Responses of Brassica napus to soil cadmium under elevated CO2 concentration based on rhizosphere microbiome, root transcriptome and metabolome.

Rising atmospheric carbon dioxide (CO2) and soil heavy metal pollution affect crop safety and production. Exposure to elevated CO2 (ECO2) increases cadmium (Cd) uptake in some crops like wheat and rice, however, it remains unclear how ECO2 affects Cd uptake by Brassica napus. Here, we investigated the responses of B. napus seedlings exposed to ECO2 and Cd through analyses of physiology, transcriptome, metabolome, and rhizosphere microbes. Compared with Cd-stress alone (Cd50_ACO2), ECO2 boosted the uptake of Cd by B. napus roots by 38.78% under coupled stresses (Cd50_ECO2). The biomass and leaf chlorophyll a content increased by 38.49% and 79.66% respectively in Cd50_ECO2 relative to Cd50_ACO2. Activities of superoxide dismutase (SOD) and peroxidase (POD) enhanced by 8.42% and 185.01%, respectively, while glutathione (GSH) and ascorbic acid (AsA) contents increased by 16.44% and 52.48%, and abundances of rhizosphere microbes changed significantly under coupled stresses (Cd50_ECO2) relative to Cd-stress alone (Cd50_ACO2). Also, the upregulation of glutathione, glutathione transferase genes, and heavy metal ATPase expression promoted the detoxification effect of rapeseed on Cd. Changes in the expression of transcription factors like MAPK, WRKY, BAK1 and PR1, as well as changes in metabolic pathways like ß-alanine, may be involved in the regulatory mechanism of stress response. These findings provide new insights for studying the regulatory mechanism of rapeseed under ECO2 on soil Cd stress, and also provide a basis for further research on Cd tolerant rapeseed varieties in the future climate context.

The duality of sulfate-reducing bacteria: Reducing methylmercury production in rhizosphere but enhancing accumulation in rice plants.

Sulfate-reducing bacteria (SRB) are known to alter methylmercury (MeHg) production in paddy soil, but the effect of SRB on MeHg dynamics in rhizosphere and rice plants remains to be fully elucidated. The present study investigated the impact of SRB on MeHg levels in unsterilized and γ-sterilized mercury-polluted paddy soils, with the aim to close this knowledge gap. Results showed that the presence of SRB reduced MeHg production by ∼22 % and ∼17 % in the two soils, but elevated MeHg contents by approximately 55 % and 99 % in rice grains, respectively. Similar trend at smaller scales were seen in roots and shoots. SRB inoculation exerted the most profound impact on amino acid metabolism in roots, with the relative response of L-arginine positively linking to MeHg concentrations in rhizosphere. The SRB-induced enrichment of MeHg in rice plants may be interpreted by the stronger presence of endophytic nitrogen-related microbes (e.g. Methylocaldum, Hyphomicrobium and Methylocystis) and TGA transcription factors interacting with glutathione metabolism and calmodulin. Our study provides valuable insights into the complex effects of SRB inoculation on MeHg dynamics in rice ecosystems, and may help to develop strategies to effectively control MeHg accumulation in rice grains.

Physiological responses of low- and high-cadmium accumulating Robinia pseudoacacia-rhizobium symbioses to cadmium stress.

The role of rhizobia in alleviating cadmium (Cd) stress in woody legumes is still unclear. Therefore, two types of black locust (Robinia pseudoacacia L.) with high and low Cd accumulation abilities were selected from 11 genotypes in China, and the effects of rhizobium (Mesorhizobium huakuii GP1T11) inoculation on the growth, CO2 and H2O gas exchange parameters, Cd accumulation, and the absorption of mineral elements of the high (SX) and low Cd-accumulator (HB) were compared. The results showed that rhizobium-inoculation significantly increased biomass, shoot Cd contents, Cd accumulation, root-to-shoot translocation factor (TF) and the absorption and accumulation of mineral elements in both SX and HB. Rhizobium-inoculation increased chlorophyll a and carotenoid contents, and the intercellular carbon dioxide concentrations in HB plants. Under Cd exposure, the high-accumulator SX exhibited a significant decrease in photosynthetic CO2 fixation (Pn) and an enhanced accumulation of Cd in leaves, but coped with Cd exposure by increasing chlorophyll synthesis, regulating stomatal aperture (Gs), controlling transpiration (Tr), and increasing the absorption and accumulation of mineral elements. In contrast, the low-accumulator HB was more sensitive to Cd exposure despite preferential accumulation of Cd in roots, with decreased chlorophyll and carotenoid contents, but significantly increased root biomass. Compared to the low-accumulator HB, non-inoculated Cd-exposed SX plants had higher chlorophyll contents, and rhizobium-inoculated Cd-exposed SX plants had higher Pn, Tr, and Gs as well as higher levels of P, K, Fe, Ca, Zn, and Cu. In conclusion, the high- and low-Cd-accumulator exhibited different physiological responses to Cd exposure. Overall, rhizobium-inoculation of black locust promoted the growth and heavy metal absorption, providing an effective strategy for the phytoremediation of heavy metal-contaminated soils by this woody legume.

Allelopathic effects of Eucalyptus extract and wood vinegar on germination and sprouting of rapeseed (Brassica rapa L.).

Eucalyptus plantations are usually characterized by low biodiversity due to allelopathy effects. Wood vinegar is considered a complex growth regulator that can promote plant growth at low concentrations. However, there is information scarcity about the co-application of eucalypt leaf water extract and wood vinegar on plants. This study aimed at clarifying whether wood vinegar can protect seed germination against suppression by eucalypt-induced allelopathy. We examined germination behavior and seedling elongation characteristics in rapeseed (Brassica rapa L.) treated with different solutions of wood vinegar and eucalypt leaf water extract. The results showed that eucalypt leaf water extracts, wood vinegar solutions, and their mixture allelopathically suppressed seed germination rate. After rapeseed sprouting, eucalypt leaf water extracts promoted root elongation, stem elongation, and fresh weight elongation. Malondialdehyde content was also lower under the influence of eucalypt leaf water extract. Mixture of high concentration of eucalypt leaf water extract and lower concentration of wood vinegar significantly promoted root elongation. Therefore, both eucalypt leaf water extract and wood vinegar are complex plant growth regulators, which can be used to inhibit or stimulate plants at different ontogenic stages. During the seed germination period, both eucalypt leaf extracts and wood vinegar could be used as weed inhibitors. Conversely, during the period of sprouting (seedling establishment), low concentrations of eucalypt leaf extracts and wood vinegar can promote growth.

Biochar application improves karstic lime soil physicochemical properties and enzymes activity and enhances sweet tea seedlings physiological performance.

Karst lime soil, commonly found in rocky desert ecosystems of Southwest China, exhibits high pH, poor water retention, and intense erosion. To prevent further soil erosion and soil losses from these ecosystems, stabilization measures based on improved green infrastructure are needed. The present study aimed at elucidating the performance of sweet tea (Lithocarpus polystachyus) seedlings grown on this soil type upon biochar application. Biochar was classified into different particle sizes, viz. 0.25-0.5 mm (medium), 0.5-1 mm (coarse), 1-2 mm (gravel), and their mixture, and added at the concentrations of 1, 2, or 5% soil mass. The pH, moisture, and porosity of soil increased upon biochar application compared to control; however, soil bulk density significantly decreased. The activity of soil phosphatase was increased by biochar particle size. Biochar particle size and concentration significantly enhanced the soil organic carbon content, but they differently affected total and plant-available nutrients in the soil. Light-saturated photosynthesis was positively affected, while stomatal conductance, leaf transpiration, and the intercellular CO2 concentrations of sweet tea leaves were negatively affected by biochar particle size and/or concentration compared to control. Leaf chlorophyll and soluble protein contents were increased by biochar application. From these results, we conclude that biochar can improve soil properties and the performance of sweet tea seedlings grown on Karst lime soil. We suggest its application at a concentration of 2% soil mass for keeping a high physiological performance of sweet tea seedlings in this environment. The selection of the ideal particle size is context-specific and depends on the target outcome.

Differences in aluminum tolerance and immobilization between two indigenous ectomycorrhizal fungi Lactarius deliciosus and Pisolithus tinctorius from Southwest China's forest stands.

Aluminum (Al) toxicity severely decreases plant growth and productivity in acidic soil globally. Ectomycorrhizal (ECM) fungi can promote host plant's Al-tolerance by acting as a physical barrier or bio-filter. However, little information is available on the role of ECM fungus on Al immobilization with respect to Al-tolerance. This present study aimed to screen a promising indigenous ECM fungus with high Al-tolerance and to understand its role in Al immobilization related to Al-tolerance. Two ECM fungal strains (Lactarius deliciosus 2 and Pisolithus tinctorius 715) isolated from forest stands in Southwest China were cultured in vitro with 0.0, 1.0 or 2.0 mM Al addition for 21 days to compare their Al accumulation and Al-tolerance. Meanwhile, fungal mycelia were incubated in 0.037 mM Al3+ solutions, and then Al3+ concentrations in the solution were determined at time 2, 5, 10, 20, 40, 60, 120, 180, and 240 min, and the Al3+ immobilization characteristics were evaluated using the pseudo-first order, pseudo-second order and intraparticle diffusion models. Results showed that 1.0 or 2.0 mM Al3+ addition significantly increased fungal biomass production by 23% or 41% in L. deliciosus 2, not in P. tinctorius 715. Fungal Al3+ concentrations in L. deliciosus 2 and P. tinctorius 715 were significantly increased by 293% and 103% under 2.0 mM than under 1.0 mM Al3+ addition. The pH values in the culture solution were significantly decreased by 0.43 after 21 d fungus growth but no changes between these two fungi under the same Al3+ addition. Fungal Al3+ immobilization showed a three-stage trend with initially a rapid rate followed a relatively slower rate until reaching equilibrium. The pseudo-second order model was the best (R2 = 0.98 and 0.99 for L. deliciosus 2 and P. tinctorius 715) to fit the experimentally observed data among the three models. Compared to P. tinctorius 715, L. deliciosus 2 also had greater intercept value, cation exchange capacity (CEC), and extracellular Al3+ proportion in fungal mycelia. Additionally, bio-concentration on Al3+, active site numbers for Al3+, boundary layer thickness, CEC, and immobilization on the cell wall in fungal mycelia were involved in ECM fungal Al-tolerance. These results show that both ECM fungi are Al-tolerant while L. deliciosus 2 is a promising indigenous ECM isolate with higher Al-tolerance in Southwest China, and they can be hence applied to the afforestation and ecological restoration in acidic soil.

Elevated CO2 and soil mercury stress affect photosynthetic characteristics and mercury accumulation of rice.

This is a novel study about responses of leaf photosynthetic traits and plant mercury (Hg) accumulation of rice grown in Hg polluted soils to elevated CO2 (ECO2). The aim of this study was to provide basic information on the acclimation capacity of photosynthesis and Hg accumulation in rice grown in Hg polluted soil under ECO2 at day, night, and full day. For this purpose, we analyzed leaf photosynthetic traits of rice at flowering and grain filling. In addition, chlorophyll content, soluble sugar and Malondialdehyde (MDA) of rice leaves were measured at flowering. Seed yield, ear number, grain number per ear, 1000-grain weight, total mercury (THg) and methylmercury (MeHg) contents were determined after harvest. Our results showed that Hg polluted soil and ECO2 had no significant effect on leaf chlorophyll content and leaf mass per area (LMA) in rice. The contents of soluble sugar and MDA in leaves increased significantly under ECO2. Mercury polluted soil treatment significantly reduced the light saturated CO2 assimilation rate (Asat) of rice leaves only at flowering, but not at grain filling. Night ECO2 greatly improved rice leaf water use efficiency (WUE). ECO2 greatly increased seed yield and ear number. In addition, ECO2 did not affect THg accumulation in rice organs, but ECO2 and Hg treatment had a significant interaction on MeHg in seeds, husks and roots.

Ethylenediurea (EDU) effects on Japanese larch: an one growing season experiment with simulated regenerating communities and a four growing season application to individual saplings.

Japanese larch (Larix kaempferi (Lamb.) Carr.) and its hybrid are economically important coniferous trees widely grown in the Northern Hemisphere. Ground-level ozone (O3) concentrations have increased since the pre-industrial era, and research projects showed that Japanese larch is susceptible to elevated O3 exposures. Therefore, methodologies are needed to (1) protect Japanese larch against O3 damage and (2) conduct biomonitoring of O3 in Japanese larch forests and, thus, monitor O3 risks to Japanese larch. For the first time, this study evaluates whether the synthetic chemical ethylenediurea (EDU) can protect Japanese larch against O3 damage, in two independent experiments. In the first experiment, seedling communities, simulating natural regeneration, were treated with EDU (0, 100, 200, and 400 mg L-1) and exposed to either ambient or elevated O3 in a growing season. In the second experiment, individually-grown saplings were treated with EDU (0, 200 and 400 mg L-1) and exposed to ambient O3 in two growing seasons and to elevated O3 in the succeeding two growing seasons. The two experiments revealed that EDU concentrations of 200-400 mg L-1 could protect Japanese larch seedling communities and individual saplings against O3-induced inhibition of growth and productivity. However, EDU concentrations ≤ 200 mg L-1 did offer only partial protection when seedling communities were coping with higher level of O3-induced stress, and only 400 mg EDU L-1 fully protected communities under higher stress. Therefore, we conclude that among the concentrations tested the concentration offering maximum protection to Japanese larch plants under high competition and O3-induced stress is that of 400 mg EDU L-1. The results of this study can provide a valuable resource of information for applied forestry in an O3-polluted world.

Effects of simulated nitrogen deposition on ectomycorrhizae community structure in hybrid larch and its parents grown in volcanic ash soil: The role of phosphorous.

With the rapid industrial development and modern agricultural practices, increasing nitrogen (N) deposition can cause nutrient imbalance in immature volcanic ash soil commonly found in Japan. Larch species, widely distributed in northeast Eurasia, are associated with ectomycorrhizal (ECM) fungi which play a critical role in nutrient acquisition for their hosts. In this study, we investigated species richness and diversity of ECM fungi associated with a hybrid larch (F1) and its parents, Dahurian larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi), under simulated N deposition (0 and 100kgha-1yr-1) with/without phosphorous (P) (0 and 50kgha-1yr-1). Seedlings planted in immature volcanic ash with low nutrient availability were subjected to the N and P treatments for fifteen months. We found that response of ECM community structure to the increased nutrient availability depended on host genotypes. Nutrient addition significantly affected ECM structure in Japanese larch, but no such significant effect was found for Dahurian larch. Effects of the nutrient addition to ECM fungal community in F1 were intermediate. F1 was tolerant to high N loading, which was due to consistent, relatively high association with Suillus sp. and Hebeloma sp. F1 showed heterosis in relative biomass, which was most apparent under high N treatments. This co-variation of ECM fungal community structure and F1 biomass in response to N loading suggest that ECM community structure might play an important role in host growth. The present findings indicate effects of N deposition on ECM fungal community structure can depend on larch species, thus it is challenging to predict general trends.

[Effects of Different Altitudes on Soil Microbial PLFA and Enzyme Activity in Two Kinds of Forests].

The soil microbial community is an important part in soil ecosystem, and it is sensitive to the ecological environment. Phospholipid-derived fatty acids ( PLFA ) analysis was used to examine variations in soil microbial community diversity and its influencing factors. The results showed that: there existed 48 PLFAs that were significant in the soil samples from six altitudes. The PLFAs of six altitudes with the highest contents were i16:0, 10Me17:0, 10Me18:0 TBSA. The citrus forest exhibited richer soil PLFAs distribution both in type and amount than those in masson pine. The microbial activity and functional diversity of masson pine were increased with increasing altitudes, and citrus forest gradually decreased, the PLFA content of different microbial groups in each altitude were significantly different. The richness index, Shannon-Wiener index and Pielou evenness index of masson pine in low elevation were holistically higher than those in high elevation. However, the highest richness index of citrus forest was in low altitude, the highest Shannon-Wiener index and Pielou evenness index were in high altitude. The PLFAs content of different microbial groups were closely correlated to the soil enzyme activities and environmental factors. The PLFAs of bacteria, actinomycetes, G⁻ (Gram- positive), G⁺ (Gram-negative) were positively correlated with Ure(urease) , Ive(invertase) , CAT( catalase activity) and forest type, the PLFAs of fungi was significantly correlated with Ure, Ive, CAT, the PLFAs of bacteria, fungi, actinomycetes, G⁻ , G⁺ were significantly negatively or less correlated with elevation. Ure, Ive, CAT, forest type and elevation are the pivotal factors controlling the soil microbial biomass and activities.

Ectomycorrhizal colonization and growth of the hybrid larch F1 under elevated CO2 and O3.

We studied the colonization of ectomycorrhizal fungi and species abundance of a hybrid larch (F1) under elevated CO2 and O3. Two-year-old seedlings were planted in an Open-Top-Chamber system with treatments: Control (O3 < 6 nmol/mol), O3 (60 nmol/mol), CO2 (600 µmol/mol), and CO2 + O3. After two growing seasons, ectomycorrhiza (ECM) colonization and root biomass increased under elevated CO2. Additionally, O3 impaired ECM colonization and species richness, and reduced stem biomass. However, there was no clear inhibition of photosynthetic capacity by O3. Concentrations of Al, Fe, Mo, and P in needles were reduced by O3, while K and Mg in the roots increased. This might explain the distinct change in ECM colonization rate and diversity. No effects of combined fumigation were observed in any parameters except the P concentration in needles. The tolerance of F1 to O3 might potentially be related to a shift in ECM community structure.

Photosynthetic response of early and late leaves of white birch (Betula platyphylla var. japonica) grown under free-air ozone exposure.

Betula platyphylla var. japonica (white birch) has heterophyllous leaves (i.e., early and late leaves) and is a typical pioneer tree species in northern Japan. Seedlings of white birch were exposed to ozone during two growing seasons, and measurements were carried out in the second year. Early leaves did not show an ozone-induced reduction in photosynthesis because of lower stomatal conductance resulting in higher avoidance capacity for ozone-induced stress. Also, an ozone-related increase in leaf nitrogen content may partly contribute to maintain the photosynthetic capacity in early leaves under elevated ozone in autumn. On the other hand, late leaves showed an ozone-induced decline of photosynthesis and early defoliation of leaves occurred. Also, smaller leaf size and higher stomatal density in late leaves were observed under elevated ozone. Differences in stress resistance to ozone may be related to differing functional roles of early and late leaves for birch species.

Photosynthetic traits of Siebold's beech and oak saplings grown under free air ozone exposure in northern Japan.

We set up a free-air ozone (O(3)) exposure system for determining the photosynthetic responses of Siebold's beech (Fagus crenata) and oak (Quercus mongolica var. crispula) to O(3) under field conditions. Ten-year-old saplings of beech and oak were exposed to an elevated O(3) concentration (60 nmol mol(-1)) during daytime from 6 August to 11 November 2011. Ozone significantly reduced the net photosynthetic rate in leaves of both species in October, by 46% for beech and 15% for oak. In beech there were significant decreases in maximum rate of carboxylation, maximum rate of electron transport in photosynthesis, nitrogen content and photosynthetic nitrogen use efficiency, but not in oak. Stomatal limitation of photosynthesis was unaffected by O(3). We therefore concluded photosynthesis in beech is more sensitive to O(3) than that in oak, and the O(3)-induced reduction of photosynthetic activity in beech was due not to stomatal closure, but to biochemical limitation.