The synergistic potential of biochar and nanoparticles in phytoremediation and enhancing cadmium tolerance in plants.

Cadmium (Cd) is classified as a heavy metal (HM) and is found into the environment through both natural processes and intensified anthropogenic activities such as industrial operations, mining, disposal of metal-laden waste like batteries, as well as sludge disposal, excessive fertilizer application, and Cd-related product usage. This rising Cd disposal into the environment carries substantial risks to the food chain and human well-being. Inadequate regulatory measures have led to Cd bio-accumulation in plants, which is increasing in an alarming rate and further jeopardizing higher trophic organisms, including humans. In response, an effective Cd decontamination strategy such as phytoremediation emerges as a potent solution, with innovations in nanotechnology like biochar (BC) and nanoparticles (NPs) further augmenting its effectiveness for Cd phytoremediation. BC, derived from biomass pyrolysis, and a variety of NPs, both natural and less toxic, actively engage in Cd removal during phytoremediation, mitigating plant toxicity and associated hazards. This review scrutinizes the application of BC and NPs in Cd phytoremediation, assessing their synergistic mechanism in influencing plant growth, genetic regulations, structural transformations, and phytohormone dynamics. Additionally, the review also underscores the adoption of this sustainable and environmentally friendly strategies for future research in employing BC-NP microaggregates to ameliorate Cd phytoremediation from soil, thereby curbing ecological damage due to Cd toxicity.

OsUGE2 Regulates Plant Growth through Affecting ROS Homeostasis and Iron Level in Rice.

BACKGROUND: The growth and development of rice (Oryza sativa L.) are affected by multiple factors, such as ROS homeostasis and utilization of iron. Here, we demonstrate that OsUGE2, a gene encoding a UDP-glucose 4-epimerase, controls growth and development by regulating reactive oxygen species (ROS) and iron (Fe) level in rice. Knockout of this gene resulted in impaired growth, such as dwarf phenotype, weakened root growth and pale yellow leaves. Biochemical analysis showed that loss of function of OsUGE2 significantly altered the proportion and content of UDP-Glucose (UDP-Glc) and UDP-Galactose (UDP-Gal). Cellular observation indicates that the impaired growth may result from decreased cell length. More importantly, RNA-sequencing analysis showed that knockout of OsUGE2 significantly influenced the expression of genes related to oxidoreductase process and iron ion homeostasis. Consistently, the content of ROS and Fe are significantly decreased in OsUGE2 knockout mutant. Furthermore, knockout mutants of OsUGE2 are insensitive to both Fe deficiency and hydrogen peroxide (H2O2) treatment, which further confirmed that OsUGE2 control rice growth possibly through Fe and H2O2 signal. Collectively, these results reveal a new pathway that OsUGE2 could affect growth and development via influencing ROS homeostasis and Fe level in rice.

Melatonin alleviated cadmium accumulation and toxicity by modulating phytohormonal balance and antioxidant metabolism in rice.

Cadmium (Cd) contamination is an eminent dilemma that jeopardizes global food safety and security, especially through its phytotoxicity in rice; one of the most edible crops. Melatonin (MET) has emerged as a protective phytohormone in stress conditions, but the defensive role and underlying mechanisms of MET against Cd toxicity in rice still remain unclear. To fulfill this knowledge gap, the present study is to uncover the key mechanisms for MET-mediated Cd-stress tolerance in rice. Cd toxicity significantly reduced growth by hindering the process of photosynthesis, cellular redox homeostasis, phytohormonal imbalance, and ultrastructural damages. Contrarily, MET supplementation considerably improved growth attributes, photosynthetic efficiency, and cellular ultrastructure as measured by gas exchange elements, chlorophyll content, reduced Cd accumulation, and ultrastructural analysis via transmission electron microscopy (TEM). MET treatment significantly reduced Cd accumulation (39.25%/31.58%), MDA (25.87%/19.45%), H2O2 (17.93%/9.56%), and O2 (29.11%/27.14%) levels in shoot/root tissues, respectively, when compared with Cd treatment. More importantly, MET manifested association with stress responsive phytohormones (ABA and IAA) and boosted the defense mechanisms of plant by enhancing the activities of ROS-scavenging antioxidant enzymes (SOD; superoxide dismutase, POD; peroxidase, CAT; catalase, APX; ascorbate peroxidase) and as well as regulating the key stress-responsive genes (OsSOD1, OsPOD1, OsCAT2, OsAPX1), thereby reinstate cellular membrane integrity and confer tolerance to ultrastructural damages under Cd-induced phytotoxicity. Overall, our findings emphasized the potential of MET as a long-term and cost-effective approach to Cd remediation in paddy soils, which can pave the way for a healthier and more environmentally conscious agricultural sector.

Interactive mode of biochar-based silicon and iron nanoparticles mitigated Cd-toxicity in maize.

Cadmium contamination poses severe environmental and health threats, necessitating effective mitigation strategies. Rice husk biochar (BC) and nanoparticle (NP) treatments are emerging strategies with limited research on their synergistic benefits. This study assesses BC, silicon NPs (nSi), and iron NPs (nFe) modifications (B-nSi, B-nFe, and B-nSi-nFe) to reduce Cd-bioavailability in soil and its toxicity in maize, not reported before. Characterization of amendments validated, nSi and nFe attachment to BC, forming new mineral crystals to adsorb Cd. We found that B-nSi-nFe induced Cd-immobilization in soil by the formation of Cd-ligand complexes with the effective retention of NPs within microporous structure of BC. B-nSi-nFe increased soil pH by 0.76 units while reducing bioavailable Cd by 49 %, than Ck-Cd. Resultantly, B-nSi-nFe reduced Cd concentrations in roots and shoots by 51 % and 75 %, respectively. Moreover, the application of B-nSi-nFe significantly enhanced plant biomass, antioxidant activities, and upregulated the expression of antioxidant genes [ZmAPX (3.28 FC), ZmCAT (3.20 FC), ZmPOD (2.58 FC), ZmSOD (3.08 FC), ZmGSH (3.17 FC), and ZmMDHAR (3.80 FC)] while downregulating Cd transporter genes [ZmNramp5 (3.65 FC), ZmHMA2 (2.92 FC), and ZmHMA3 (3.40 FC)] compared to Ck-Cd. Additionally, confocal microscopy confirmed the efficacy of B-nSi-nFe in maintaining cell integrity due to reduced oxidative stress. SEM and TEM observations revealed alleviation of Cd toxicity to stomata, guard cells, and ultracellular structures with B-nSi-nFe treatment. Overall, this study demonstrated the potential of B-nSi-nFe for reducing Cd mobility in soil-plant system, mitigating Cd-toxicity in plants and improving enzymatic activities in soil.

Ethylene accelerates copper oxide nanoparticle-induced toxicity at physiological, biochemical, and ultrastructural levels in rice seedlings.

The enormous use of metal-based nanoparticles (NPs) in different sectors may result in enhanced accumulation in agricultural soil, which could impose negative effects on crop productivity. Hence, strategies are needed to explore the mechanisms of copper oxide nanoparticle (CuO NP)-induced toxicity in crops. The present study aimed to investigate the involvement of ethylene in CuO NP-induced toxicity in rice seedlings. Here, our results indicate that 450 mg L-1 of CuO NPs induced toxic effects in rice seedlings. Thus, it was evidenced by the reduced plant biomass accumulation, enhanced oxidative stress indicators, and cellular ultrastructural damages. More importantly, the exogenous supply of ethylene biosynthesis and signaling antagonists cobalt (Co) and silver (Ag) respectively provided tolerance and improved the defense system of rice seedlings against CuO NP toxicity. The ethylene antagonists could significantly reduce the extent of ultrastructural and stomatal damage by controlling the ROS accumulation in rice seedlings under CuO NP stress. Furthermore, Co and Ag augmented the antioxidant defense system against CuO NP-induced toxicity. Contrary to that, all oxidative damage attributes were further enhanced exogenous application of ethylene biosynthesis precursor [1-aminocyclopropane-1-carboxylic acid (ACC)] in the presence of CuO NPs. In addition, ACC could increase the CuO NP-induced stomatal and ultrastructural damages by reducing the ROS-scavenging ability in rice seedlings. Taken together, these results indicate the involvement of ethylene in CuO NP-induced toxicity in rice seedlings.

Ethylene participates in zinc oxide nanoparticles induced biochemical, molecular and ultrastructural changes in rice seedlings.

Nowadays, the applications of engineered nanoparticles (ENPs) have been significantly increased, thereby negatively affecting crop production and ultimately contaminating the food chain worldwide. Zinc oxide nanoparticles (ZnO NPs) induced oxidative stress has been clarified in previous studies. But until now, it has not been investigated that how ethylene mediates or participates in ZnO NPs-induced toxicity and related cellular ultrastructural changes in rice seedlings. Here, we reported that 500 mg/L of ZnO NPs reduced the fresh weight (54.75% and 55.64%) and dry weight (40.33% and 47.83%) in shoot and root respectively as compared to control. Furthermore, ZnO NPs (500 mg/L) reduced chlorophyll content (72% Chla, 70% Chlb), induced the stomatal closure and ultrastructural damages by causing oxidative stress in rice seedlings. These cellular damages were significantly increased by exogenous applications of ethylene biosynthesis precursor (ACC) in the presence of ZnO NPs. In contrary, ZnO NPs induced damages on the above-mentioned attributes were reversed through the exogenous supply of ethylene signaling and biosynthesis antagonists such as silver (Ag) and cobalt (Co) respectively. Interestingly, ZnO NPs accelerate ethylene biosynthesis by up-regulating the transcriptome of ethylene biosynthesis responsive genes. The antioxidant enzymes activities and related gene expressions were further increased in ethylene signaling and biosynthesis associated antagonists (Ag and Co) treated seedlings as compared to sole ZnO NPs treatments. In contrary, the above-reported attributes were further decreased by ACC together with ZnO NPs. In a nutshell, ethylene effectively contributes in ZnO NPs induced toxicity and causing ultrastructural and stomatal damage in rice seedlings. Such findings could have potential implications in producing genetic engineered crops, which will be able to tolerate nanoparticles toxicity in the environment.

Dichromate-induced ethylene biosynthesis, perception, and signaling regulate the variance in root growth inhibition among Shaheen basmati and basmati-385 rice varieties.

Heavy metals, including a hexavalent form of chromium (Cr(VI)) increasing accumulation in agricultural soil, cause a significant reduction in quality, yield, and growth of rice varieties worldwide. Screening for the selection of tolerant varieties is essential for conventional and molecular breeding. Shaheen basmati (SB) and basmati-385 (B-385) rice varieties, a subspecies of indica, show different sensitivity to Cr(VI), but the underlying mechanisms of this different sensitivity remain elusive. In the current study, we examine the sensitivity of SB and B-385 based on the root, which is the primary organ that encounters water and soil containing Cr(VI), elongation assay, and ethylene's possible role (a stress-responsive phytohormone) in the process. Our results show that SB's seedlings exhibit hypersensitivity as a higher root elongation inhibition than B-385 under different Cr(VI) concentrations. Hypersensitive SB consistently expresses a higher level of ethylene biosynthesis and signaling-related genes than B-385. Moreover, ethylene signaling antagonist (silver, Ag) and biosynthesis inhibitor (aminoethoxy vinyl glycine, AVG) alleviate the difference in Cr(VI)-induced root growth inhibition between SB and B-385, respectively. Taken together, we conclude that ethylene mediates difference in sensitivity based on the difference in root growth inhibition in different rice varieties. The difference in Cr(VI)-induced root growth inhibition in SB and B-385. (A) Root growth of SB is slightly more as compared to B-385 in control conditions in the Hoagland solutions. (B) Seedlings of SB showed hypersensitivity to 200 µM Cr(VI) compared to B-385 in terms of primary root growth inhibition, which was higher in SB than B-385. Interestingly, Cr(VI)-induced relative transcript level of ethylene biosynthesis, perception, and signaling-related genes was significantly higher in hypersensitive SB than B-385. Current results in association with previous literature show that Cr(VI)-induced ethylene biosynthesis is regulating Cr(VI)-induced ethylene perception, signaling, and associated Cr(VI)-induced ethylene-mediated primary root growth inhibition. Conclusively, the difference in ethylene quantities in both varieties mediates the difference in root growth inhibition between SB and B-385 (C and E). The difference in Cr(VI)-induce root growth inhibition between SB and B-385 was significantly alleviated by ethylene signaling inhibitor (10 µM Ag, as AgNO3) and ethylene biosynthesis inhibitor (10 µM AVG) treatment in the presence of 200 µM Cr(VI), respectively. (D) Ethylene biosynthesis precursor (10 µM ACC) treatment-mediated induced root growth inhibition difference between SB and B-385 was not significant, which may be because of enough quantity of the Cr(VI)-mediated ethylene accumulation or unknown limiting factor. Arrows mean addition and an increase in expression, and T-line means suppression or inhibition. The width of the pointers (arrows) is proportional to the gene expression level.

The WRKY6 transcription factor affects seed oil accumulation and alters fatty acid compositions in Arabidopsis thaliana.

In rapeseed, the oil content of the seed not only supplies energy for seed germination and seedling development but also provides essential dietary nutrients for humans and livestock. Recent studies have revealed that many transcription factors (TFs) regulate the accumulation of fatty acids (FAs) during seed development. WRKY6, a WRKY6 family TF, was reported to serve a function in the plant senescence processes, pathogen defense mechanisms and abiotic stress responses. However, the precise role of WRKY6 in influencing FA accumulation in seeds is still unknown. In this study, we demonstrate that WRKY6 has a high expression level in developing seeds and plays an essential role in regulating the accumulation of FAs in developing seeds of Arabidopsis. Mutation of WRKY6 resulted in significant increase in seed size, accompanied by an increase in FA content and changes in FA composition. Ultrastructure analyses showed that the absence of WRKY6 resulted in more and higher percentage of oil body in the cell of mature seeds. Quantitative real-time PCR analysis revealed changes in the expression of several genes related to photosynthesis and FA biosynthesis in wrky6 mutants at 10 or 16 days after pollination. These results reveal a novel function of WRKY6 influencing seed oil content and FAs compositions. This gene could be used as a promising gene resource to improve FA accumulation and seed yield in Brassica napus through genetic manipulation.

Zinc finger protein 5 (ZFP5) associates with ethylene signaling to regulate the phosphate and potassium deficiency-induced root hair development in Arabidopsis.

KEY MESSAGE: Zinc finger protein transcription factor ZFP5 positively regulates root hair elongation in response to Pi and potassium deficiency by mainly activating the expression of EIN2 in Arabidopsis. Phosphate (Pi) and potassium (K+) are major plant nutrients required for plant growth and development, and plants respond to low-nutrient conditions via metabolic and morphology changes. The C2H2 transcription factor ZFP5 is a key regulator of trichome and root hair development in Arabidopsis. However, its role in regulating root hair development under nutrient deprivations remains unknown. Here, we show that Pi and potassium deficiency could not restore the short root hair phenotype of zfp5 mutant and ZFP5 RNAi lines to wild type level. The deprivation of either of these nutrients also induced the expression of ZFP5 and the activity of an ethylene reporter, pEBS:GUS. The significant reduction of root hair length in ein2-1 and ein3-1 as compared to wild-type under Pi and potassium deficiency supports the involvement of ethylene in root hair elongation. Furthermore, the application of 1-aminocyclopropane-1-carboxylic acid (ACC) significantly enhanced the expression level of ZFP5 while the application of 2-aminoethoxyvinyl glycine (AVG) had the opposite effect when either Pi or potassium was deprived. Further experiments reveal that ZFP5 mainly regulates transcription of ETHYLENE INSENSITIVE 2 (EIN2) to control deficiency-mediated root hair development through ethylene signaling. Generally, these results suggest that ZFP5 regulates root hair elongation by interacting with ethylene signaling mainly through regulates the expression of EIN2 in response to Pi and potassium deficiency in Arabidopsis.

NbGIS regulates glandular trichome initiation through GA signaling in tobacco.

KEY MESSAGE: A novel gene NbGIS positively regulates glandular trichome initiation through GA Signaling in tobacco. NbMYB123-like regulates glandular trichome initiation by acting downstream of NbGIS in tobacco. Glandular trichome is a specialized multicellular structure which has capability to synthesize and secrete secondary metabolites and protects plants from biotic and abiotic stresses. Our previous results revealed that a C2H2 zinc-finger transcription factor GIS and its sub-family genes act upstream of GL3/EGL3-GL1-TTG1 transcriptional activator complex to regulate trichome initiation in Arabidopsis. In this present study, we found that NbGIS could positively regulate glandular trichome development in Nicotiana benthamiana (tobacco). Our result demonstrated that 35S:NbGIS lines exhibited much higher densities of trichome on leaves, main stems, lateral branches and sepals than WT plants, while NbGIS:RNAi lines had the opposite phenotypes. Furthermore, our results also showed that NbGIS was required in response to GA signal to control glandular trichome initiation in Nicotiana benthamiana. In addition, our results also showed that NbGIS significantly influenced GA accumulation and expressions of marker genes of the GA biosynthesis, might result in the changes of growth and maturation in tobacco. Lastly, our results also showed that NbMYB123-like regulated glandular trichome initiation in tobacco by acting downstream of NbGIS. These findings provide new insights to discover the molecular mechanism by which C2H2 transcriptional factors regulates glandular trichome initiation through GA signaling pathway in tobacco.

Ethylene mediates dichromate-induced inhibition of primary root growth by altering AUX1 expression and auxin accumulation in Arabidopsis thaliana.

The hexavalent form of chromium [Cr(VI)] causes a major reduction in yield and quality of crops worldwide. The root is the first plant organ that interacts with Cr(VI) toxicity, which inhibits primary root elongation, but the underlying mechanisms of this inhibition remain elusive. In this study, we investigate the possibility that Cr(VI) reduces primary root growth of Arabidopsis by modulating the cell cycle-related genes and that ethylene signalling contributes to this process. We show that Cr(VI)-mediated inhibition of primary root elongation was alleviated by the ethylene perception and biosynthesis antagonists silver and cobalt, respectively. Furthermore, the ethylene signalling defective mutants (ein2-1 and etr1-3) were insensitive, whereas the overproducer mutant (eto1-1) was hypersensitive to Cr(VI). We also report that high levels of Cr(VI) significantly induce the distribution and accumulation of auxin in the primary root tips, but this increase was significantly suppressed in seedlings exposed to silver or cobalt. In addition, genetic and physiological investigations show that AUXIN-RESISTANT1 (AUX1) participates in Cr(VI)-induced inhibition of primary root growth. Taken together, our results indicate that ethylene mediates Cr(VI)-induced inhibition of primary root elongation by increasing auxin accumulation and polar transport by stimulating the expression of AUX1.

Biochemical responses and ultrastructural changes in ethylene insensitive mutants of Arabidopsis thialiana subjected to bisphenol A exposure.

Bisphenol A (BPA), an important raw material in plastic industry, has become a serious environmental contaminant due to its wide spread use in different products and increasing release into the environment. BPA is known to cause adverse effects in living organisms including plants. Several studies reported that BPA affects growth and development in plants, mainly through oxidative stress. Plants are known to generally cope with stress mainly through hormonal regulation and adaptation, but little is known about the role of plant hormones in plants under BPA stress. The present study was conducted to investigate the role of ethylene in BPA induced oxidative stress in plants using Arabidopsis thaliana as a test plant. The response of ethylene insensitive mutants of Arabidopsis (ein2-1 and etr1-3) to BPA exposure was studied in comparison to the wild type Arabidopsis (WT). In all three genotypes, exposure to BPA adversely affected cellular structures, stomata and light-harvesting pigments. An increase in reactive oxygen species (ROS) lipid peroxidation and other oxidative stress markers indicated that BPA induced toxicity through oxidative stress. However, the overall results revealed that WT Arabidopsis had more pronounced BPA induced damages while ein2-1 and etr1-3 mutants withstood the BPA induced stress more efficiently. The activity of antioxidant enzymes and expression of antioxidants related genes revealed that the antioxidant defense system in both mutants was more efficiently activated than in WT against BPA induced oxidative stress, which further evidenced the involvement of ethylene in regulating BPA induced oxidative stress. It is concluded that ethylene perception and signaling may be involved in BPA induced oxidative stress responses in plants.

AtGIS, a C2H2 zinc-finger transcription factor from Arabidopsis regulates glandular trichome development through GA signaling in tobacco.

Glandular trichome is specialized multicellular structures that have capability to synthesize and secrete secondary metabolites and protect plants from biotic and abiotic stresses. Our previous results revealed that the C2H2 zinc-finger transcription factors (GIS) acts upstream of GL3/EGL3-GL1-TTG1transcriptional activator complex to regulate trichome initiation through GA signal in Arabidopsis. In the present study, we are reporting that ectopic expression of AtGIS could regulate glandular trichome development through GA signaling in tobacco. X-gluc staining of various organs from transgenic plants showed that AtGIS expressed mainly in the glandular trichomes. Statistical analysis demonstrated that over expression of GIS increased significantly glandular trichome production on the leaf, stem, branch, and sepal in tobacco. After PAC treatment, reduction of glandular trichome production in transgenic plants was more severe with compared to wild type plants. Furthermore, GA treatment could induce expression of AtGIS. More importantly, our results also demonstrated that overexpressed AtGIS significantly affect the main components of trichome exudates, such as significantly increase the content of nicotine, Cembratriene-4, 6-diol. Taken together, these results suggest that ectopic expression of AtGIS regulates glandular trichome development and may play a key role in compounds secretion in tobacco.

A zinc finger protein gene ZFP5 integrates phytohormone signaling to control root hair development in Arabidopsis.

Although root hair development in Arabidopsis thaliana has been extensively studied, it remains unknown whether the zinc finger proteins, the largest family of transcription factors in plants, are involved in this process. Here we report that the C2H2 zinc finger protein ZINC FINGER PROTEIN 5 (ZFP5) is a key regulator of root hair initiation and morphogenesis in Arabidopsis. ZFP5 is mainly expressed in root and preferentially in root hair cells. Using both zfp5 mutants and ZFP5 RNAi lines, we show that reduction in ZFP5 function leads to fewer and much shorter root hairs compared to wild-type. Genetic and molecular experiments demonstrate that ZFP5 exerts its effect on root hair development by directly promoting expression of the CAPRICE (CPC) gene. Furthermore, we show that ZFP5 expression is induced by cytokinin, and that ZFP5 mediates cytokinin and ethylene effects on the formation and growth of root hairs. These results suggest that ZFP5 integrates various plant hormone cues to control root epidermal cell development in Arabidopsis.

The responses of trichome mutants to enhanced ultraviolet-B radiation in Arabidopsis thaliana.

To gain a better understanding of the protective function of the trichome in Arabidopsis against UV-B radiation, we performed a study using several Arabidopsis trichome mutants (gl1, gis, gis2, zfp8, try82, and gl3), overexpressing trichome positive regulator lines (35S:GIS and 35S:GIS2), and wild-types (WT) under simulated enhanced UV-B radiation conditions. The flowering time, height, diameter of rosette, leaf size, trichome density, and expression levels of GL3 gene were measured. Significant decreases in height, diameter of rosette, leaf size, and a notable delay in flowering time were observed in all mutants and wild-types after exposure to UV-B. Moreover, the trichome density showed a significant increase, suggesting a clear induction of trichome formation by UV-B. Comparing the mutants and WT, we found that the mutants that had more trichomes showed a lower sensitivity to UV-B than the WT, whereas the mutants that had fewer trichomes were more sensitive to UV-B. These results indicated that the trichome plays a key shielding role against UV-B radiation. qRT-PCR analysis indicated that UV-B radiation induced expression of GL3 and an increase in GL3 transcript level correlated with the increase in trichome density and, suggesting a possible role of GL3 by integrating the environmental signal to control trichome initiation.