Tiny but Mighty: Nanoscale Materials in Plant Disease Management.

Nanoscale materials are promising tools for managing plant diseases and are becoming important players in the current agritech revolution. However, adopting modern methodologies requires a broad understanding of their effectiveness in solving target problems and their effects on the environment and food chain. Furthermore, it is paramount that such technologies are mechanistically and economically feasible for growers to adopt in order to be sustainable in the long run. This Feature Article summarizes the latest findings on the role of nanoscale materials in managing agricultural plant pathogens. Herein, we discussed the benefits and limitations of using nanoscale materials in plant disease management and their potential impacts on the environment and global food security.

Biologically synthesized zinc and copper oxide nanoparticles using Cannabis sativa L. enhance soybean (Glycine max) defense against fusarium virguliforme.

In this study, zinc and copper oxide nanoparticles (NPs) were synthesized using hemp (Cannabis sativa L.) leaves (ZnONP-HL and CuONP-HL), and their antifungal potential was assessed against Fusarium virguliforme in soybean (Glycine max L.). Hemp was selected because it is known to contain large quantities of secondary metabolites that can potentially enhance the reactivity of NPs through surface property modification. Synthesizing NPs with biologically derived materials allows to avoid the use of harsh and expensive synthetic reducing and capping agents. The ZnONP-HL and CuONP-HL showed average grain/crystallite size of 13.51 nm and 7.36 nm, respectively. The biologically synthesized NPs compared well with their chemically synthesized counterparts (ZnONP chem, and CuONP chem; 18.75 nm and 10.05 nm, respectively), confirming the stabilizing role of hemp-derived biomolecules. Analysis of the hemp leaf extract and functional groups that were associated with ZnONP-HL and CuONP-HL confirmed the presence of terpenes, flavonoids, and phenolic compounds. Biosynthesized NPs were applied on soybeans as bio-nano-fungicides against F. virguliforme via foliar treatments. ZnONP-HL and CuONP-HL at 200 µg/mL significantly (p < 0.05) increased (∼ 50%) soybean growth, compared to diseased controls. The NPs improved the nutrient (e.g., K, Ca, P) content and enhanced photosynthetic indicators of the plants by 100-200%. A 300% increase in the expression of soybean pathogenesis related GmPR genes encoding antifungal and defense proteins confirmed that the biosynthesized NPs enhanced disease resistance against the fungal phytopathogen. The findings from this study provide novel evidence of systemic suppression of fungal disease by nanobiopesticides, via promoting plant defense mechanisms.

Phytosynthesis of Zinc Oxide Nanoparticles Using Ceratonia siliqua L. and Evidence of Antimicrobial Activity.

Carob (Ceratonia siliqua L.) is a tree crop cultivated extensively in the eastern Mediterranean regions but that has become naturalized in other regions as well. The present study focused on the green synthesis of zinc oxide nanoparticles (ZnONPs) from Carob and their evaluation for antimicrobial activity in bacteria and fungi. The synthesized ZnONPs showed strong antibacterial activity against Staphylococcus aureus ATCC 25 923 (92%). The NPs inhibited the growth of pathogenic yeast strains, including Candida albicans ATCC90028, Candida krusei ATCC6258, and Candida neoformans ATCC14116, by 90%, 91%, and 82%, respectively, compared to the control. Fungal inhibition zones with the ZnONPs were 88.67% and 90%, respectively, larger for Aspergillus flavus 15UA005 and Aspergillus fumigatus ATCC204305, compared to control fungal growth. This study provides novel information relevant for plant-based development of new and potentially antimicrobial ZnONPs based on extracts. In particular, the development and application of phytogenic nanoparticles enhances the biocompatibility of nano-scale materials, thereby allowing to tune effects to prevent adverse outcomes in non-target biological systems.

Clue of zinc oxide and copper oxide nanoparticles in the remediation of cadmium toxicity in Phaseolus vulgaris L. via the modulation of antioxidant and redox systems.

The present study represents new evidence of the successful use of metal oxide nanoparticles in the remediation of heavy metals. Zinc oxide nanoparticles (ZnO NP) and copper oxide nanoparticles (CuO NP) were assessed to alleviate cadmium (Cd) toxicity in Phaseolus vulgaris L. seedlings and plants. Monitoring physiological and metabolic parameters allowed to elucidate Cd mechanism and process whereby it exerts phytotoxic effects on bean. The response of P. vulgaris seedlings is NP dose-dependent (10 mg/L, 50 mg/L, 100 mg/L, and 200 mg/L). Similarly, applied concentrations triggered a differential response of growing plants in terms of length and biomass. Our physiological data allowed to select 100 mg/L as the most appropriate concentration to apply, in order to avoid any risk of phytotoxicity. The regulatory mechanisms by which ZnO NP and CuO NP act are for the first time compared in the embryonic axes of bean seedlings under Cd stress. Both NP were able to reduce the hypergeneration of hydrogen peroxide (H2O2). They also acted via enhancing ROS scavenging enzymatic capacity, and activity of antioxidant enzymes CAT, APX, GPOX, GPX, and GR, and inhibited the activity of ROS producing enzymes such as GOX and NOX. Another mechanistic effect of NP consisted of the modulation of redox enzymes Trx, NTR, Fd, and FNR evolved in cellular homeostasis and maintaining reduced status in cells. Taken together, ZnO NP triggered more significant metabolic regulations allowing to mitigate the oxidative damage caused by Cd.

Cu and CuO Nanoparticles Affected the Germination and the Growth of Barley (Hordeum vulgare L.) Seedling.

The application of Cu and CuO nanofertilizers in horticulture has been a promising strategy to promote plants' growth. In our study, increasing concentrations (10, 25, 50, 100, 250, 500, 1000, 2000 mg/L) were assessed for their inhibitory or stimulatory effects on barley (Hordeum vulgare L.) seedlings. Our results showed an enhancement of seed germination parameters (FGP, t50, cumulative germination rate, AUC), and seedling growth parameters (roots and shoots' lengths, fresh biomasses and dry biomasses) by the low concentrations of Cu NPs and CuO NPs, while concentrations above 500 mg/L displayed inhibiting effects. CuO NPs treatment showed a significant similitude with CuSO4, which confirms that CuO NPs act mainly via released Cu ions. However, Cu NPs exhibited a different behavior since the nanosized particles contribute together with Cu ions in barley response to Cu NPs. This provides an overall picture of the way these nanoparticles may behave in plant systems.

Assessment of the risks of copper- and zinc oxide-based nanoparticles used in Vigna radiata L. culture on food quality, human nutrition and health.

The present article aims to assess the phytotoxic effects of copper and zinc oxide nanoparticles (Cu NPs, ZnO NPs) on mung bean (Vigna radiata L.) and their possible risk on food quality and safety. We also study the molecular mechanisms underlying the toxicity of nanosized Cu and ZnO. Seeds of mung bean were germinated under increasing concentrations of Cu NPs and ZnO NPs (10, 100, 1000, 2000 mg/L). We analyzed levels of free amino acids, total soluble sugars, minerals, polyphenols and antioxidant capacity. Our results showed that depending on the concentrations used of Cu NPs and ZnO NPs, the physiology of seed germination and embryo growth were modified. Both free metal ions and nanoparticles themselves may impact plant cellular and physiological processes. At 10 mg/L, an improvement of the nutritive properties, in terms of content in free amino acids, total soluble sugars, essential minerals, antioxidant polyphenols and flavonoids, was shown. However, higher concentrations (100-2000 mg/L) caused an alteration in the nutritional balance, which was revealed by the decrease in contents and quality of phenolic compounds, macronutrients (Na, Mg, Ca) and micronutrients (Cu, Fe, Mn, Zn, K). The overall effects of Cu and ZnO nanoparticles seem to interfere with the bioavailability of mineral and organic nutrients and alter the beneficial properties of the antioxidant phytochemicals, mineral compounds, phenolic acids and flavonoids. This may result in a potential hazard to human food and health, at some critical doses of nanofertilizers. This study may contribute in the guidelines to the safe use of nanofertilizers or nanosafety, for more health benefit and less potential risks.

Response to Salinity in Legume Species: An Insight on the Effects of Salt Stress during Seed Germination and Seedling Growth.

The process of soil salinization and the preponderance of saline water sources all over the world represent one of the most harmful abiotic stress to plant growth. This pointed to the importance of obtaining plants which are tolerant or resistant to salt, considering that projection of climate change for the coming years indicate an increase in temperature and rain scarcity. In the current study, the effect of NaCl was investigated on germinating seeds of Lathyrus sativus L., Vicia sativa L., Vigna radiata L. R.Wilczek and Vigna unguiculata L. Walp., by combining physiological, biochemical, biostatistical and ultrastructural analyses. Our results revealed that germination was not influenced by high NaCl concentrations, while seedling growth was affected even at low NaCl concentrations, probably due to an alteration in water uptake and in organic matter biosynthesis. Nevertheless, the synthesis of antioxidant enzymes, phenolic acids and flavonoids was registered in all species, which tended to cope with the increasing salt stress, allowing a response mechanism such as cytoplasm detoxification and cellular turgor maintenance. Besides, the ultrastructural analysis evidenced plasmolyzed cells close to cells with a normal ultrastructure with no appreciable differences among the species. This research deeply investigates the mechanism of salt-stress response focusing on species never tested before for their possible tolerance to salinity.

Efficiency of the Green Synthesized Nanoparticles as New Tools in Cancer Therapy: Insights on Plant-Based Bioengineered Nanoparticles, Biophysical Properties, and Anticancer Roles.

The aim of this work is to review the current knowledge on the efficiency of plant-based synthesized nanoparticles in medical field, particularly in the prevention, diagnosis, and therapy of cancer. For this, we examine the advantages of nanotechnological tools. Besides, a particular attention was given to understand the mechanism by which plant-based bioengineered nanoparticles can interact with components of cancerous cells. Green biosynthesized nanoparticles seem to be novel tool for prognostic biomarkers for cancer diagnosis and drug delivery in tumor cells. They can act either by leading to the damage of tumor cells, or by the protection of healthy cells, via mechanisms involving the specific properties of nanoparticles themselves and the antioxidative and antitumor properties found in plants. However, special attention should be given to the choice of plant species, extracts, and the toxic dose of some phytocompounds during the biosynthesis process. An increase in metal or trace element release from metal and metal oxide biosynthesized nanoparticles can lead to greater oxidative stress, which is associated with higher risk of cancer. Hence, plant-based nanosystems should be more developed to increase their specific targeting of the cancerous cells, in order to preserve the healthy ones.

Cadmium effects on embryo growth of pea seeds during germination: Investigation of the mechanisms of interference of the heavy metal with protein mobilization-related factors.

This work aims to give more insight into mechanisms of action of cadmium (Cd) on germinating pea seeds (Pisum sativum L. var. douce province), specifically the different ways by which Cd cations may interfere with the principal factors involved during germination process, notably storage proteins mobilization, amino acids freeing and proteolytic activities. Obtained results revealed that the process of hydrolysis of main storage proteins showed a significant disruption, which resulted in the decrease of the release of free amino acids, thus imposing a lack in nitrogen supply of essential nutrients to growing embryo under Cd stress. This hypothesis was evidenced by Cd-induced changes occurring in main purified protein fractions; Albumins, Legumins and Vicilins, during their breakdown. Besides, at enzymatic level, the activities of main proteases responsible for this hydrolysis were altered. Indeed, assays using synthetic substrates and specific protease inhibitors followed by protease activity measurements demonstrated that Cd inhibited drastically the total azocaseinolytic activity (ACA) and activities of different proteolytic classes: cysteine-, aspartic-, serine- and metallo-endopeptidases (EP), leucine- and proline-aminopeptidases (LAP and PAP, respectively), and glycine-carboxypeptidases (Gly-CP). The data here presented may suggest that the vulnerability of the embryonic axes towards Cd toxicity could be explained as a result of eventual disruption of metabolic pathways that affect mobilization of reserves and availability of nutrients. In vitro studies suggest that Cd cations may act either directly on the catalytic sites of the proteolytic enzymes, which may cause their deactivation, or indirectly via the generation of oxidative stress and overproduction of free radicals that can interact with enzymes, by altering their activity and structure.

Protective role of exogenous phytohormones on redox status in pea seedlings under copper stress.

The present work aims to provide insight on the role of phytohormone application in developing efficient practical defense strategies to improve plants tolerance under heavy metal contamination. For this purpose, pea (Pisum sativum L.) seeds were germinated in an aqueous solution of 200µM CuCl2 up to the 3rd day and then continued to germinate in the presence of distilled water (stress cessation) or were subjected to following combinations: Cu+1µM IAA and Cu+1µM GA3 for 3 additional days. The results showed that copper excess induced oxidative stress in germinating seeds, which resulted in changes of the redox state of glutathione and cysteine, and proteomics revealed Cu-induced modifications of thiols (SH) and carbonyls (CO) (indicators of protein oxidation). However, application of IAA or GA3 in the germination medium after 3days of Cu exposure alleviated toxicity on seedlings, despite the persistence of Cu up to 6th day. This improving effect seems to be mediated by a cell Cu accumulation decrease and a protein reduced status recovery, since phytohormones modulate thioredoxin/ferredoxin systems in favor of protecting proteins against oxidation. In addition, an IAA and GA3 protective effect was evidenced by a cellular homeostasis amelioration resulting from the balance conservation between the regeneration and consumption processes of glutathione and cysteine reduced forms. The exogenous effectors also induced modifications of profiles of SH and CO, suggesting changes in the regulation and expression of proteins that could be involved in defense mechanism against Cu stress.

Redox biology response in germinating Phaseolus vulgaris seeds exposed to copper: Evidence for differential redox buffering in seedlings and cotyledon.

In agriculture, heavy metal contamination of soil interferes with processes associated with plant growth, development and productivity. Here, we describe oxidative and redox changes, and deleterious injury within cotyledons and seedlings caused by exposure of germinating (Phaseolus vulgaris L. var. soisson nain hâtif) seeds to copper (Cu). Cu induced a marked delay in seedling growth, and was associated with biochemical disturbances in terms of intracellular oxidative status, redox regulation and energy metabolism. In response to these alterations, modulation of activities of antioxidant proteins (thioredoxin and glutathione reductase, peroxiredoxin) occurred, thus preventing oxidative damage. In addition, oxidative modification of proteins was detected in both cotyledons and seedlings by one- and two-dimensional electrophoresis. These modified proteins may play roles in redox buffering. The changes in activities of redox proteins underline their fundamental roles in controlling redox homeostasis. However, observed differential redox responses in cotyledon and seedling tissues showed a major capacity of the seedlings' redox systems to protect the reduced status of protein thiols, thus suggesting quantitatively greater antioxidant protection of proteins in seedlings compared to cotyledon. To our knowledge, this is the first comprehensive redox biology investigation of the effect of Cu on seed germination.

Application of advanced X-ray methods in life sciences.

BACKGROUND: Synchrotron radiation (SR) sources provide diverse X-ray methods for the investigation of structure-function relationships in biological macromolecules. SCOPE OF REVIEW: Recent developments in SR sources and in the X-ray tools they offer for life sciences are reviewed. Specifically, advances in macromolecular crystallography, small angle X-ray solution scattering, X-ray absorption and fluorescence spectroscopy, and imaging are discussed with examples. MAJOR CONCLUSIONS: SR sources offer a range of X-ray techniques that can be used in a complementary fashion in studies of biological systems at a wide range of resolutions from atomic to cellular scale. Emerging applications of X-ray techniques include the characterization of disordered proteins, noncrystalline and nonequilibrium systems, elemental imaging of tissues, cells and organs, and detection of time-resolved changes in molecular structures. GENERAL SIGNIFICANCE: X-ray techniques are in the center of hybrid approaches that are used to gain insight into complex problems relating to biomolecular mechanisms, disease and possible therapeutic solutions. This article is part of a Special Issue entitled "Science for Life". Guest Editors: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Effects of copper on reserve mobilization in embryo of Phaseolus vulgaris L.

The present research reports a biochemical and micro-submicroscopic analysis of copper effect on reserve mobilization during germination of Phaseolus vulgaris L. var. soisson nain hatif seeds. Dry embryonic cells are rich in protein bodies and little starch grains. In Cu-treated embryos copper inhibited 50% of albumin and globulin mobilization after 72 h imbibition. The severe alterations in treated embryo cells, observed by electron microscope, were probably the cause of the inability to utilize the amino acids freed by protein mobilization and so possibly the cause of the inhibition of P. vulgaris embryonic axis elongation.

Responses of proteolytic enzymes in embryonic axes of germinating bean seeds under copper stress.

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.

Role of the ubiquitin-proteasome pathway and some peptidases during seed germination and copper stress in bean cotyledons.

The role of the ubiquitin (Ub)-proteasome pathway and some endo- and aminopeptidases (EPs and APs, respectively) was studied in cotyledons of germinating bean seeds (Phaseolus vulgaris L.). The Ub system appeared to be important both in the early (3 days) and late (9 days) phases of germination. In the presence of copper, an increase in protein carbonylation and a decrease in reduced -SH pool occurred, indicating protein damage. This was associated with an enhancement in accumulation of malondialdehyde, a major product of lipid peroxidation, and an increase in content of hydrogen peroxide (H2O2), showing oxidative stress generation. Moreover, copper induced inactivation of the Ub-proteasome (EC 3.4.25) pathway and inhibition of leucine and proline aminopeptidase activities (EC 3.4.11.1 and EC 3.4.11.5, respectively), thus limiting their role in modulating essential metabolic processes, such as the removal of regulatory and oxidatively-damaged proteins. By contrast, total trypsin and chymotrypsin-like activities (EC 3.4.21.4 and EC 3.4.21.1, respectively) increased after copper exposure, in parallel with a decrease in their inhibitor capacities (i.e. trypsin inhibitor and chymotrypsin inhibitor activity), suggesting that these endoproteases are part of the protective mechanisms against copper stress.

Proteolytic activities in Phaseolus vulgaris cotyledons under copper stress.

The changes in the protease activities of bean cotyledons were investigated in response to copper stress. Assays using synthetic substrates and specific protease inhibitors followed by activity measurements and electrophoresis analysis allowed to study the classes of enzymes involved in the storage protein mobilization during the germination of bean (Phaseolus vulgaris L) seeds, and then identify which ones were affected in the presence of 200 µM CuCl2 in the imbibition medium. Copper treatment affected embryo growth and total protease activity. The results of SDS-gelatin-PAGE show that Cu excess led to a decrease in protease activity of 45 to 66 kDa. Moreover, cysteine-, aspartic- and metallo-protease activities were markedly lowered under copper stress, while serine-protease one was enhanced as well as its activity dependent abundance in comparison with control. However, the relative distribution of major cysteine protease in H2O-germinated seeds was significantly diminished after Cu exposure. Thus, copper excess can disturb the nitrogen freeing from reserve tissues at enzymatic level; differential responses of protease classes are discussed, notably, cysteine protease in the way of storage protein mobilization and serine protease in protective mechanism one.

Copper excess impairs mobilization of storage proteins in bean cotyledons.

Germination represents a limiting stage of plant life cycle. One of the underlying metabolic activities following imbibition of seed is the reserve mobilization. Seeds of bean (Phaseolus vulgaris L. var. soisson nain hatif) were germinated by soaking in distilled water or 200 µM CuCl(2). Storage proteins breakdown and amino acids freeing from reserve tissues were investigated. Compared to the control, Cu caused a reduction in germination rate, embryo growth, and in mobilization of cotyledonary biomass. The failure in albumin and globulin hydrolysis after the exposure to the pollutant was argued by (1) higher contents of remaining proteins than control ones, (2) persistence of some polypeptide bands resolved by polyacrylamide gel electrophoresis of albumin and globulin-rich fractions, and (3) decrease in the availability of amino acids. Nitrogen starvation in embryonic axis should be associated with the Cu-imposed delay in growth.