Insights into the alleviation of cadmium toxicity in rice by nano-zinc and Serendipita indica: Modulation of stress-responsive gene expression and antioxidant defense system activation.

The adversities of cadmium (Cd) contamination are quite distinguished among other heavy metals (HMs), and so is the efficacy of zinc (Zn) nutrition in mitigating Cd toxicity. Rice (Oryza sativa) crop, known for its ability to absorb HMs, inadvertently facilitates the bioaccumulation of Cd, posing a significant risk to both the plant itself and to humans consuming its edible parts, and damaging the environment as well. The use of nanoparticles, such as nano-zinc oxide (nZnO), to improve the nutritional quality of crops and combat the harmful effects of HMs, have gained substantial attention among scientists and farmers. While previous studies have explored the individual effects of nZnO or Serendipita indica (referred to as S.i) on Cd toxicity, the synergistic action of these two agents has not been thoroughly investigated. Therefore, the gift of nature, i.e., S. indica, was incorporated alongside nZnO (50 mg L-1) against Cd stress (15 µM L-1) and their alliance manifested as phenotypic level modifications in two rice genotypes (Heizhan43; Hz43 and Yinni801; Yi801). Antioxidant activities were enhanced, specifically peroxidase (61.5 and 122.5% in Yi801 and Hz43 roots, respectively), leading to a significant decrease in oxidative burst; moreover, Cd translocation was reduced (85% for Yi801 and 65.5% for Hz43 compared to Cd alone treatment). Microstructural study showed a decrease in number of vacuoles and starch granules with ameliorative treatments. Overall, plants treated with nZnO displayed gene expression pattern (particularly of ZIP genes), different from the ones with alone or combined S.i and Cd. Inferentially, the integration of nZnO and S.i holds great promise as an effective strategy for alleviating Cd toxicity in rice plants. By immobilizing Cd ions in the soil and promoting their detoxification, this novel approach contributes to environmental restoration and ensures food safety worldwide.

Phosphorus and Serendipita indica synergism augments arsenic stress tolerance in rice by regulating secondary metabolism related enzymatic activity and root metabolic patterns.

The multifarious problems created by arsenic (As), for collective environment and human health, serve a cogent case for searching integrative agricultural approaches to attain food security. Rice (Oryza sativa L.) acts as a sponge for heavy metal(loid)s accretion, specifically As, due to anaerobic flooded growth conditions facilitating its uptake. Acclaimed for their positive impact on plant growth, development and phosphorus (P) nutrition, 'mycorrhizas' are able to promote stress tolerance. Albeit, the metabolic alterations underlying Serendipita indica (S. indica; S.i) symbiosis-mediated amelioration of As stress along with nutritional management of P are still understudied. By using biochemical, RT-qPCR and LC-MS/MS based untargeted metabolomics approach, rice roots of ZZY-1 and GD-6 colonized by S. indica, which were later treated with As (10 µM) and P (50 µM), were compared with non-colonized roots under the same treatments with a set of control plants. The responses of secondary metabolism related enzymes, especially polyphenol oxidase (PPO) activities in the foliage of ZZY-1 and GD-6 were enhanced 8.5 and 12-fold, respectively, compared to their respective control counterparts. The current study identified 360 cationic and 287 anionic metabolites in rice roots, and the commonly enriched pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was biosynthesis of phenylalanine, tyrosine and tryptophan, which validated the results of biochemical and gene expression analyses associated with secondary metabolic enzymes. Particularly under As+S.i+P comparison, both genotypes exhibited an upregulation of key detoxification and defense related metabolites, including fumaric acid, L-malic acid, choline, 3,4-dihydroxybenzoic acid, to name a few. The results of this study provided the novel insights into the promising role of exogenous P and S. indica in alleviating As stress.

Nitrogen metabolic rate and differential ammonia volatilization regulate resistance against opportunistic fungus Alternaria alternata in tobacco.

Nutritional correlations between plants and pathogens can crucially affect disease severity. As an essential macronutrient, the availability of nitrogen (N) and the types of N content play a fundamental part not only in energy metabolism and protein synthesis but also in pathogenesis. However, a direct connection has not yet been established between differences in the level of resistance and N metabolism. Pertinently, former studies hold ammonia (NH3) accountable for the development of diseases in tobacco (Nicotiana tabacum L.) and in some post-harvest fruits. With a purpose of pinpointing the function of NH3 volatilization on Alternaria alternata (Fries) Keissl pathogenesis and its correlation with both N metabolism and resistance differences to Alternaria alternata infection in tobacco, leaf tissue of two tobacco cultivars with susceptibility (Changbohuang; CBH), or resistance (Jingyehuang; JYH) were analyzed apropos of ammonia compensation point, apoplastic NH4 + concentration, pH value as well as activities of key enzymes and N status. At the leaf age of 40 to 60 d, the susceptible cultivar had a significantly higher foliar apoplastic ammonium (NH4 +) concentration, pH value and NH3 volatilization potential compared to the resistant one accompanied by a significant reduction in glutamine synthetase (GS), which in particular was a primary factor causing the NH3 volatilization. The NH4 + concentration in CBH was 1.44 times higher than that in JYH, and CBH had NH3 compensation points that were 7.09, 6.15 and 4.35-fold higher than those of JYH at 40, 50 and 60 d, respectively. Moreover, the glutamate dehydrogenase (GDH) activity had an upward tendency related to an increased NH4 + accumulation in both leaf tissues and apoplast but not with the NH3 compensation point. Collectively, our results strongly suggest that the accumulation of NH3 volatilization, rather than NH4 + and total N, was the primary factor inducing the Alternaria alternata infection in tobacco. Meanwhile, the susceptible cultivar was characterized by a higher N re-transfer ability of NH3 volatilization, in contrast to the disease-resistant cultivar, and had a stronger capability of N assimilation and reutilization. This study provides a deeper understanding of the pathogenicity mechanism induced by Alternaria alternata, which is useful for breeding Alternaria alternata-resistant varieties of tobacco, at the same time, our research is also conducive to control tobacco brown spot caused by Alternaria alternata in the field.

Spatio-Temporal Variation in the Phyllospheric Microbial Biodiversity of Alternaria Alternata-Infected Tobacco Foliage.

Phyllospheric microbial composition of tobacco (Nicotiana tabacum L.) is contingent upon certain factors, such as the growth stage of the plant, leaf position, and cultivar and its geographical location, which influence, either directly or indirectly, the growth, overall health, and production of the tobacco plant. To better understand the spatiotemporal variation of the community and the divergence of phyllospheric microflora, procured from healthy and diseased tobacco leaves infected by Alternaria alternata, the current study employed microbe culturing, high-throughput technique, and BIOLOG ECO. Microbe culturing resulted in the isolation of 153 culturable fungal isolates belonging to 33 genera and 99 bacterial isolates belonging to 15 genera. High-throughput sequencing revealed that the phyllosphere of tobacco was dominantly colonized by Ascomycota and Proteobacteria, whereas, the most abundant fungal and bacterial genera were Alternaria and Pseudomonas. The relative abundance of Alternaria increased in the upper and middle healthy groups from the first collection time to the third, whereas, the relative abundance of Pseudomonas, Sphingomonas, and Methylobacterium from the same positions increased during gradual leaf aging. Non-metric multi-dimensional scaling (NMDs) showed clustering of fungal communities in healthy samples, while bacterial communities of all diseased and healthy groups were found scattered. FUNGuild analysis, from the first collection stage to the third one in both groups, indicated an increase in the relative abundance of Pathotroph-Saprotroph, Pathotroph-Saprotroph-Symbiotroph, and Pathotroph-Symbiotroph. Inclusive of all samples, as per the PICRUSt analysis, the predominant pathway was metabolism function accounting for 50.03%. The average values of omnilog units (OUs) showed relatively higher utilization rates of carbon sources by the microbial flora of healthy leaves. According to the analysis of genus abundances, leaf growth and leaf position were the important drivers of change in structuring the microbial communities. The current findings revealed the complex ecological dynamics that occur in the phyllospheric microbial communities over the course of a spatiotemporal varying environment with the development of tobacco brown spots, highlighting the importance of community succession.

Stress signaling convergence and nutrient crosstalk determine zinc-mediated amelioration against cadmium toxicity in rice.

Consumption of rice (Oryza sativa L.) is one of the major pathways for heavy metal bioaccumulation in humans over time. Understanding the molecular responses of rice to heavy metal contamination in agriculture is useful for eco-toxicological assessment of cadmium (Cd) and its interaction with zinc (Zn). In certain crops, the impacts of Cd stress or Zn nutrition on the biophysical chemistry and gene expression have been widely investigated, but their molecular interactions at transcriptomic level, particularly in rice roots, are still elusive. Here, hydroponic investigations were carried out with two rice genotypes (Yinni-801 and Heizhan-43), varying in Cd contents in plant tissues to determine their transcriptomic responses upon Cd15 (15 µM) and Cd15+Zn50 (50 µM) treatments. High throughput RNA-sequencing analysis confirmed that 496 and 2407 DEGs were significantly affected by Cd15 and Cd15+Zn50, respectively, among which 1016 DEGs were commonly induced in both genotypes. Multitude of DEGs fell under the category of protein kinases, such as calmodulin (CaM) and calcineurin B-like protein-interacting protein kinases (CBL), indicating a dynamic shift in hormonal signal transduction and Ca2+ involvement with the onset of treatments. Both genotypes expressed a mutual regulation of transcription factors (TFs) such as WRKY, MYB, NAM, AP2, bHLH and ZFP families under both treatments, whereas genes econding ABC transporters (ABCs), high affinity K+ transporters (HAKs) and Glutathione-S-transferases (GSTs), were highly up-regulated under Cd15+Zn50 in both genotypes. Zinc addition triggered more signaling cascades and detoxification related genes in regulation of immunity along with the suppression of Cd-induced DEGs and restriction of Cd uptake. Conclusively, the effective integration of breeding techniques with candidate genes identified in this study as well as economically and technologically viable methods, such as Zn nutrient management, could pave the way for selecting cultivars with promising agronomic qualities and reduced Cd for sustainable rice production.

Improvement of morpho-physiological, ultrastructural and nutritional profiles in wheat seedlings through astaxanthin nanoparticles alleviating the cadmium toxicity.

Heavy metal accumulation in arable lands and water bodies has become one of the serious global issues among multitude of food security challenges. In particular, cadmium (Cd) concentration has been increasing substantially in the environment that negatively affects the growth and yield of important agricultural crops, especially wheat (Triticum aestivum L.). No doubt, nanotechnology is a revolutionary science but the comprehension of nanoparticle-plants interaction and its potential alleviatory role against metal stress is still elusive. Here, we investigated the mechanistic role of astaxanthin nanoparticles (AstNPs) in Cd stress amelioration and their interaction with wheat under Cd-spiked conditions. The AstNPs fabrication was confirmed through ultraviolet visible spectroscopy, where the particles showed characteristic peak at 423 nm. However, Fourier transform infrared, X-ray diffraction, scanning electron microscopy and transmission electron microscopy analyses confirmed the presence of stabilized spherical-shaped nanocrystals of AstNPs within the size range of 12.03-30.37 nm. The hydroponic application of AstNPs (100 mg L-1) to Cd-affected wheat plants increased shoot height (59%), shoot dry weight (31%), nitrogen concentration (42%), and phosphorus concentration (26%) as compared to non-treated Cd affected seedlings. Moreover, AstNPs-treated plants showed reduction in acropetal Cd translocation (29%) in contrast to plants treated with Cd only. Under Cd-spiked conditions, AstNPs-treated plants displayed an improved nutrient profile (P, N, K+ and Ca2+) with a relative decrease in Na+ content in comparison with non-treated plants. Interestingly, it was found that AstNPs restricted the translocation of Cd to aerial plant parts by negatively regulating Cd transporter genes (TaHMA2 and TaHMA3), and relieved plants from oxidative burst by activating antioxidant machinery via triggering expressions of TaSOD and TaPOD genes. Consequently, it was observed that the application of AstNPs helped in maintaining the nutrient acquisition and ionic homeostasis in Cd-affected wheat plants, which subsequently improved the physiochemical profiles of plants under Cd-stress. This study suggests that AstNPs plausibly serve as stress stabilizers for plants under heavy metal-polluted environment.

Physio-ultrastructural footprints and iTRAQ-based proteomic approach unravel the role of Piriformospora indica-colonization in counteracting cadmium toxicity in rice.

Due to its immense capability to concentrate in rice grain and ultimately in food chain, cadmium (Cd) has become the cause of an elevated concern among agriculturists, scientists and the environmental activists. Symbiotic association of Piriformospora indica (P. indica) has been characterized as a potential aid in combating heavy metal stress in plants for sustainable crop production but our scant knowledge regarding ameliorative tendency of P. indica against Cd, specifically in rice, necessitates an in-depth investigation. This study aimed at elaborating the underlying mechanisms involved in P. indica-mediated tolerance against Cd stress in two rice genotypes, IR8 and ZX1H, varying in Cd accumulation pattern. Either colonized or un-inoculated with P. indica, seedlings of both genotypes were subjected to Cd stress. The results showed that P. indica colonization significantly supported plant biomass, photosynthetic attributes and chlorophyll contents in Cd stressed plants. P. indica colonization sustained chloroplast integrity and reduced Cd translocation (46% and 64%), significantly lowering malondialdehyde (MDA) content (11.3% and 50.4%) compared to uninoculated roots under Cd stress in IR8 and ZX1H, respectively. A genotypic difference was evident when a 2-fold enhancement in root peroxidase (POD) activity was recorded in P. indica colonized IR8 plants as compared to ZX1H. The root proteomic analysis was performed using isobaric tags for relative and absolute quantification (iTRAQ) and the results showed that P. indica alleviates Cd stress in rice via down-regulation of key glycolysis cycle enzymes in a bid to reduce energy consumption by the plants and possibly re-directing it to Cd defense response pathways; and up-regulation of glutamine synthetase, a key enzyme in the L-Arg-dependent pathway for nitric oxide (NO) production, which acts as a stress signaling molecule, thus conferring tolerance by reduction of NO-mediated modification of essential proteins in response to Cd stress. Conclusively, both the tested genotypes benefited from P. indica symbiosis at varying levels by an enhanced detoxification capacity and signaling efficiency in response to stress. Hence, a step forward towards the employment of an environmentally sound and self-renewing approach holding the hope for a healthy future.

Zinc alleviates cadmium toxicity by modulating photosynthesis, ROS homeostasis, and cation flux kinetics in rice.

Understanding of cadmium (Cd) uptake mechanism and development of lower Cd crop genotypes are crucial for combating its phytotoxicity and meeting 70% increase in food demand by 2050. Bio-accumulation of Cd continuously challenges quality of life specifically in regions without adequate environmental planning. Here, we investigated the mechanisms operating in Cd tolerance of two rice genotypes (Heizhan-43 and Yinni-801). Damage to chlorophyll contents and PSII, histochemical staining and quantification of reactive oxygen species (ROS), cell viability and osmolyte accumulation were studied to decipher the interactions between Cd and zinc (Zn) by applying two Cd and two Zn levels (alone as well as combined). Cd2+ and Ca2+ fluxes were also measured by employing sole Cd100 (100 µmol L-1) and Zn50 (50 µmol L-1), and their combination with microelectrode ion flux estimation (MIFE) technique. Cd toxicity substantially reduced chlorophyll contents and maximal photochemical efficiency (Fv/Fm) compared to control plants. Zn supplementation reverted the Cd-induced toxicity by augmenting osmoprotectants and interfering with ROS homeostasis under combined treatments, particularly in Yinni-801 genotype. Fluorescence microscopy indicated a unique pattern of live and dead root cells, depicting more damage with Cd10, Cd15 and Cd15+Zn50. Our results confer that Cd2+ impairs the uptake of Ca2+ whereas, Zn not only competes with Cd2+ but also Ca2+, thereby modifying ion homeostasis in rice plants. This study suggests that exogenous application of Zn is beneficial for rice plants in ameliorating Cd toxicity in a genotype and dose dependent manner by minimizing ROS generation and suppressing collective oxidative damage. The observations confer that Yinni-801 performed better than Heizhan-43 genotype mainly under combined Zn treatments with low-Cd, presenting Zn fortification as a solution to increase rice production.

iTRAQ-based comparative proteomic analysis reveals high temperature accelerated leaf senescence of tobacco (Nicotiana tabacum L.) during flue-curing.

Tobacco (Nicotiana tabacum) is extensively cultivated all over the world for its economic value. During curing and storage, senescence occurs, which is associated with physiological and biochemical changes in postharvest plant organs. However, the molecular mechanisms involved in accelerated senescence due to high temperatures in tobacco leaves during curing need further elaboration. We studied molecular mechanisms of senescence in tobacco leaves exposed to high temperature during curing (Fresh, 38 °C and 42 °C), revealed by isobaric tags for relative and absolute quantification (iTRAQ) for the proteomic profiles of cultivar Bi'na1. In total, 8903 proteins were identified, and 2034 (1150 up-regulated and 1074 down-regulated) differentially abundant proteins (DAPs) were obtained from tobacco leaf samples. These DAPs were mainly involved in posttranslational modification, protein turnover, energy production and conversion. Sugar- and energy-related metabolic biological processes and pathways might be critical regulators of tobacco leaves exposed to high temperature during senescence. High-temperature stress accelerated tobacco leaf senescence mainly by down-regulating photosynthesis-related pathways and degrading cellular constituents to maintain cell viability and nutrient recycling. Our findings provide a valuable inventory of novel proteins involved in senescence physiology and elucidate the protein regulatory network in postharvest organs exposed to high temperatures during flue-curing.

Resemblance and Difference of Seedling Metabolic and Transporter Gene Expression in High Tolerance Wheat and Barley Cultivars in Response to Salinity Stress.

To elucidate inter-specific similarity and difference of tolerance mechanism against salinity stress between wheat and barley, high tolerant wheat cv. Suntop and sensitive cv. Sunmate and tolerant barley cv. CM72 were hydroponically grown in a greenhouse with 100 mM NaCl. Glutathione, secondary metabolites, and genes associated with Na+ transport, defense, and detoxification were examined to discriminate the species/cultivar difference in response to salinity stress. Suntop and CM72 displayed damage to a lesser extent than in Sunmate. Compared to Sunmate, both Suntop and CM72 recorded lower electrolyte leakage and reactive oxygen species (ROS) production, higher leaf relative water content, and higher activity of PAL (phenylalanine ammonia-lyase), CAD (cinnamyl alcohol dehydrogenase), PPO (polyphenol oxidase), SKDH (shikimate dehydrogenase), and more abundance of their mRNA under salinity stress. The expression of HKT1, HKT2, salt overly sensitive (SOS)1, AKT1, and NHX1 was upregulated in CM72 and Suntop, while downregulated in Sunmate. The transcription factor WRKY 10 was significantly induced in Suntop but suppressed in CM72 and Sunmate. Higher oxidized glutathione (GSSG) content was accumulated in cv. CM72 and Sunmate, but increased glutathione (GSH) content and the ratio of GSH/GSSG were observed in leaves and roots of Suntop under salinity stress. In conclusion, glutathione homeostasis and upregulation of the TaWRKY10 transcription factor played a more important role in wheat salt-tolerant cv. Suntop, which was different from barley cv. CM72 tolerance to salinity stress. This new finding could help in developing salinity tolerance in wheat and barley cultivars.

Comparative Proteomic Analysis by iTRAQ Reveals that Plastid Pigment Metabolism Contributes to Leaf Color Changes in Tobacco (Nicotiana tabacum) during Curing.

Tobacco (Nicotiana tabacum), is a world's major non-food agricultural crop widely cultivated for its economic value. Among several color change associated biological processes, plastid pigment metabolism is of trivial importance in postharvest plant organs during curing and storage. However, the molecular mechanisms involved in carotenoid and chlorophyll metabolism, as well as color change in tobacco leaves during curing, need further elaboration. Here, proteomic analysis at different curing stages (0 h, 48 h, 72 h) was performed in tobacco cv. Bi'na1 with an aim to investigate the molecular mechanisms of pigment metabolism in tobacco leaves as revealed by the iTRAQ proteomic approach. Our results displayed significant differences in leaf color parameters and ultrastructural fingerprints that indicate an acceleration of chloroplast disintegration and promotion of pigment degradation in tobacco leaves due to curing. In total, 5931 proteins were identified, of which 923 (450 up-regulated, 452 down-regulated, and 21 common) differentially expressed proteins (DEPs) were obtained from tobacco leaves. To elucidate the molecular mechanisms of pigment metabolism and color change, 19 DEPs involved in carotenoid metabolism and 12 DEPs related to chlorophyll metabolism were screened. The results exhibited the complex regulation of DEPs in carotenoid metabolism, a negative regulation in chlorophyll biosynthesis, and a positive regulation in chlorophyll breakdown, which delayed the degradation of xanthophylls and accelerated the breakdown of chlorophylls, promoting the formation of yellow color during curing. Particularly, the up-regulation of the chlorophyllase-1-like isoform X2 was the key protein regulatory mechanism responsible for chlorophyll metabolism and color change. The expression pattern of 8 genes was consistent with the iTRAQ data. These results not only provide new insights into pigment metabolism and color change underlying the postharvest physiological regulatory networks in plants, but also a broader perspective, which prompts us to pay attention to further screen key proteins in tobacco leaves during curing.

Cadmium-zinc cross-talk delineates toxicity tolerance in rice via differential genes expression and physiological / ultrastructural adjustments.

Understanding the physiological and molecular response of crop genotypes could be useful in eco-toxicological evaluation with cadmium (Cd) and could be a strategy to solve heavy metal contamination in agriculture. This study corroborates unique patterns of Cd accumulation and molecular mechanisms adopted by plants to acquire Cd tolerance and counteractive effects of zinc (Zn) against Cd toxicity. Two rice (Oryza sativa) genotypes (Heizhan 43 and Yinni 801) differing in cadmium tolerance and its accumulation in plant tissues were investigated hydroponically using two Cd levels [Cd10 (10 µM L-1) and Cd15 (15 µM L-1)] and two Zn levels [Zn25 (25 µM L-2) and Zn50 (50 µM L-1)] and their combinations. Cadmium toxicity rendered substantial reduction in plant height, biomass, chlorophyll contents and photosynthesis as compared to the control plants after 15 days of treatment. Supplementation of Zn evidently ameliorated Cd toxicity by minimizing the reduction in plant growth, chlorophyll contents and photosynthetic attributes (Pn, gs, Ci, and Tr). Comparatively, lower accumulation of Cd in Yinni 801 under combined treatments revealed a preferential uptake of Zn in this genotype. A cross-talk among Cd, Zn, Fe, Ca and K correlated with fluctuating gs, Ci and Tr. Both genotypes also differed in morphological alterations of cell membrane, chloroplasts and appearance of enlarged plastoglobuli along with distorted mitochondria. An increased ascorbate peroxidase activity in roots of Yinni 801 presented a defensive strategy. Relative expression of Cd and Zn ion transporter genes also confirmed the genotypic background of phenotypic divergence. The OsLCT1 and OsHMA2 expression was significant in Heizhan 43, indicating possible translocation of Cd from shoot to grains contrary to Yinni 801, which accumulated Cd in shoot and showed stunted growth. Zn supplementation promises tolerance to Cd in Yinni 801 by differential expression of putative genes for Cd translocation with minimum ultrastructural modifications by maintaining physiological functions in contrast to Heizhan 43.