Ethylene mediates brassinosteroid‐induced stomatal closure via Gα protein‐activated hydrogen peroxide and nitric oxide production in Arabidopsis

Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24‐epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Gα protein, and hydrogen peroxide (H₂O₂) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H₂O₂ and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1‐301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Gα mediate the inductive effects of EBR on H₂O₂ and NO production. EBR‐triggered H₂O₂ and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1‐1 mutant and the cGα line (constitutively overexpressing the G protein α‐subunit AtGPA1). Exogenously applied H₂O₂ or sodium nitroprusside (SNP) rescued the defects of etr1‐3 and gpa1 or etr1 and gpa1 mutants in EBR‐induced stomatal closure, whereas the stomata of eto1‐1/AtrbohF and cGα/AtrbohF or eto1‐1/nia1‐2 and cGα/nia1‐2 constructs had an analogous response to H₂O₂ or SNP as those of AtrbohF or Nia1‐2 mutants. Moreover, we provided evidence that Gα plays an important role in the responses of guard cells to ethylene. Gα activator CTX largely restored the lesion of the etr1‐3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR‐induced stomatal closure. Lastly, we demonstrated that Gα‐activated H₂O₂ production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H₂O₂ production in mutant Nia1‐2. Exogenously applied SNP rescued the defect of AtrbohF in EBR‐induced stomatal closure, but H₂O₂ did not reverse the lesion of EBR‐induced stomatal closure in Nia1‐2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Gα, and then promotes AtrbohF‐dependent H₂O₂ production and subsequent Nia1‐catalyzed NO accumulation, and finally closes stomata.     

An eukaryotic translation initiation factor,AteIF5A‐2, affects cadmium accumulation and sensitivity in Arabidopsis

Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild‐type Columbia‐0 (Col‐0) with a knockdown mutant of AteIF5A‐2, fbr12‐3 under Cd stress conditions. The results showed that the mutant fbr12‐3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A‐2 makes the mutant more Cd sensitive. Real‐time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12‐3 compared with Col‐0. As a result, an increase in MDA and H₂O₂ content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.     

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl₂. The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants.     

Antioxidant enzyme activities and hormonal status in response to Cd stress in the wetland halophyte Kosteletzkya virginica under saline conditions

Salt marshes constitute major sinks for heavy metal accumulation but the precise impact of salinity on heavy metal toxicity for halophyte plant species remains largely unknown. Young seedlings of Kosteletzkya virginica were exposed during 3 weeks in nutrient solution to Cd 5 µM in the presence or absence of 50 mM NaCl. Cadmium (Cd) reduced growth and shoot water content and had major detrimental effect on maximum quantum efficiency (F_v/F_m), effective quantum yield of photosystem II (Y(II)) and electron transport rates (ETRs). Cd induced an oxidative stress in relation to an increase in O₂^?? and H₂O₂ concentration and lead to a decrease in endogenous glutathione (GSH) and α‐tocopherol in the leaves. Cd not only increased leaf zeatin and zeatin riboside concentration but also increased the senescing compounds 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and abscisic acid (ABA). Salinity reduced Cd accumulation already after 1 week of stress but was unable to restore shoot growth and thus did not induce any dilution effect. Salinity delayed the Cd‐induced leaf senescence: NaCl reduced the deleterious impact of Cd on photosynthesis apparatus through an improvement of F_v/F_m, Y(II) and ETR. Salt reduced oxidative stress in Cd‐treated plants through an increase in GSH, α‐tocopherol and ascorbic acid synthesis and an increase in glutathione reductase (EC 1.6.4.2) activity. Additional salt reduced ACC and ABA accumulation in Cd+NaCl‐treated leaves comparing to Cd alone. It is concluded that salinity affords efficient protection against Cd to the halophyte species K. virginica, in relation to an improved management of oxidative stress and hormonal status.     

Enhancement of cadmium accumulation in sweet sorghum as affected by nitrate

• The Cadmium (Cd)‐polluted soils are is an increasing concern worldwide. Phytoextraction of Cd pollutants by high biomass plants, such as sweet sorghum, is considered an environmentally‐friendly, cost‐effective and sustainable strategy for remediating this problem. Nitrogen (N) is a macronutrient essential for plant growth, development and stress resistance. Nevertheless, how nitrate, as an important form of N, affects Cd uptake, translocation and accumulation in sweet sorghum is still unclear.
• In the present study, a series of nitrate levels (N1, 0.5 mm ; N2, 2 mm ; N3, 4 mm ; N4, 8 mm and N5, 16 mm ) with or without added 5 μm CdCl₂ treatment in sweet sorghum was investigated hydroponically.
• The results indicate that Cd accumulation in the aboveground parts of sweet sorghum was enhanced by optimum nitrate supply, resulting from both increased dry weight and Cd concentration. Although root‐to‐shoot Cd translocation was not enhanced by increased nitrate, some Cd was transferred from cell walls to vacuoles in leaves. Intriguingly, expression levels of Cd uptake and transport genes, SbNramp1, SbNramp5 and SbHMA3, were not closely related to increased Cd as affected by nitrate supply. The expression of SbNRT1.1B in relation to nitrate transport showed an inverted ‘U’ shape with increasing nitrate levels under Cd stress, which was in agreement with trends in Cd concentration changes in aboveground tissues.
• Based on the aforementioned results, nitrate might regulate Cd uptake and accumulation through expression of SbNRT1.1B rather than SbNramp1, SbNramp5 or SbHMA3, the well‐documented genes related to Cd uptake and transport in sweet sorghum.

     

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl₂ over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

Salt affects plant Cd-stress responses by modulating growth and Cd accumulation

Cadmium contamination is a serious environmental problem for modern agriculture and human health. Salinity affects plant growth and development, and interactions between salt and cadmium have been reported. However, the molecular mechanisms of salinity–cadmium interactions are not fully understood. Here, we show that a low concentration of salt alleviates Cd-induced growth inhibition and increases Cd accumulation in Arabidopsis thaliana. Supplementation with low concentrations of salt reduced the reactive oxygen species level in Cd-stressed roots by increasing the contents of proline and glutathione and down-regulating the expression of RCD1, thereby protecting the plasma membrane integrity of roots under cadmium stress. Salt supplementation substantially reduces the Cd-induced elevation of IAA oxidase activity, thereby maintaining auxin levels in Cd-stressed plants, as indicated by DR5::GUS expression. Salt supply increased Cd absorption in roots and increased Cd accumulation in leaves, implying that salt enhances both Cd uptake in roots and the root-to-shoot translocation of Cd. The elevated Cd accumulation in plants in response to salt was found to be correlated with the elevated levels of phytochelatin the expression of heavy metal transporters AtHMA1-4, especially AtHMA4. Salt alleviated growth inhibition caused by Cd and increased Cd accumulation also was observed in Cd accumulator Solanum nigrum.     

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild‐type and leaf vein mutants

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (K_leaf), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col‐0) and four vein mutants (dot3‐111 and dot3‐134, and cvp1‐3 and cvp2‐1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean K_leaf. Across all genotypes, there was a strong correlation of K_leaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence K_leaf, indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing K_leaf.     

Growth responses of <Emphasis Type="Italic">Sagittaria sagittifolia</Emphasis> L. plants to water contamination with cadmium

Cadmium accumulation, the relative content of different chemical forms of Cd, as well as the toxic effect of Cd on nutrient element uptake, physiological parameters, and ultrastructure of Sagittaria sagittifolia L. seedlings were determined after the seedlings were exposed to different Cd concentrations for 4 days. The results showed that S. sagittifolia had the ability to accumulate large amounts of Cd. In the root, stem, and bulb, the predominant chemical Cd forms were NaCl extractable. With an increase in the Cd²⁺ concentration, the chlorophyll content, the relative membrane penetrability (RMP) of root cells, peroxidase (POD) activity, superoxide dismutase (SOD) activity in leaves, malondiadehyde (MDA) content and the superoxide anion (O₂⁻) generation rate in roots all decreased following an initial increase. On the other hand, catalase (CAT) activity, SOD activity in roots, MDA content, and the generation rate of O₂⁻ in leaves all increased gradually. The toxic effect of Cd²⁺ was more severe on roots than on leaves at the same concentration. Cadmium affected the mineral nutrition balance; mainly, it promoted the uptake of Ca, Cu, Mn, and Fe, while inhibited Mg, Na, and K uptake. The physiological toxic effect of Cd²⁺ was close to the ultrastructural damage induced by Cd contamination. A significant correspondence was observed between the Cd dose and its toxic effect. Cadmium could destroy the normal ultrastructure, disturb the ion balance, and interfere with cell metabolism.     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Effect of Cd on growth, photosynthetic gas exchange, and chlorophyll fluorescence of wild and Cd-sensitive mutant rice

Growth, photosynthetic gas exchange, and chlorophyll fluorescence characteristics were investigated in wild type (WT) and Cd-sensitive mutant rice (Oryza sativa L.) plants using 50 μM Cd treatment for 12 d followed by a 3-d recovery. Under Cd stress, net dry mass and pigment contents were significantly lower in the mutant plants than in the WT. The mutant had lower net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) than WT rice, however, it had higher intercellular CO₂ concentration (C _i), indicating that non-stomatal factors accounted for the inhibition of P _N. Maximal photochemical efficiency of photosystem 2 (F_v/F_m), effective quantum yield of PS2 (Φ_PS2), and photochemical quenching (q_P) decreased much in the mutant under Cd stress. Cd content in roots and leaves of the mutant was significantly higher than those in the WT. Hence Cd toxicity was associated with the marked increases in Cd contents of plant tissue. After the recovery for 3 d, the WT rice had higher capacity to recover from Cd injury than the mutant.     

The Nicotiana attenuata GLA1 lipase controls the accumulation of Phytophthora parasitica‐induced oxylipins and defensive secondary metabolites

Nicotiana attenuata plants silenced in the expression of GLYCEROLIPASE A1 (ir‐gla1 plants) are compromised in the herbivore‐ and wound‐induced accumulation of jasmonic acid (JA). However, these plants accumulate wild‐type (WT) levels of JA and divinyl‐ethers during Phytophthora parasitica infection. By profiling oxylipin‐enriched fractions with targeted and untargeted liquid chromatography‐tandem time‐of‐flight mass spectrometry approaches, we demonstrate that the accumulation of 9‐hydroxy‐10E,12Z‐octadecadienoic acid (9‐OH‐18:2) and additional C₁₈ and C₁₉ oxylipins is reduced by ca. 20‐fold in P. parasitica‐infected ir‐gla1 leaves compared with WT. This reduced accumulation of oxylipins was accompanied by a reduced accumulation of unsaturated free fatty acids and specific lysolipid species. Untargeted metabolic profiling of total leaf extracts showed that 87 metabolites accumulated differentially in leaves of P. parasitica‐infected ir‐gla1 plants with glycerolipids, hydroxylated‐diterpene glycosides and phenylpropanoid derivatives accounting together for ca. 20% of these 87 metabolites. Thus, P. parasitica‐induced oxylipins may participate in the regulation of metabolic changes during infection. Together, the results demonstrate that GLA1 plays a distinct role in the production of oxylipins during biotic stress responses, supplying substrates for 9‐OH‐18:2 and additional C₁₈ and C₁₉ oxylipin formation during P. parasitica infection, whereas supplying substrates for the biogenesis of JA during herbivory and mechanical wounding.     

Cadmium-induced oxidative damage and antioxidative defense mechanisms in <Emphasis Type="Italic">Vigna mungo</Emphasis> L.

Cadmium (Cd)-induced oxidative stress and antioxidant defense mechanisms were analyzed in roots and leaves of Vigna mungo L. Seeds were germinated in perlite-vermiculite and irrigated with Hoagland nutrient solution. At day 6, seedlings were exposed to 40 μM Cd under semi-hydroponic conditions for a period of 12 days. Growth anomalies and abnormal chromatin condensation were observed in Cd-treated plants, in comparison with control ones. Cd accumulation was observed in roots of treated plants. The analyses of antioxidative defense and oxidative parameters in roots, stems and leaves showed different tissue-specific responses. Superoxide dismutase (SOD) and guaiacol peroxidase (GPx) activities and the level of lipid peroxidation (MDA content) decreased in roots. However, they increased in leaves. Catalase activity and chlorophyll content, on the other hand, decreased over exposure to Cd stress. Total glutathione, non-protein thiols, reduced glutathione (GSH) and phytochelatins increased significantly, while oxidized glutathione (GSSG) decreased, as compared with control plants. The present data suggest that the presence of Cd in soil and water can cause oxidative damage that may be detrimental for optimum production of nutritional mung.     

Mutants impaired in vacuolar metal mobilization identify chloroplasts as a target for cadmium hypersensitivity in Arabidopsis thaliana

Cadmium (Cd) is highly toxic to plants causing growth reduction and chlorosis. It binds thiols and competes with essential transition metals. It affects major biochemical processes such as photosynthesis and the redox balance, but the connection between cadmium effects at the biochemical level and its deleterious effect on growth has seldom been established. In this study, two Cd hypersensitive mutants, cad1‐3 impaired in phytochelatin synthase (PCS1), and nramp3nramp4 impaired in release of vacuolar metal stores, have been compared. The analysis combines genetics with measurements of photosynthetic and antioxidant functions. Loss of AtNRAMP3 and AtNRAMP4 function or of PCS1 function leads to comparable Cd sensitivity. Root Cd hypersensitivities conferred by cad1‐3 and nramp3nramp4 are cumulative. The two mutants contrast in their tolerance to oxidative stress. In nramp3nramp4, the photosynthetic apparatus is severely affected by Cd, whereas it is much less affected in cad1‐3. In agreement with chloroplast being a prime target for Cd toxicity in nramp3nramp4, the Cd hypersensitivity of this mutant is alleviated in the dark. The Cd hypersensitivity of nramp3nramp4 mutant highlights the critical role of vacuolar metal stores to supply essential metals to plastids and maintain photosynthetic function under Cd and oxidative stresses.