a comprehensive approach to studying the effect tomato fruits derived from plants exposed to multi-walled carbon nanotubes (CNTs) have on gastrointestinal epithelial barrier integrity and their impact on the human commensal intestinal microbiota using an in vitro cell culture and batch human fecal suspension models. The effects of CNTs on selected pure cultures of Salmonella enterica Typhimurium and Lactobacillus acidophilus were also evaluated. This study demonstrated that CNT-containing fruits or the corresponding residual level of pure CNTs (0.001 µg ml-1) was not sufficient to initiate a significant change in transepithelial resistance and on gene expression of the model T-84 human intestinal epithelial cells. However, at 10 µg ml-1 concentration CNTs were able to penetrate the cell membrane and change the gene expression profile of exposed cells. Moreover, extracts from CNT-containing fruits had minimal to no effect on human intestinal microbiota as revealed by culture-based analysis and 16S rRNA sequencing.

Effects of carbon-based nanomaterials on seed germination, biomass accumulation and salt stress response of bioenergy crops.

Bioenergy crops are an attractive option for use in energy production. A good plant candidate for bioenergy applications should produce a high amount of biomass and resist harsh environmental conditions. Carbon-based nanomaterials (CBNs) have been described as promising seed germination and plant growth regulators. In this paper, we tested the impact of two CBNs: graphene and multi-walled carbon nanotubes (CNTs) on germination and biomass production of two major bioenergy crops (sorghum and switchgrass). The application of graphene and CNTs increased the germination rate of switchgrass seeds and led to an early germination of sorghum seeds. The exposure of switchgrass to graphene (200 mg/l) resulted in a 28% increase of total biomass produced compared to untreated plants. We tested the impact of CBNs on bioenergy crops under salt stress conditions and discovered that CBNs can significantly reduce symptoms of salt stress imposed by the addition of NaCl into the growth medium. Using an ion selective electrode, we demonstrated that the concentration of Na+ ions in NaCl solution can be significantly decreased by the addition of CNTs to the salt solution. Our data confirmed the potential of CBNs as plant growth regulators for non-food crops and demonstrated the role of CBNs in the protection of plants against salt stress by desalination of saline growth medium.

Assessment of Effects of the Long-Term Exposure of Agricultural Crops to Carbon Nanotubes.

Carbon-based nanoparticles (CBNs) are nanomaterials that have been shown to be plant growth regulators. Here, we investigated the effects of long-term exposure to multi-walled carbon nanotubes (MWCNTs) on the growth of three important crops (barley, soybean, and corn). The tested species were cultivated in hydroponics supplemented with 50 µg/mL MWCNTs. After 20 weeks of continuous exposure to the nanomaterials, no significant toxic effects on plant development were observed. Several positive phenotypical changes were recorded, in addition to the enhancement of photosynthesis in MWCNT-exposed crops. Raman spectroscopy with point-by-point mapping proved that the MWCNTs in the hydroponic solution moved into all tested species and were distributed in analyzed organs (leaves, stems, roots, and seeds). Our results confirmed the significant potential of CBN in plant agriculture. However, the documented presence of MWCNTs in different organs of all exposed crops highlighted the importance of detailed risk assessment of nanocontaminated plants moving into the food chain.

Multiwalled Carbon Nanotubes Dramatically Affect the Fruit Metabolome of Exposed Tomato Plants.

Here, we reported that multiwalled carbon nanotubes (MWCNT) added to hydroponics system can enhance fruit production of exposed tomato plants. We quantified the exact amount of MWCNT accumulated inside of fruits collected by MWCNT-exposed plants using an advanced microwave induced heating technique (MIH). We found that absorption of MWCNT by tomato fruits significantly affected total fruit metabolome as was confirmed by LC-MS. Our data highlight the importance of comprehensive toxicological risk assessment of plants contaminated with carbon nanomaterials.

Graphene and carbon nanotubes activate different cell surface receptors on macrophages before and after deactivation of endotoxins.

Nanomaterial synthesis and handling in a non-sterile environment can result in the final product becoming contaminated with bacterial endotoxin or lipopolysaccharides (LPB). During toxicological testing, the effects caused by endotoxin-contaminated nanomaterials can be misinterpreted in the end-point analysis (such as cytotoxicity and immune responses) and could result in erroneous conclusions. The objective of this study was twofold: (i) to test different carbon-based nanomaterials (CBNs) [pristine graphene and multi-wall carbon nanotubes (MWCNTs)] for the presence of endotoxin and develop strategies for depyrogenation, and (ii) to compare the immune response exhibited by macrophages after exposure to native CBNs versus depyrogenated CBNs. The gel-clot limulus amebocyte lysate (LAL) and chromogenic-based LAL assays were used to detect endotoxins. Results revealed that the CBNs contained greater amounts of endotoxin than are approved by major regulatory agencies (0.5 EU ml-1 ). Three repeated cycles of autoclaving reduced the endotoxin in the test materials. Macrophages were incubated with pyrogenated and depyrogenated pristine graphene and MWCNTs to test differences in phagocytosis, cytotoxicity, and expression of genes involved in macrophage activation. The uptake of depyrogenated CBNs was significantly reduced as compared with pyrogenated CBNs. Exposure of macrophages to depyrogenated CBNs resulted in a distinct pattern of gene expression for TLR signaling, NOD-like receptor signaling, and downstream signal transduction molecules. Furthermore, macrophages exposed to both types of CBNs showed the downregulation of TLR5 and NLRC4 inflammasomes. The results of this study reaffirm that assessment of endotoxin and other bacterial contamination is critical when evaluating the cellular toxicity of nanomaterials. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells.

In recent years, by extensive achievements in understanding the mechanisms and the pathways affected by cancer, the focus of cancer research is shifting from developing new chemotherapy methods to using natural compounds with therapeutic properties to reduce the adverse effects of synthetic drugs on human health. We used fruit extracts from previously generated human type I InsP 5-ptase gene expressing transgenic tomato plants for assessment of the anti-cancer activity of established genetically modified tomato lines. Cellular assays (MTT, Fluorescent microscopy, Flow Cytometry analysis) were used to confirm that InsP 5-ptase fruit extract was more effective for reducing the proliferation of breast cancer cells compared to wild-type tomato fruit extract. Metabolome analysis of InsP 5-ptase expressing tomato fruits performed by LC-MS identified tomato metabolites that may play a key role in the increased anti-cancer activity observed for the transgenic fruits. Total transcriptome analysis of cancer cells (MCF-7 line) exposed to an extract of transgenic fruits revealed a number of differently regulated genes in the cells treated with transgenic extract compared to untreated cells or cells treated with wild-type tomato extract. Together, this data demonstrate the potential role of the plant derived metabolites in suppressing cell viability of cancer cells and further prove the potential application of plant genetic engineering in the cancer research and drug discovery.

Carbon nanotubes as carriers of Panax ginseng metabolites and enhancers of ginsenosides Rb1 and Rg1 anti-cancer activity.

A major benefit to nanomaterial based-medicine is the ability to provide nanosized vehicles for sporadic metabolites. Here, we describe how the conjugation of valuable ginseng secondary metabolites (ginsenoside Rb1 or Rg1) with carbon nanotubes (CNT) can enhance their anti-proliferative and anti-cancer effects. Ginsenoside-CNT conjugate (Rb-CNT or Rg-CNT) permitted the ginsenosides to be used at a low dose, yet achieve a higher incidence of cancer killing. We were able to demonstrate that the ginsenoside-CNT conjugate can decrease cell viability up to 62% in breast cancer cells (MCF-7) and enhance antiproliferation of drug-resistant pancreatic cancer cells (PANC-1) by 61%. The interaction of the ginsenoside-CNT conjugate with breast cancer cells was studied using Raman Spectroscopy mapping. Total transcriptome profiling (Affymetrix platform) of MCF-7 cells treated with the ginsenoside-CNT conjugate shows that a number of cellular, apoptotic and response to stimulus processes were affected. Therefore, our data confirmed the potential use of CNT as a drug delivery system.

Comparative study of plant responses to carbon-based nanomaterials with different morphologies.

The relationship between the morphology of carbon-based nanomaterials (CBNs) and the specific response of plants exposed to CBNs has not been studied systematically. Here, we prove that CBNs with different morphologies can activate cell growth, germination, and plant growth. A tobacco cell culture growth was found to increase by 22%-46% when CBNs such as helical multi-wall carbon nanotubes (MWCNTs), few-layered graphene, long MWCNTs, and short MWCNTs were added to the growth medium at a concentration of 50 µg ml(-1). The germination of exposed tomato seeds, as well as the growth of exposed tomato seedlings, were significantly enhanced by the addition of all tested CBNs. The presence of CBNs inside exposed seeds was confirmed by transmission electron microscopy and Raman spectroscopy. The effects of helical MWCNTs on gene expression in tomato seeds and seedlings were investigated by microarray technology and real time-PCR. Helical MWCNTs affected a number of genes involved in cellular and metabolic processes and response to stress factors. It was shown that the expression of the tomato water channel gene in tomato seeds exposed to helical MWCNTs was upregulated. These established findings demonstrate that CBNs with different morphologies can cause the same biological effects and share similar mechanisms in planta.

Genetic reduction of inositol triphosphate (InsP3) increases tolerance of tomato plants to oxidative stress.

MAIN CONCLUSION: We demonstrate here that the reduction of InsP 3 , the key component of the phosphoinositol pathway, results in changes in ROS-scavenging machinery and, subsequently, increases the tolerance of tomato plants to light stress. Different plant stress signaling pathways share similar elements and, therefore, 'cross-talk' between the various pathways can exist. Links between the phosphoinositol signaling pathway and light signaling were recently found. Tomato plants expressing InsP 5-ptase and exhibiting reduction in the level of inositol 1,4,5-triphosphate (InsP3) demonstrated enhanced tolerance to stress caused by continuous light exposure. To understand the molecular basis of observed stress tolerance in tomato lines with decreased amount of InsP3, we monitored the expression of enzymatic antioxidants as well as important factors in light signaling associated with non-enzymatic antioxidants (secondary metabolites). Here, we demonstrated that InsP 5-ptase transgenic plants accumulate less hydroxide peroxide and maintain higher chlorophyll content during stress caused by continuous light exposure. This observation can be explained by documented activation of multiple enzymatic antioxidants (LeAPX1, SICAT2, LeSOD) at levels of gene expression and enzymatic activities during continuous light exposure. In addition, we noticed the up-regulation of photoreceptors LePHYB and LeCHS1, key enzymes in flavonoid biosynthesis pathway, transcription factors LeHY5, SIMYB12, and early light-inducible protein (LeELIP) genes in transgenic tomato seedlings exposed to blue or red light. Our study confirmed the existence of a correlation between phosphoinositol signaling pathway modification, increased tolerance to stress caused by continuous light exposure, activation of ROS-scavenging enzymes, and up-regulation of molecular activators of non-enzymatic antioxidants in InsP 5-ptase expressing tomato lines.