Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

The glutathione-dependent alarm triggers signalling responses involved in plant acclimation to cadmium.

Cadmium (Cd) uptake from polluted soils inhibits plant growth and disturbs physiological processes, at least partly due to disturbances in the cellular redox environment. Although the sulfur-containing antioxidant glutathione is important in maintaining redox homeostasis, its role as an antioxidant can be overruled by its involvement in Cd chelation as a phytochelatin precursor. Following Cd exposure, plants rapidly invest in phytochelatin production, thereby disturbing the redox environment by transiently depleting glutathione concentrations. Consequently, a network of signalling responses is initiated, in which the phytohormone ethylene is an important player involved in the recovery of glutathione levels. Furthermore, these responses are intricately connected to organellar stress signalling and autophagy, and contribute to cell fate determination. In general, this may pave the way for acclimation (e.g. restoration of glutathione levels and organellar homeostasis) and plant tolerance in the case of mild stress conditions. This review addresses connections between these players and discusses the possible involvement of the gasotransmitter hydrogen sulfide in plant acclimation to Cd exposure.

Arabidopsis root growth and development under metal exposure presented in an adverse outcome pathway framework.

Due to human activities, soils become more and more polluted with metals, which imposes risks for human health and wildlife welfare. As most of the metals end up in the food chain through accumulation in plants, we need to establish science-based environmental criteria and risk management policies. To meet these necessities, a thorough understanding is required of how these metals accumulate in and affect plants. Many studies have been conducted towards this aim, but strikingly, only a few entries can be found in ecotoxicological databases, especially on Arabidopsis thaliana, which serves as a model species for plant (cell) physiology and genetic studies. As experimental conditions seem to vary considerably throughout literature, extrapolation or comparison of data is rather difficult or should be approached with caution. Furthermore, metal-polluted soils often contain more than one metal, yet limited studies investigated the impact of metal mixtures on plants. This review aims to compile all data concerning root system architecture under Cu, Cd and Zn stress, in single or multi-metal exposure in A. thaliana, and link it to metal-induced responses at different biological levels. Global incorporation into an adverse outcome pathway framework is presented.

Essential trace metals in plant responses to heat stress.

Essential trace metals function as structural components or cofactors in many proteins involved in a wide range of physiological processes in plants. Hence, trace metal deficiency can significantly hamper plant growth and development. On the other hand, excess concentrations of trace metals can also induce phytotoxicity, for example via an enhanced production of reactive oxygen species. Besides their roles in plant growth under favourable environmental conditions, trace metals also contribute to plant responses to biotic and abiotic stresses. Heat is a stress factor that will become more prevalent due to increasing climate change and is known to negatively affect crop yield and quality, posing a severe threat to food security for future generations. Gaining insight into heat stress responses is essential to develop strategies to optimize plant growth and quality under unfavourable temperatures. In this context, trace metals deserve particular attention as they contribute to defence responses and are important determinants of plant nutritional value. Here, we provide an overview of heat-induced effects on plant trace metal homeostasis and the involvement of trace metals and trace metal-dependent enzymes in plant responses to heat stress. Furthermore, avenues for future research on the interactions between heat stress and trace metals are discussed.

Biostimulation of Vigna unguiculata subsp. sesquipedalis-Cultivar Sesquipedalis (Yardlong Bean)-by Brevibacillus sp. B65 in Organoponic Conditions.

In the current research, the effects of fluid inoculum of Brevibacillus sp. B65, a plant growth promoting microorganism (PGPM), on growth of V. unguiculata subsp. sesquipedalis cultivated in organoponic conditions were evaluated in comparison with traditional inorganic and organic fertilizers. Plant growth promotion of Yardlong bean was assessed through the effects of four different treatments on plant growth and development traits, as well as on crop yield. The four treatments were NPK-inorganic fertilizer (T1), organic matter alone (T2), fluid inoculum of B65 alone (T3) and inoculum supplemented with organic matter (T4). The inoculum of B65 supplemented with organic matter improved different traits of plant growth and development such as seed germination, root development, plant and leaves growth, flowering, as well as crop yield. The main impact of the inoculation mixture was on seed emergence. In the present research, it was demonstrated that biostimulation of Vigna unguiculata subsp. sesquipedalis through inoculation of PGPM Brevibacillus sp. B65 supplemented with organic matter, may replace traditional organic and inorganic fertilization strategies. The nature of the positive influence of strain B65 on the legume is not well understood yet; however, it could be attributed to bacterial phytostimulation through auxin and ethylene production, as well as P mobilization. Additionally, organic matter supplementation demonstrated a stimulating effect on B65 traits. This is of utmost importance and will have a main impact on the sustainable development of agronomical practices.

Chemical and Pharmacological Potential of Coccoloba cowellii, an Endemic Endangered Plant from Cuba.

Coccoloba cowellii Britton (Polygonaceae) is an endemic and critically endangered plant that only grows in Camagüey, a province of Cuba. In this study, a total of 13 compounds were identified in a methanolic leaf extract, employing a dereplication of the UHPLC-HRMS data by means of feature-based molecular networking (FBMN) analysis in the Global Natural Products Social Molecular Network (GNPS), together with the interpretation of the MS/MS data and comparison with the literature. The major constituents were glucuronides and glycosides of myricetin and quercetin, as well as epichatechin-3-O-gallate, catechin, epicatechin and gallic acid, all of them being reported for the first time in C. cowellii leaves. The leaf extract was also tested against various microorganisms, and it showed a strong antifungal effect against Candida albicans ATCC B59630 (azole-resistant) (IC50 2.1 µg/mL) and Cryptococcus neoformans ATCC B66663 (IC50 4.1 µg/mL) with no cytotoxicity (CC50 > 64.0 µg/mL) on MRC-5 SV2 cells, determined by the resazurin assay. Additionally, the extract strongly inhibited COX-1 and COX-2 enzyme activity using a cell-free experiment in a dose-dependent manner, being significantly more active on COX-1 (IC50 4.9 µg/mL) than on COX-2 (IC50 10.4 µg/mL). The constituents identified as well as the pharmacological activities measured highlight the potential of C. cowellii leaves, increasing the interest in the implementation of conservation strategies for this species.

The Roots of Plant Frost Hardiness and Tolerance.

Frost stress severely affects agriculture and agroforestry worldwide. Although many studies about frost hardening and resistance have been published, most of them focused on the aboveground organs and only a minority specifically targets the roots. However, roots and aboveground tissues have different physiologies and stress response mechanisms. Climate models predict an increase in the magnitude and frequency of late-frost events, which, together with an observed loss of soil insulation, will greatly decrease plant primary production due to damage at the root level. Molecular and metabolic responses inducing root cold hardiness are complex. They involve a variety of processes related to modifications in cell wall composition, maintenance of the cellular homeostasis and the synthesis of primary and secondary metabolites. After a summary of the current climatic models, this review details the specificity of freezing stress at the root level and explores the strategies roots developed to cope with freezing stress. We then describe the level to which roots can be frost hardy, depending on their age, size category and species. After that, we compare the environmental signals inducing cold acclimation and frost hardening in the roots and aboveground organs. Subsequently, we discuss how roots sense cold at a cellular level and briefly describe the following signal transduction pathway, which leads to molecular and metabolic responses associated with frost hardening. Finally, the current options available to increase root frost tolerance are explored and promising lines of future research are discussed.

Cadmium inhibits cell cycle progression and specifically accumulates in the maize leaf meristem.

It is well known that cadmium (Cd) pollution inhibits plant growth, but how this metal impacts leaf growth processes at the cellular and molecular level is still largely unknown. In the current study, we show that Cd specifically accumulates in the meristematic tissue of the growing maize leaf, while Cd concentration in the elongation zone rapidly declines as the deposition rates diminish and cell volumes increase due to cell expansion. A kinematic analysis shows that, at the cellular level, a lower number of meristematic cells together with a significantly longer cell cycle duration explain the inhibition of leaf growth by Cd. Flow cytometry analysis suggests an inhibition of the G1/S transition, resulting in a lower proportion of cells in the S phase and reduced endoreduplication in expanding cells under Cd stress. Lower cell cycle activity is also reflected by lower expression levels of key cell cycle genes (putative wee1, cyclin-B2-4, and minichromosome maintenance4). Cell elongation rates are also inhibited by Cd, which is possibly linked to the inhibited endoreduplication. Taken together, our results complement studies on Cd-induced growth inhibition in roots and link inhibited cell cycle progression to Cd deposition in the leaf meristem.

Dihydrofolate Reductase/Thymidylate Synthase Fine-Tunes the Folate Status and Controls Redox Homeostasis in Plants.

Folates (B9 vitamins) are essential cofactors in one-carbon metabolism. Since C1 transfer reactions are involved in synthesis of nucleic acids, proteins, lipids, and other biomolecules, as well as in epigenetic control, folates are vital for all living organisms. This work presents a complete study of a plant DHFR-TS (dihydrofolate reductase-thymidylate synthase) gene family that implements the penultimate step in folate biosynthesis. We demonstrate that one of the DHFR-TS isoforms (DHFR-TS3) operates as an inhibitor of its two homologs, thus regulating DHFR and TS activities and, as a consequence, folate abundance. In addition, a novel function of folate metabolism in plants is proposed, i.e., maintenance of the redox balance by contributing to NADPH production through the reaction catalyzed by methylenetetrahydrofolate dehydrogenase, thus allowing plants to cope with oxidative stress.

Identifying the Pressure Points of Acute Cadmium Stress Prior to Acclimation in Arabidopsis thaliana.

The toxic metal cadmium (Cd) is a major soil pollutant. Knowledge on the acute Cd-induced stress response is required to better understand the triggers and sequence of events that precede plant acclimation. Therefore, we aimed to identify the pressure points of Cd stress using a short-term exposure set-up ranging from 0 h to 24 h. Acute responses related to glutathione (GSH), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and the oxidative challenge were studied at metabolite and/or transcript level in roots and leaves of Arabidopsis thaliana either exposed or not to 5 µM Cd. Cadmium rapidly induced root GSH depletion, which might serve as an alert response and modulator of H2O2 signalling. Concomitantly, a stimulation of root ACC levels was observed. Leaf responses were delayed and did not involve GSH depletion. After 24 h, a defined oxidative challenge became apparent, which was most pronounced in the leaves and concerted with a strong induction of leaf ACC synthesis. We suggest that root GSH depletion is required for a proper alert response rather than being a merely adverse effect. Furthermore, we propose that roots serve as command centre via a.o. root-derived ACC/ethylene to engage the leaves in a proper stress response.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Selection of Appropriate Reference Genes for Gene Expression Analysis under Abiotic Stresses in Salix viminalis.

Salix viminalis is a fast growing willow species with potential as a plant used for biomass feedstock or for phytoremediation. However, few reference genes (RGs) for quantitative real-time polymerase chain reaction (qPCR) are available in S. viminalis, thereby limiting gene expression studies. Here, we investigated the expression stability of 14 candidate reference genes (RGs) across various organs exposed to five abiotic stresses (cold, heat, drought, salt, and poly-metals). Four RGs ranking algorithms, namely geNormPLUS, BestKeeper, NormFinder, and GrayNorm were applied to analyze the qPCR data and the outputs were merged into consensus lists with RankAggreg, a rank aggregation algorithm. In addition, the optimal RG combinations were determined with geNormPLUS and GrayNorm. The genes that were the most stable in the roots were TIP41 and CDC2. In the leaves, TIP41 was the most stable, followed by EF1b and ARI8, depending on the condition tested. Conversely, GAPDH and ß-TUB, two genes commonly used for qPCR data normalization were the least stable across all organs. Nevertheless, both geNormPLUS and GrayNorm recommended the use of a combination of genes rather than a single one. These results are valuable for research of transcriptomic responses in different S. viminalis organs.

Cadmium and Plant Development: An Agony from Seed to Seed.

Anthropogenic pollution of agricultural soils with cadmium (Cd) should receive adequate attention as Cd accumulation in crops endangers human health. When Cd is present in the soil, plants are exposed to it throughout their entire life cycle. As it is a non-essential element, no specific Cd uptake mechanisms are present. Therefore, Cd enters the plant through transporters for essential elements and consequently disturbs plant growth and development. In this review, we will focus on the effects of Cd on the most important events of a plant's life cycle covering seed germination, the vegetative phase and the reproduction phase. Within the vegetative phase, the disturbance of the cell cycle by Cd is highlighted with special emphasis on endoreduplication, DNA damage and its relation to cell death. Furthermore, we will discuss the cell wall as an important structure in retaining Cd and the ability of plants to actively modify the cell wall to increase Cd tolerance. As Cd is known to affect concentrations of reactive oxygen species (ROS) and phytohormones, special emphasis is put on the involvement of these compounds in plant developmental processes. Lastly, possible future research areas are put forward and a general conclusion is drawn, revealing that Cd is agonizing for all stages of plant development.

Relationship between Mg, B and Mn status and tomato tolerance against Cd toxicity.

Distinct tomato genotypes possess different tolerance degree to cadmium (Cd), but the mechanisms behind this phenomenon are scarcely understood. To this end, the physiological, biochemical, anatomical, nutritional and molecular mechanisms associated to the plant tolerance against Cd toxicity were investigated in five tomato accessions with contrasting sensitivity to Cd exposure. Firstly, the data revealed that larger biomass loss was not always coupled to higher Cd concentration, indicating that other events, in addition to the internal Cd accumulation, impact tomato performance at early stages of Cd exposure. Secondly, the results indicated that the fine regulation of nutrient status, particularly magnesium (Mg), boron (B) and manganese (Mn), is associated to the mitigation of Cd toxicity. Magnesium status was coupled to the modulation of root development, resulting in changes in root hair formation and biomass allocation. Boron accumulation in leaves was linked to Cd toxicity, suggesting that tolerance mechanisms involved strategies to decrease or even avoid B excess in photosynthetic tissues. Disturbances in Mn status, i.e. Mn excess in leaves and Mn deficiency in roots, were also related to tomato sensitivity to Cd exposure. Thirdly, plant capacity to maintain leaf blade expansion is a relevant strategy for a better tomato development after short-term Cd exposure. Fourthly, tomato tolerance to Cd-induced stress does not depend on CAT activity enhancements in such conditions. In conclusion, tomato ability to quickly manage its nutritional status is necessary for alleviation of the Cd effects at early stages of exposure to this metal. The better understanding about tolerance mechanisms and mode of action of Cd toxicity in plants can help in the establishment of strategies to mitigate its impacts on crops.

Changes in the Proteome of Medicago sativa Leaves in Response to Long-Term Cadmium Exposure Using a Cell-Wall Targeted Approach.

Accumulation of cadmium (Cd) shows a serious problem for the environment and poses a threat to plants. Plants employing various cellular and molecular mechanisms to limit Cd toxicity and alterations of the cell wall structure were observed upon Cd exposure. This study focuses on changes in the cell wall protein-enriched subproteome of alfalfa (Medicago sativa) leaves during long-term Cd exposure. Plants grew on Cd-contaminated soil (10 mg/kg dry weight (DW)) for an entire season. A targeted approach was used to sequentially extract cell wall protein-enriched fractions from the leaves and quantitative analyses were conducted with two-dimensional difference gel electrophoresis (2D DIGE) followed by protein identification with matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time of flight (TOF/TOF) mass spectrometry. In 212 spots that showed a significant change in intensity upon Cd exposure a single protein was identified. Of these, 163 proteins are predicted to be secreted and involved in various physiological processes. Proteins of other subcellular localization were mainly chloroplastic and decreased in response to Cd, which confirms the Cd-induced disturbance of the photosynthesis. The observed changes indicate an active defence response against a Cd-induced oxidative burst and a restructuring of the cell wall, which is, however, different to what is observed in M. sativa stems and will be discussed.

A novel, highly conserved metallothionein family in basidiomycete fungi and characterization of two representative SlMTa and SlMTb genes in the ectomycorrhizal fungus Suillus luteus.

The basidiomycete Suillus luteus is an important member of the ectomycorrhizal community that thrives in heavy metal polluted soils covered with pioneer pine forests. This study aimed to identify potential heavy metal chelators in S. luteus. Two metallothionein (MT) coding genes, SlMTa and SlMTb, were identified. When heterologously expressed in yeast, both SlMTa and SlMTb can rescue the Cu sensitive mutant from Cu toxicity. In S. luteus, transcription of both SlMTa and SlMTb is induced by Cu but not Cd or Zn. Several putative Cu-sensing and metal-response elements are present in the promoter sequences. These results indicate that SlMTa and SlMTb function as Cu-thioneins. Homologs of the S. luteus MTs are present in 49 species belonging to 10 different orders of the subphylum Agaricomycotina and are remarkably conserved. The length of the proteins, number and distribution of cysteine residues indicate a novel family of fungal MTs. The ubiquitous and highly conserved features of these MTs suggest that they are important for basic cellular functions in species in the subphylum Agaricomycotina.

Placental mitochondrial DNA and CYP1A1 gene methylation as molecular signatures for tobacco smoke exposure in pregnant women and the relevance for birth weight.

BACKGROUND: Maternal smoking during pregnancy results in an increased risk of low birth weight through perturbations in the utero-placental exchange. Epigenetics and mitochondrial function in fetal tissues might be molecular signatures responsive to in utero tobacco smoke exposure. METHODS: In the framework of the ENVIRONAGE birth cohort, we investigated the effect of self-reported tobacco smoke exposure during pregnancy on birth weight and the relation with placental tissue markers such as, (1) relative mitochondrial DNA (mtDNA) content as determined by real-time quantitative PCR, (2) DNA methylation of specific loci of mtDNA (D-loop and MT-RNR1), and (3) DNA methylation of the biotransformation gene CYP1A1 (the last two determined by bisulfite-pyrosequencing). The total pregnant mother sample included 255 non-smokers, 65 former-smokers who had quit smoking before pregnancy, and 62 smokers who continued smoking during pregnancy. RESULTS: Smokers delivered newborns with a birth weight on average 208 g lower [95% confidence interval (CI) -318 to -99, p = 0.0002] than mothers who did not smoke during pregnancy. In the smoker group, the relative mtDNA content was lower (-21.6%, 95% CI -35.4 to -4.9%, p = 0.01) than in the non-smoker group; whereas, absolute mtDNA methylation levels of MT-RNR1 were higher (+0.62%, 95% CI 0.21 to 1.02%, p = 0.003). Lower CpG-specific methylation of CYP1A1 in placental tissue (-4.57%, 95% CI -7.15 to -1.98%, p < 0.0001) were observed in smokers compared with non-smokers. Nevertheless, no mediation of CYP1A1 methylation nor any other investigated molecular signature was observed for the association between tobacco smoke exposure and birth weight. CONCLUSIONS: mtDNA content, methylation of specific loci of mtDNA, and CYP1A1 methylation in placental tissue may serve as molecular signatures for the association between gestational tobacco smoke exposure and low birth weight.

Cd-induced Cu deficiency responses in Arabidopsis thaliana: are phytochelatins involved?

Cadmium (Cd) exposure can disturb the homeostasis of essential elements. In Arabidopsis thaliana, Cd induces a squamosa promoter binding protein-like 7 (SPL7)-dependent Cu deficiency response. We investigated how Cd induces a Cu deficiency response. The Cu deficiency response consists of the active SPL7 transcription factor binding to GTAC motifs in promoters of among others several Cu transporters, a Cu chaperone, and cupro-miRNAs to regulate Cu homeostasis. We demonstrated that the addition of supplemental Cu to Cd-exposed A. thaliana plants diminished the Cu deficiency response in roots, while it even disappeared in leaves. Exposure of plants to Cd in combination with extra Cu reduced Cd levels in both roots and leaves resulting in an improved cellular oxidative state. Furthermore, we demonstrated a role for phytochelatins (PCs) in the Cd-induced Cu deficiency response, because it was reduced in roots of cad1-3 mutant plants exposed to Cd. In conclusion, a working mechanism is provided in which it is suggested that Cd increases PC levels that can complex both Cd and Cu. This results in cellular Cu deficiency and subsequently the activation of SPL7 and hence the induction of the Cu deficiency response.