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Forest soil microbes play a crucial role in regulating atmospheric-soil carbon fluxes. Environmental heterogeneity across forest types and regions may lead to differences in soil CO 2 and CH 4 emissions. However, the microbial mechanisms underlying these emission variations are currently unclear. In this study, we measured CO 2 and CH 4 emissions of Robinia pseudoacacia forests along a north-south transect in the Loess Plateau. Using metagenomic sequencing, we investigated the structural and functional profiles of soil carbon cycling microbial communities. Results indicated that the forest CO 2 emissions of Robinia pseudoacacia was significantly higher in the north region than in the south region, while the CH 4 emission was oppositely. This is mainly attributed to changes in gene abundance driven by soil pH and moisture in participating carbon degradation and methane oxidation processes across different forest regions. The gene differences in carbon fixation processes between regions primarily stem from the Calvin cycle, where the abundance of rbcL, rbcS, and prkB genes dominates microbial carbon fixation in forest soils. Random forest models revealed key genes involved in predicting forest soil CO 2 emissions, including SGA1 and amyA for starch decomposition, TYR for lignin decomposition, chitinase for chitin decomposition, and pectinesterase for pectin decomposition. Microbial functional characterization revealed that interregional differences in CH 4 emissions during methane metabolism may originate from methane oxidation processes, and the associated gene abundances (glyA, ppc, and pmoB) were key genes for predicting CH 4 emissions from forest soils. Our results provide new insights into the microbial mechanisms of CO 2 and CH 4 emissions from forest soils, which will be crucial for accurate prediction of the forest soil carbon cycle in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Both sulfur (S) supply and legume-rhizobium symbiosis can significantly contribute to enhancing the efficiency of phytoremediation of heavy metals (HMs). However, the regulatory mechanism determining the performance of legumes at lead (Pb) exposure have not been elucidated. Here, we cultivated black locust (Robinia pseudoacacia L.), a leguminous woody pioneer species at three S supply levels (i.e., deficient, moderate, and high S) with rhizobia inoculation and investigated the interaction of these treatments upon Pb exposure. Our results revealed that the root system of Robinia has a strong Pb accumulation and anti-oxidative capacity that protect the leaves from Pb toxicity. Compared with moderate S supply, high S supply significantly increased Pb accumulation in roots by promoting the synthesis of reduced S compounds (i.e., thiols, phytochelatin), and also strengthened the antioxidant system in leaves. Weakened defense at deficient S supply was indicated by enhanced oxidative damage. Rhizobia inoculation alleviated the oxidative damage of its Robinia host by immobilizing Pb to reduce its absorption by root cells. Together with enhanced Pb chelation in leaves, these mechanisms strengthen Pb detoxification in the Robinia-rhizobia symbiosis. Our results indicate that appropriate S supply can improve the defense of legume-rhizobia symbiosis against HM toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exploring nutrient limitation in forest soil holds significant implications for forest tending and management. However, current research on nutrient limitation status of microorganisms in Robinia pseudoacacia plantations within the Loess Plateau remains insufficient. To investigate soil microbial nutrient limitation of R. pseu-doacacia plantations on the Loess Plateau, we selected R. pseudoacacia plantations with different afforestation time series (15, 25, 35, and 45 years) and a pile of barren slope cropland (control) in Yongshou County, Shaanxi Province as the research objects. We analyzed the contents of soil organic matter, total nitrogen, and total phosphorus, and the activities of β-1,4-glucosidase (BG), cellobiose hydrolase (CBH), leucine aminopeptidase (LAP), β-1,4-N-acetylglucoside (NAG) and phosphatase (AP). We analyzed the soil nutrient limitation by stoichiometry and enzyme metrology. The results showed a shift in soil pH from alkaline to acidic during vegetation restoration process, and that total phosphorus exhibited a gradual decrease over the course of 0 to 25 years. Soil orga-nic matter, total nitrogen and enzyme activities exhibited an increasing trend during the same time frame. However, between 25 and 45 years of age, soil total phosphorus, soil organic matter, total nitrogen, AP and LAP gradually declined while NAG, BG, and CBH initially increased and then decreased. Notably, the values of (BG+CBH)/(LAP+NAG), (BG+CBH)/AP and (LAP+NAG)/AP in R. pseudoacacia plantations were higher than the global average throughout the process of vegetation restoration. In the study area, the vector length was less than 1 and gradually increased, indicating that a progressive increase in microbial carbon limitation during the process of vegetation restoration. The vector angle exceeded 45° and exhibited an overall decreasing trend, suggesting that soil microorganisms were constrained by phosphorus (P) with a gradual deceleration of P limitation, without any nitrogen (N) limitation. The restoration of R. pseudoacacia plantation resulted in significant change in soil physical and chemical properties, while the time series of afforestation also influenced nutrient limitation of soil microorganisms.

The increase in atmospheric CO 2 concentration is a significant factor in triggering global warming. CO 2 is essential for plant photosynthesis, but excessive CO 2 can negatively impact photosynthesis and its associated physiological and biochemical processes. The tetraploid Robinia pseudoacacia L., a superior and improved variety, exhibits high tolerance to abiotic stress. In this study, we investigated the physiological and proteomic response mechanisms of the tetraploid R. pseudoacacia under high CO 2 treatment. The results of our physiological and biochemical analyses revealed that a 5% high concentration of CO 2 hindered the growth and development of the tetraploid R. pseudoacacia and caused severe damage to the leaves. Additionally, it significantly reduced photosynthetic parameters such as P n, G s, T r, and C i, as well as respiration. The levels of chlorophyll (Chl a and b) and the fluorescent parameters of chlorophyll ( Fm , Fv / Fm , q P, and ETR ) also significantly decreased. Conversely, the levels of ROS (H 2 O 2 and O 2 ·- ) were significantly increased, while the activities of antioxidant enzymes (SOD, CAT, GR, and APX) were significantly decreased. Furthermore, high CO 2 induced stomatal closure by promoting the accumulation of ROS and NO in guard cells. Through a proteomic analysis, we identified a total of 1652 DAPs after high CO 2 treatment. GO functional annotation revealed that these DAPs were mainly associated with redox activity, catalytic activity, and ion binding. KEGG analysis showed an enrichment of DAPs in metabolic pathways, secondary metabolite biosynthesis, amino acid biosynthesis, and photosynthetic pathways. Overall, our study provides valuable insights into the adaptation mechanisms of the tetraploid R. pseudoacacia to high CO 2 .

In Romania, huge quantities of gangue material from the mining activity practiced in the past were improperly stored and led to the pollution of the environment. Thus, this work is framed to manage the sterile dump of the "Radeș" mine (Alba, Romania) through a 12-week phytoremediation process. The efficient use of Robinia pseudoacacia was studied through the implementation, at the laboratory level, of a phytoremediation experiment based on various variants prepared by mixtures of gangue material, uncontaminated soil, and dehydrated sludge. The prepared variants, all planted with R. pseudoacacia, were watered with tap water, potassium monobasic phosphate, and enzyme solution. The bioconcentration and translocation factors for lead showed values ˂ 1, which indicates a potential presence of an exclusion system for Pb or a reduced Pb bioavailability since the R. pseudoacacia accumulates high concentrations of metals absorbed on and inside the roots. For copper, both factors had values > 1 indicating the suitability of R. pseudoacacia to readily translocate copper into the epigean organs. In the investigated experimental conditions, the highest efficiency in the removal of copper (93.0%) and lead (66.4%) by plants was obtained when gangue material was not mixed with other materials and wetted with enzymatic solution., (© 2024. The Author(s).)

Transpiration is a significant part of water cycle in forest ecosystems, influenced by meteorological factors and potentially constrained by soil moisture. We used Granier-type thermal dissipation probes to monitor xylem sap flow dynamics of three tree species ( Quercus liaotungensis , Platycladus orientalis , and Robinia pseudoacacia ) in a semi-arid loess hilly region, and to continuously monitor the key meteorological factors and soil water content (SWC). We established the SWC thresholds delineating soil moisture-limited and -unlimited sap flow responses to transpiration drivers. The results showed that mean sap flux density ( J s ) of Q. liaotungensis and R. pseudoacacia was significantly higher during period with higher soil moisture compared to lower soil moisture, while the difference in J s for P. orientalis between the two periods was not significant. We used an exponential saturation function to fit the relationship between the J s of each tree species and the integrated transpiration variable (VT) which reflected solar radiation and vapor pressure deficit. The difference in the fitting curve parameters indicated that there were distinct response patterns between J s and VT under different soil moisture conditions. There was a threshold in soil moisture limitation on sap flow for each species, which was identified as 0.129 m 3 ·m -3 for Q. liaotungensis , 0.116 m 3 ·m -3 for P. orientalis , and 0.108 m 3 ·m -3 for R. pseudoacacia . Below the thresholds, J s was limited by soil moisture. Above these points, the normalized sensitivity index (NSI) for Q. liaotungensis and P. orientalis reached saturation, while that of R. pseudoacacia did not reach saturation but exhibited a significant reduction in moisture limitation. Among the three species, P. orientalis was the most capable of overcoming soil moisture constraints.

Interaction of different environmental constrains pose severe threats to plants that cannot be predicted from individual stress exposure. In this context, mercury (Hg), as a typical toxic and hazardous heavy metal, has recently attracted particular attention. Nitrogen (N 2 )-fixing legumes can be used for phytoremediation of Hg accumulation, whereas N availability could greatly affect its N 2 -fixation efficiency. However, information on the physiological responses to combined Hg exposure and excess N supply of woody legume species is still lacking. Here, we investigated the interactive effects of rhizobia inoculation, Hg exposure (+Hg), and high N (+N) supply, individually and in combination (+N*Hg), on photosynthesis and biochemical traits in Robinia pseudoacacia L. seedlings of two provenances, one from Northeast (DB) and one from Northwest (GS) China. Our results showed antagonistic effects of combined + N*Hg exposure compared to the individual treatments that were provenance-specific. Compared to individual Hg exposure, combined + N*Hg stress significantly increased foliar photosynthesis (+50.6%) of inoculated DB seedlings and resulted in more negative (-137.4%) δ 15 N abundance in the roots. Furthermore, combined + N*Hg stress showed 47.7% increase in amino acid N content, 39.4% increase in NR activity, and 14.8% decrease in MDA content in roots of inoculated GS seedlings. Inoculation with rhizobia significantly promoted Hg uptake in both provenances, reduced MDA contents of leaves and roots, enhanced photosynthesis and maintained the nutrient balance of Robinia. Among the two Robinia provenances investigated, DB seedlings formed more nodules, had higher biomass and Hg accumulation than GS seedlings. For example, total Hg concentrations in leaves and roots and total biomass of inoculated DB seedlings were 1.3,1.9 and 3.4 times higher than in inoculated GS seedlings under combined + N*Hg stress, respectively. Therefore, the DB provenance is considered to possess a higher potential for phytoremediation of Hg contamination compared to the GS provenance in environments subjected to N deposition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Urea is the most frequently used nitrogen (N) fertilizer worldwide. However, the mechanisms in plants to cope with excess urea are largely unknown, especially for woody legumes that can meet their N demand by their own N 2 -fixation capacity. Here, we studied the immediate consequences of different amounts of urea application and exposure duration on photosynthesis, N metabolism, and the activity of antioxidative enzymes of Robinia pseudoacacia seedlings. For this purpose, seedlings were grown for 3 months under normal N availability with rhizobia inoculation and, subsequently, 50 mg N kg -1 was applied to the soil twice with urea as additional N source. Our results show that excess urea application significantly promoted photosynthesis, which increased by 80.3% and 84.7% compared with CK after the 1st and 2nd urea applications, respectively. The increase in photosynthesis translated into an increase in root and nodule biomass of 88.7% and 82.0%, respectively, while leaf biomass decreased by 4.8% after the first application of urea. The N content in leaves was 92.6% higher than in roots, but excess urea application increased the N content of protein and free amino acids in roots by 25.0%, and 43.3%, respectively. Apparently, enhanced root growth and N storage in the roots constitute mechanisms to prevent the negative consequences of excess N in the shoot upon urea application. Nitrate reductase (NR) activity of leaves and roots increased by 74.4% and 26.3%, respectively. Glutathione reductase (GR) activity in leaves and roots was enhanced by 337% and 34.0%, respectively, but then decreased rapidly to the initial level before fertilization. This result shows that not only N metabolism, but also antioxidative capacity was transiently promoted by excess urea application. Apparently, excess urea application initially poses oxidative stress to the plants that is immediately counteracted by enhanced scavenging of reactive oxygen species via enhanced GR activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Soil organic carbon (SOC) stabilization is vital for the mitigation of global climate change and retention of soil carbon stocks. However, there are knowledge gaps on how SOC sources and stabilization respond to vegetation restoration. Therefore, we investigated lignin phenol and amino sugar biomarkers, SOC physical fractions and chemical structure in one farmland and four stands of a Robinia pseudoacacia plantation. We observed that the content of SOC increased with afforestation, but the different biomarkers had different contributions to SOC. Compared to farmland, the contribution of lignin phenols to SOC decreased in the plantations, whereas there was no difference among the four stand ages, likely resulting from the balance between increasing lignin derivation input and increasing lignin degradation. Conversely, vegetation restoration increased the content of microbial necromass carbon (MNC) and the contribution of MNC to SOC, mainly because microbial residue decomposition was inhibited by decreasing the activity of leucine aminopeptidase, while microbial necromass preservation was promoted by adjusting soil variables (soil water content, clay, pH and total nitrogen). In addition, vegetation restoration increased the particulate organic carbon (POC), mineral-associated organic carbon (MAOC) pools and the O-alkyl C intensify. Overall, vegetation restoration affected SOC composition by regulating lignin phenols and microbial necromass and also altered SOC stabilization by increasing the physically stable MAOC pool during late afforestation. The results of this study suggest that more attention should be given to SOC sequestration and stability during late vegetation restoration., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nitrogen (N) deposition in the context of human activities continuously affects the carbon cycle of ecosystems. The effect of N deposition on soil organic carbon is related to the differential responses of different carbon fractions. To investigate the changes in soil organic carbon fraction and its influencing factors in the context of short-term N deposition, four N addition gradients:0 (CK), 1.5 (N1), 3 (N2), and 6 (N3) g·(m 2 ·a) -1 were set up in acacia plantations based on field N addition experiments, and the soil physicochemical properties, microbial biomass, and enzyme activities were measured in June and September. The results showed that:① exogenous N input reduced soil pH, promoted the increase in soluble organic carbon content, and increased soil nitrogen effectiveness. ② Short-term N addition significantly reduced soil organic carbon content, and the response of each component of organic carbon to N addition was different. Among them, the content of easily oxidized organic carbon was significantly reduced and reached the lowest value under the N2 treatment, with 54.4% and 48.2% reduction compared with that of the control, respectively, and the content of inert organic carbon increased, although the increase was not significant. Nitrogen addition reduced the soil carbon pool activity and improved the stability of the soil carbon pool. Soil carbon pool activity reached its lowest under the N3 and N2 treatments, with a decrease of 53.3% and 52.80%, respectively, compared to that of the control. ③Random forest modeling indicated that the soil microbial biomass stoichiometry ratio, microbial biomass carbon, and AP were the key factors driving the changes in soil organic carbon activity under short-term N addition, explaining 65.96% and 66.68% of the changes in oxidizable organic carbon and inert organic carbon, respectively. Structural equation modeling validated the results of the random forest modeling, and soil microbial biomass stoichiometric ratios significantly influenced carbon pool activity. Short-term nitrogen addition changed soil microbial biomass and its stoichiometric ratio in the acacia plantation forest mainly through two pathways, i.e., increasing soil nitrogen effectiveness and promoting soil acidification and inhibiting extracellular carbon hydrolase activity, thus changing the soil carbon fraction ratio and participating in the soil organic carbon cycling process.

Symbiotic nitrogen fixation (SNF) is a critical mechanism of ecosystem recovery, and in forests of the eastern United States, the most common tree species that supports SNF is black locust (Robinia pseudoacacia L.). Despite its prevalence, black locust's fixation strategy-whether it maintains fixation at a constant rate (obligate fixation) or reduces its fixation rate (facultative fixation)-is unknown. Here, we examined how nitrogen and light control SNF by black locust, by growing seedlings under two nitrogen levels and across four levels of light. Seedlings were harvested after 12 weeks to determine biomass changes, nodule activity, and photosynthetic rates. Black locust seedlings increased biomass growth with increasing light, but only in the absence of nitrogen addition, while seedling root:shoot (biomass) modestly declined with increasing light regardless of nitrogen level. We found that black locust behaved like a facultative fixer, and regulated fixation by excising or maintaining nodules, and by controlling nodule biomass and activity. Specifically, nitrogen addition reduced seedling investment in nodule biomass (g g -1 ) by 63%, and reduced seedling allocation to nitrogen fixation (µmol C 2 H 4 g -1  h -1 ) by 66%. In contrast, light affected nitrogen fixation through two indirect pathways. First, light increased plant growth, and hence nitrogen demands, which caused an increase in nitrogen fixation proportional to biomass. Second, light increasd photosynthetic activity, which was positively associated with nodule activity, but only in the absence of nitrogen addition. Our findings for how black locust regulates SNF can improve predictions of ecosystem SNF under the changing environmental conditions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L -1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1-1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.

To select elite Robinia pseudoacacia L. germplasm resources for production, 13 phenotypes and three physiological indicators of 214 seedlings from 20 provenances were systematically evaluated and analyzed. The leaf phenotypic and physiological coefficients of variation among the genotypes ranged from 3.741% to 19.599% and from 8.260% to 42.363%, respectively. The Kentucky provenance had the largest coefficient of variation (18.541%). The average differentiation coefficients between and within provenances were 34.161% and 38.756%, respectively. These close percentages showed that R. pseudoacacia presented high genetic variation among and within provenances, which can be useful for assisted migration and breeding programs. Furthermore, based on the results of correlations, principal component analysis and cluster analysis, breeding improvements targeting R. pseudoacacia's ornamental value, food value, and stress resistance of were performed. Forty and 30 excellent individuals, accounting for 18.692% and 14.019%, respectively, of the total resources. They were ultimately screened, after comprehensively taking into considering leaf phenotypic traits including compound leaf length, leaflet number and leaflet area and physiological characteristics including proline and soluble protein contents. These selected individuals could provide a base material for improved variety conservation and selection., Competing Interests: We declare that (1) the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper; (2) This work was supported by Youth Science Foundation of Henan Province (202300410136), National Nature Science Foundation of China (31570677), the National Key R&D Program of China (2017YFD0600503), and the National Forestry and Grassland Administration of Science and Technology Development Center Project (2016007); (3) All authors agree with the decision to submit the report for publication; and (4) This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Flavonoids participate in several plant processes such as growth and physiological protection in adverse environments. In this study, we investigated the combined effects of eCO 2 and cadmium (Cd)-contaminated soils on the total flavonoid and monomer contents in the leaves of Robinia pseudoacacia L. seedlings. Elevated CO 2 , Cd, and eCO 2 + Cd increased the total flavonoids in the leaves relative to the control, and eCO 2 mostly increased (p < 0.05) the total flavonoid content under Cd exposure. Elevated CO 2 increased (p < 0.05) robinin, rutin, and acacetin contents in the leaves of 45-day seedlings and decreased (p < 0.05) the content of robinin and acacetin at 90 and 135 d under Cd exposure except for robinin at day 45 under Cd1 and acacetin on day 135 under Cd1. Quercetin content decreased (p < 0.05) under the combined conditions relative to Cd alone. Kaempferol in the leaves was only detected under eCO 2 on day 135. The responses of total chlorophyll, total soluble sugars, starch, C, N, S, and the C/N ratio in the leaves to eCO 2 significantly affected the synthesis of total flavonoids and monomers under Cd exposure. Overall, rutin was more sensitive to eCO 2 + Cd than the other flavonoids. Cadmium, CO 2, and time had significant interactive effects on the synthesis of flavonoids in the leaves of R. pseudoacacia L. seedlings. Elevated CO 2 may improve the protection and defense system of seedlings grown in Cd-contaminated soils by promoting the synthesis of total flavonoids, although robinin, rutin, quercetin, and acacetin yields may reduce with time. Additionally, increased Cd in the leaves suggested that eCO 2 could improve the phytoremediation of Cd-contaminated soils., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Sodium sulfide (Na 2 S) is usually used as an amendment in industrial sewage treatment. To evaluate the effects of Na 2 S on the growth of Robinia pseudoacacia (black locust), heavy metal immobilization, and soil microbial activity, the R. pseudoacacia biomass and nutrient content and the soil heavy metal bioavailability, enzyme activity, and arbuscular mycorrhizal (AM) fungal community were measured by a single-factor pot experiment. The Pb-Zn-contaminated soil was collected from a Pb-Zn mine that had been remediated by R. pseudoacacia for five years. Three pollution levels (unpolluted, mildly polluted, and severely polluted) were evaluated by the pollution load index. Na 2 S application increased the shoot biomass under severe and mild contamination. In soil, Na 2 S application decreased the bioavailable Pb and Zn contents under severe and mild contamination, which resulted in a decrease in the Pb and Zn content in R. pseudoacacia. However, Na 2 S application did not affect the total Pb content per plant and enhanced the total Zn content per plant because of the higher biomass of the plants under Na 2 S application. Increased phosphatase activity and increased available phosphorous content may promote the uptake of phosphorus in R. pseudoacacia. Moreover, Na 2 S application is beneficial to the diversity of AM fungi under mild and severe pollution. Overall, Na 2 S application has great potential for enhancing soil heavy metal immobilization, enhancing soil microbial activity, and improving the growth of R. pseudoacacia in polluted soils. Therefore, Na 2 S is suitable for use in Pb-Zn remediation to ameliorate environmental heavy metal pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

As the driver of plantation ecosystem function, microorganisms can decompose plant residues and soil organic matter. To identify dynamics of microbial communities in litter and soil and its influence by vegetation and soil at regional scales, the plantations of Robinia pseudoacacia at different successional stages (13, 19, 29, and 44 y) was selected on the Loess Plateau. High-throughput sequencing of the 16S rRNA gene was used to examine bacterial communities in litter and soil, and changes in vegetation, litter, and soil characteristics were analyzed. With increase of stand age, coverage and biomass of understory vegetation increased significantly and peaked at 44-y. Concentrations of carbon (C), nitrogen (N), and phosphorus (P) in litter and soil increased significantly, whereas pH values decreased significantly. Composition and diversity of bacterial communities in litter and soil were significantly different. Diversity and richness of litter bacterial communities were higher than that of soils. Relative abundances of Actinobacteria and Proteobacteria in litter were higher than that in soil; relative abundance of Acidobacteria exhibited the reverse trend. The diversity and richness index of vegetation significantly affected that of litter bacterial communities. Soil C/P significantly affected the Simpson and Shannon index of soil bacterial communities. The C/P and pH of litter and soil were significantly correlated with bacterial composition, primarily including Actinobacteria, Acidobacteria, and Gemmatimonadetes. Diversity of litter bacterial communities was more sensitive to the diversity and richness of vegetation flora than that of soil in the succession of R. pseudoacacia. Canopy density, vegetation, and litter and soil nutrients might directly or indirectly affect bacterial communities. Carbon, phosphorus, and pH may be critical factors influencing the composition of bacterial communities in litter and soil., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Significant improvements to the centrifuge water-extraction method of measuring the percentage loss volume of water (PLV) and corresponding vulnerability curves (VCs) are reported. Cochard and Sperry rotors are both incapable of measuring the VCs of species with long vessels because of premature embolism induced by hypothetical nanoparticles that can be drawn into segments during flow measurement. In contrast, water extraction pushes nanoparticles out of the sample. This study focuses on a long-vessel species, Robinia pseudoacacia, for which many VCs have been constructed by different methods, and the daily water relations have been quantified. PLV extraction curves have dual Weibull curves, and this paper focuses on the second Weibull curve because it involves the extraction of water from vessels, as proven by staining methods. We demonstrate an improved water extraction method after evaporation correction that has accuracy to within 0.5%, shows good agreement with two traditional methods that are slower and less accurate, and is immune to nanoparticle artefacts. Using Poiseuille's Law and the geometry of vessels, we argue that the percentage loss of conductivity (PLC) equals 2PLV-PLV2 in a special case where all vessels, regardless of size, have the same vulnerability curve. In this special case, this equation predicts the data reasonably well., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The box tree moth, Cydalima perspectalis, is an invasive pest in Europe causing damage on Buxus species. In this study, we aimed to develop a "bisexual" lure to attract both female and male moths. Based on a previous screening bioassay we tested methyl salicylate, phenylacetaldehyde and eugenol as potential attractants in different combinations. The trapping results showed that both binary and ternary blends attracted male and female moths. Catches with these blends were comparable to catches with the synthetic pheromone. Subsequently we carried out single sensillum recordings, which proved the peripheral detection of the above-mentioned compounds on male and female antennae. To identify synergistic flower volatiles, which can be also attractive and can increase the trap capture, we collected flower headspace volatiles from 12 different flowering plant species. Several components of the floral scents evoked good responses from antennae of both females and males in gas chromatography-electroantennographic detection. The most active components were tentatively identified by gas chromatography coupled mass spectrometry as benzaldehyde, cis-ß-ocimene, (±)-linalool and phenethyl alcohol. These selected compounds in combination did not increase significantly the trap capture compared to the methyl salicylate- phenyacetaldehyde blend. Based on these results we discovered the first attractive blend, which was able to attract both adult male and female C. perspectalis in field conditions. These results will yield a good basis for the optimization and development of a practically usable bisexual lure against this invasive pest.

Honey is a natural sweetener composed mostly of sugars, but it contains also pollen grains, proteins, free amino acids, and minerals. The amounts and proportions of these components depend on the honey type and bee species. Despite the low content of honey protein, they are becoming a popular study object, and have recently been used as markers of the authenticity and quality of honey. Currently, the most popular methods of protein isolation from honey are dialysis against distilled water, lyophilization of dialysate, or various precipitation protocols. In this work, we propose a new method based on saturated phenol. We tested it on three popular polish honey types and we proved its compatibility with both 1D and 2D polyacrylamide gel electrophoresis (PAGE) and MS (mass spectrometry) techniques. The elaborated technique is also potentially less expensive and less time-consuming than other previously described methods, while being equally effective.

Few studies have explored the long-term effects of elevated atmospheric CO 2 combined with lead (Pb) contamination on plants. The objective of this study was to examine the effects of 3 years of elevated CO 2 (700 ± 23 μmol mol -1 ) on Pb accumulation and plant defenses in leaves of Robinia pseudoacacia L. seedlings in exposed to Pb (500 mg kg -1 soil). Elevated CO 2 increased Pb accumulation in leaves and Pb removal rate in soils. In plants exposed to Pb stress, total chlorophyll and carotenoid contents in leaves were lower under elevated CO 2 than under ambient CO 2 , but seedling height and width increased under elevated CO 2 relative to ambient CO 2 . Elevated CO 2 significantly (p < .01) stimulated malondialdehyde content in leaves under Pb exposure. Superoxide dismutase and catalase activity increased significantly (p < .01), peroxidase activity decreased significantly (p < .01), and glutathione, cystine, and phytochelatin contents increased under elevated CO 2  + Pb relative to Pb alone. Elevated CO 2 stimulated the production of soluble sugars, proline, flavonoids, saponins, and phenolics in plants exposed to Pb stress. Ove rall, long-term elevation of CO 2 increased Pb-induced oxidative damage in seedlings, but enhanced the phytoextraction of Pb from contaminated soils., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heavy metals can cause serious contamination of soils, especially in mining regions. A detailed understanding of the effects of heavy metals on plants and root-associated microbial communities could help to improve phytoremediation systems. In this study, black locust (Robinia pseudoacacia) seedlings with or without rhizobial inoculation were planted in soils contaminated with different levels of heavy metals. Bacterial communities in rhizosphere and bulk soil samples were analyzed using 16S rRNA gene sequencing on the Illumina MiSeq platform and shotgun metagenome sequencing on the Illumina HiSeq platform. Soil bacterial communities varied significantly depending on the level of soil contamination, and planting also had some influence. Although inoculation of Mesorhizobium loti HZ76 (a natural microsymbiont of R. pseudoacacia) was a relatively minor factor, it did influence the soil bacterial community. Under the selective pressure, plant growth promotion-related biomarkers in the rhizosphere increased after inoculation compared with non-inoculated controls, especially those associated with Mesorhizobium, Variovorax, Streptomyces, and Rhodococcus genera. Genes encoding ATP-binding cassette transporters were up-regulated in the rhizosphere after inoculation compared with genes related to sulfur/nitrogen metabolism. These results provide insight into soil bacterial communities and their functions in the R. pseudoacacia rhizosphere in response to rhizobial inoculation and heavy metal contamination. This knowledge may prove useful for improving phytoremediation of metal-contaminated soils., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The relationships of CO 2 assimilation under saturated-light conditions (A sat ) with exposure- (AOTX, Accumulated Ozone exposure over a hourly Threshold of X ppb) and flux-based (POD Y , Phytotoxic Ozone Dose over a hourly threshold Y nmol·m -2 ·s -1 ) O 3 metrics was studied on three common urban trees, Fraxinus chinensis (FC), Platanus orientalis (PO) and Robinia pseudoacacia (RP). Parameterizations for a stomatal multiplicative model were proposed for the three species. RP was the species showing lower species-specific maximum stomatal conductance (g max ) and experiencing lower cumulative O 3 uptake along the experiment, but in contrast it was the most sensitive to O 3 . POD Y was slightly better than AOTX metric at estimating relative A sat (R-A sat )for PO and RB but not for FC. The best fittings obtained for the regressions between R-A sat and AOTX for FC, PO and RP were 0.904, 0.868, and 0.876, when the thresholds of X were 60ppb, 55ppb and 30ppb, respectively. However, AOT40 performed also well for all of them, with R 2 always >0.83. For POD Y , the highest R 2 values for FC, PO and RB were 0.863, 0.897 and 0.911 at thresholds Y=7, 5 and 1nmolO 3 m -2 s -1 , respectively. Given the potentially higher O 3 removal capacity of FC and PO by stomatal uptake and their lower sensitivity to this pollutant than RP, the former two species would be appropriate for urban gardens and areas where O 3 levels are high. Parameterization and modeling of stomatal conductance for the main urban tree species may provide reliable estimations of the stomatal uptake of O 3 and other gaseous pollutants by vegetation, which may support decision making on the most suitable species for green urban planning in polluted areas., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This research was carried out on plants Taraxacum officinale, Plantago lanceolata, Betula pendula and Robinia pseudoacacia growing in urban biotopes with different levels of heavy metal contamination in the city of Dąbrowa Górnicza (southern Poland). Based on the pollution index, the highest heavy metal contamination was determined in the site 4 (connected with industry emitters) and 6 (high traffic). The metal accumulation index (MAI) values ranged within the biotopes in Dąbrowa Górnicza between 7.3 and 20.6 for R. pseudoacacia, 4.71-23.1 for P. lanceolata, 4.68-28.1 for T. officinale and 10.5-27.2 for B. pendula. Increasing tendency in proline content in biotopes connected with high traffic was found in the leaves of investigated plants (except R. pseudoacacia). Similar tendency was observed for ascorbic acid content in the foliage of the plants as well as in T. officinalle in stands connected industrial emission. Non-protein thiols content increased especially in the leaves of R. pseudoacacia in biotopes with high traffic emissions as well as in T. officinale in stands connected with industry. The mean values of APTI (Air Pollution Tolerance Index) within the city of Dąbrowa Górnicza for investigated plants were found in the following ascending order P. lanceolata < R. pseudoacacia < B. pendula < T. officinale. Among the investigated plants B. pendula and T. officinale may be postulated as appropriate plants in urban areas with considerable soil and air contamination, especially with heavy metals. The results indicate that species deemed tolerant according to APTI are suitable plants in barriers areas to combat atmospheric pollution., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Polyploidy is an important phenomenon in plants because of its roles in agricultural and forestry production as well as in plant tolerance to environmental stresses. Tetraploid black locust (Robinia pseudoacacia L.) is a polyploid plant and a pioneer tree species due to its wide ranging adaptability to adverse environments. To evaluate the ploidy-dependent differences in leaf mitochondria between diploid and tetraploid black locust under salinity stress, we conducted comparative proteomic, physiological, biochemical and ultrastructural profiling of mitochondria from leaves., Results: Mitochondrial proteomic analysis was performed with 2-DE and MALDI-TOF-MS, and the ultrastructure of leaf mitochondria was observed by transmission electron microscopy. According to 2-DE analysis, 66 proteins that responded to salinity stress significantly were identified from diploid and/or tetraploid plants and classified into 9 functional categories. Assays of physiological characters indicated that tetraploids were more tolerant to salinity stress than diploids. The mitochondrial ultrastructure of diploids was damaged more severely under salinity stress than that of tetraploids., Conclusions: Tetraploid black locust possessed more tolerance of, and ability to acclimate to, salinity stress than diploids, which may be attributable to the ability to maintain mitochondrial structure and to trigger different expression patterns of mitochondrial proteins during salinity stress.

Secondary metabolites play important roles in plant interactions with the environment. The co-occurrence of heavy metal contamination of soils and rising atmospheric CO2 has important effects on plant. It is important to explore the ways in which production of plant secondary metabolites is affected by heavy metals under elevated atmospheric CO2. We examined the effects of elevated CO2 on secondary metabolite contents in Robinia pseudoacacia seedlings grown in Cd- and lead (Pb)-contaminated soils. The increase in secondary metabolites was greater under Cd + Pb exposure than under exposure to individual metals regardless of elevated CO2 with the exception of condensed tannins in leaves and total alkaloids in stems. Except for phenolic compounds and condensed tannins, elevated CO2 was associated with increased secondary metabolite contents in leaves and stems of plants exposed to Cd, Pb, and Cd + Pb compared to plants exposed to ambient CO2 + metals. Changes in saponins in leaves and alkaloids in stems were greater than changes in the other secondary metabolites. Significant interactive effects of CO2, Cd, and Pb on secondary metabolites were observed. Saponins in leaves and alkaloids in stems were more sensitive than other secondary metabolites to elevated CO2 + Cd + Pb. Elevated CO2 could modulate plant protection and defense mechanisms in R. pseudoacacia seedlings exposed to heavy metals by altering the production of secondary metabolites. The increased Cd and Pb uptake under elevated CO2 suggested that R. pseudoacacia may be used in the phytoremediation of heavy metal-contaminated soils under global environmental scenarios., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Plant secondary metabolites play a pivotal role in growth regulation, antioxidant activity, pigment development, and other processes. As the global climate changes, increasing atmospheric temperatures and contamination of soil by heavy metals co-occur in natural ecosystems, which alters the pH of rhizosphere soil and influences the bioavailability and mobility of metals. Elevated temperatures in combination with heavy metals are expected to affect plant secondary metabolites, but this issue has not been extensively examined. Here, we investigated secondary metabolites in Robiniapseudoacacia seedlings exposed to elevated temperatures using a passive warming device in combination with Cd- and Pb-contaminated soils. Heavy metals significantly stimulated the accumulation of saponins, phenolic compounds, and flavonoids in leaves and stems; alkaloid compounds increased in leaves and decreased in stems, and condensed tannins fluctuated. Elevated temperatures, alone and in combination with Cd and Pb, caused increases in secondary metabolites in the plant tissues. Phenolic compounds showed the greatest changes among the secondary metabolites and significant interactive effects of temperature and metals were observed. These results suggest that slightly elevated temperature could enhance protective and defense mechanisms of Robinia pseudoacacia seedlings exposed to heavy metals by stimulating the production of secondary metabolites., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Phytoremediation is an emerging technology that employs higher plants to cleanup contaminated environments, including metal-polluted soils. Because it produces a biomass rich in extracted toxic metals, further treatment of this biomass is necessary. The aim of our study was to assess the five-year potential of the following native wild plants to produce biomass and remove heavy metals from a polluted site: poplar (Populus ssp.), ailanthus (Ailanthus glandulosa L.), false acacia (Robinia pseudoacacia L.), ragweed (Artemisia artemisiifolia L.), and mullein (Verbascum thapsus L). Average soil contamination with Pb, Cd, Zn, Cu, Ni, Cr, and As in the root zone was 22,948.6 mg kg-1, 865.4 mg kg-1, 85,301.7 mg kg-1, 3,193.3 mg kg-1, 50.7 mg kg-1, 41.7 mg kg-1,and 617.9 mg kg-1, respectively. We measured moisture and ash content, concentrations of Pb, Cd, Zn, Cu, Ni, Cr, and As in the above-ground parts of the plants and in ash produced by combustion of the plants, plus gross calorific values. The plants' phytoextraction and phytostabilisation potential was evaluated based on their bioconcentration factor (BCF) and translocation factor (TF). Mullein was identified as a hyperaccumulator for Cd. It also showed a higher gross calorific value (19,735 kJ kg-1) than ragweed (16,469 kJ kg-1).The results of this study suggest that mullein has a great potential for phytoextraction and for biomass generation, and that ragweed could be an effective tool of phytostabilisation.

In this study, HPLC-MS and ICP-MS methods were used for the determination of histidine and nickel in Morus L., Robinia pseudoacacia L., and Populus nigra L. leaves taken from industrial areas including Gaziantep and Bursa cities. In the determination of histidine by HPLC-MS, all of the system parameters such as flow rate of mobile phase, fragmentor potential, injection volume and column temperature were optimized and found to be 0.2 mL min(-1), 70 V, 15 µL, and 20°C, respectively. Under the optimum conditions, histidine was extracted from plant sample by distilled water at 90°C for 30 min. Concentrations of histidine as mg kg(-1) were found to be between 2-9 for Morus L., 6-13 for Robinia pseudoacacia L., and 2-10 for Populus nigra L. Concentrations of nickel were in the ranges of 5-10 mg kg(-1) for Morus L., 3-10 mg kg(-1) for Robinia pseudoacacia L., and 0.6-4 mg kg(-1) for Populus nigra L. A significant linear correlation (r = 0.78) between histidine and Ni was observed for Populus nigra L., whereas insignificant linear correlation for Robinia pseudoacacia L. (r = 0.22) were seen. Limits of detection (LOD) and quantitation (LOQ) were found to be 0.025 mg Ni L(-1) and 0.075 mg Ni L(-1), respectively.

Water entry at germination for black locust (Robinia pseudoacacia L.) seeds which are known as hard seeds with impermeable seed coat to water, was examined using micro-magnetic resonance imaging (MRI). The MRI apparatus equipped with a low-field (1 T; Tesla) permanent magnet was used, which is open access, easy maintenance, operable and transportable. The excellent point of the apparatus is that T 1-enhancement of water signals absorbed in dry seeds against steeping free water is stronger than the apparatuses with high-field superconducting magnets, which enabled clear detection of water entry. Water hardly penetrated into the seeds for more than 8 h but approximately 60 % of seeds germinated by incubating on wet filter papers for several days. Hot water treatments above 75 °C for 3 min effectively induced water gap; scarification was 70 % at 100 °C and 75 °C, declined to 15 % at 50 °C and decreased further at room temperature. Water entered into the scarified seeds exclusively through the lens, spread along the dorsal side of the seeds and reached the hypocotyl, whereas water migrated slowly through hilum side to radicle within 3 h.

In the present paper, we investigated the effects of afforestation on nitrogen fractions and microbial communities. A total of 24 soil samples were collected from farmland (FL) and three afforested lands, namely Robinia pseudoacacia L (RP), Caragana korshinskii Kom (CK), and abandoned land (AL), which have been arable for the past 40 years. Quantitative PCR and Illumina sequencing of 16S rRNA genes were used to analyze soil bacterial abundance, diversity, and composition. Additionally, soil nitrogen (N) stocks and fractions were estimated. The results showed that soil N stock, N fractions, and bacterial abundance and diversity increased following afforestation. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla of soil bacterial compositions. Overall, soil bacterial compositions generally changed from Actinobacteria (Acidobacteria)-dominant to Proteobacteria-dominant following afforestation. Soil N fractions, especially for dissolved organic nitrogen (DON), were significantly correlated with most bacterial groups and bacterial diversity, while potential competitive interactions between Proteobacteria (order Rhizobiales) and Cyanobacteria were suggested. In contrast, nitrate nitrogen (NO3(-)-N) influenced soil bacterial compositions less than other N fractions. Therefore, the present study demonstrated that bacterial diversity and specific species respond to farmland-to-forest conversion and hence have the potential to affect N dynamic processes in the Loess Plateau.

Transcription factors play a key role to enable plants to cope with abiotic stresses. DREB2 regulates the expression of several stress-inducible genes and constitutes major hubs in the water stress signalling webs. We cloned and characterized a novel gene encoding the FpDREB2A transcription factor from Fraxinus pennsylvanica, and a yeast activity assay confirmed its DRE binding and transcription activation. Overexpression of FpDREB2A in R. pseudoacacia showed enhanced resistance to drought stress. The transgenic plant survival rate was significantly higher than that of WT in soil drying and re-watering treatments. Transgenic lines showed a dramatic change in root architecture, and horizontal and vertical roots were found in transgenic plants compared to WT. The vertical roots penetrated in the field soil to more than 60 cm deep, while horizontal roots expanded within the top 20-30 cm of the soil. A physiological test demonstrated that chlorophyll contents were more gradually reduced and that soluble sugars and proline levels elevated more sharply but malondialdehyde level stayed the same (P < 0.05). Plant hormone levels of abscisic acid and IAA were higher than that of WT, while gibberellins and zeatin riboside were found to be lower. The root transcriptomes were sequenced and annotated into 2011 differential expression genes (DEGs). The DEGs were categorized in 149 pathways and were found to be involved in plant hormone signalling, transcription factors, stimulus responses, phenylalanine, carbohydrate and other metabolic pathways. The modified pathways in plant hormone signalling are thought to be the main cause of greater horizontal and vertical root development, in particular., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Present work examines phytoextraction potential of four black locust families (half-sibs 54, 56, 115, and 135) grown hydroponically. Plants were treated with 6 ppm of cadmium (Cd), 100 ppm of nickel (Ni), and 40 ppm of lead (Pb) added in Hoagland nutrient solution, accompanying with simultaneously applied all three metals. Responses to metals exposure among families were different, ranging from severe to slight reduction of root and shoot biomass production of treated plants. Calculated tolerance indices are indicating tested families as highly tolerant (Ti > 60). Family 135 had the lowest tolerance index, pointing that it was highly susceptible to applied metals. Comparing photosynthetic activities of tested families it has been noticed that they were highly sensitive to stress induced by heavy metals. Net photosynthetic rate of nickel treated plants was the most affected by applied concentration. Cadmium and nickel concentrations in stems and leaves of black locust families exceeded 100 mg Cd kg(-1) and 1000 mg Ni kg(-1), in both single and multipollution context. On the contrary, accumulation of lead in above ground biomass was highly affected by multipollution treatment. Tf and BCF significantly varied between investigated treatments and families of black locust. Concerning obtained results of heavy metals accumulation and tolerance of black locust families can be concluded that tested families might be a promising tool for phytoextraction purposes, but it takes to be further confirmed in field trials.

Sequential Fenton oxidation (FO) and hydrothermal treatment were performed to improve the effect of pretreatment and enzymatic hydrolysis of mixed hardwood. The molar ratio of the Fenton reagent (FeSO4·7H2O and H2O2) was 1:25, and the reaction time was 96h. During the reaction, little or no weight loss of biomass was observed. The concentration of Fe(2+) was determined and was found to increase continuously during FO. Hydrothermal treatment at 190-210°C for 10-80min was performed following FO. Sequential FO and hydrothermal treatment showed positive effects on pretreatment and enzymatic hydrolysis. Xylose concentration in the hydrolysate was as high as 14.16g/L when FO-treated biomass was treated at 190°C, while its concentration in the raw material was 3.72g/L. After 96h of enzymatic hydrolysis, cellulose conversion in the biomass obtained following sequential treatment was 69.58-79.54%. In contrast, the conversion in the raw material (without FO) was 64.41-67.92%., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Arbuscular mycorrhizal fungi (AMF) are considered as a potential biotechnological tool for improving phytostabilization efficiency and plant tolerance to heavy metal-contaminated soils. However, the mechanisms through which AMF help to alleviate metal toxicity in plants are still poorly understood. A greenhouse experiment was conducted to evaluate the effects of two AMF species (Funneliformis mosseae and Rhizophagus intraradices) on the growth, Pb accumulation, photosynthesis and antioxidant enzyme activities of a leguminous tree (Robinia pseudoacacia L.) at Pb addition levels of 0, 500, 1000 and 2000 mg kg(-1) soil. AMF symbiosis decreased Pb concentrations in the leaves and promoted the accumulation of biomass as well as photosynthetic pigment contents. Mycorrhizal plants had higher gas exchange capacity, non-photochemistry efficiency, and photochemistry efficiency compared with non-mycorrhizal plants. The enzymatic activities of superoxide dismutase (SOD), ascorbate peroxidases (APX) and glutathione peroxidase (GPX) were enhanced, and hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were reduced in mycorrhizal plants. These findings suggested that AMF symbiosis could protect plants by alleviating cellular oxidative damage in response to Pb stress. Furthermore, mycorrhizal dependency on plants increased with increasing Pb stress levels, indicating that AMF inoculation likely played a more important role in plant Pb tolerance in heavily contaminated soils. Overall, both F. mosseae and R. intraradices were able to maintain efficient symbiosis with R. pseudoacacia in Pb polluted soils. AMF symbiosis can improve photosynthesis and reactive oxygen species (ROS) scavenging capabilities and decrease Pb concentrations in leaves to alleviate Pb toxicity in R. pseudoacacia. Our results suggest that the application of the two AMF species associated with R. pseudoacacia could be a promising strategy for enhancing the phytostabilization efficiency of Pb contaminated soils.

Black locust (Robinia pseudoacacia) is a tree in the subfamily Faboideae, native to North America, that has been naturalized and widely planted in temperate Europe and Asia. Black locust has important ecological and economic value, but its quality needs improvement. Hybridization programs are important for black locust breeding, but the low rate of fruit set after controlled pollination limits both its breeding and that of other monoclinous plant species that share this problem. In this study, we investigated gene expression in emasculated black locust flowers using the cDNA-amplified fragment length polymorphism technique to determine why the rate of fruit set is low after controlled pollination. Flowers that were emasculated after being frozen in liquid nitrogen were used as controls. Changes in the flower transcriptome were more dramatic at 5 h after emasculation than at 48 h. Injury caused by emasculation decreased the expression levels of genes associated with metabolism, growth regulation, signal transduction, and photosynthesis, and it increased the expression of genes related to stress-response metabolism, signal transduction, and promotion of senescence. The changes in the expression levels of these genes had negative effects on sugar metabolism, protein metabolism, lipid metabolism, energy metabolism, matter transport, signal transduction, osmotic regulation, pH regulation, and photosynthesis. Thus, emasculation accelerated flower senescence, resulting in low fruit set.

During periods of water deficit, growing roots may shrink, retaining only partial contact with the soil. In this study, known mathematical models were used to calculate the root-soil air gap and water flow resistance at the soil-root interface, respectively, of Robinia pseudoacacia L. under different water conditions. Using a digital camera, the root-soil air gap of R. pseudoacacia was investigated in a root growth chamber; this root-soil air gap and the model-inferred water flow resistance at the soil-root interface were compared with predictions based on a separate outdoor experiment. The results indicated progressively greater root shrinkage and loss of root-soil contact with decreasing soil water potential. The average widths of the root-soil air gap for R. pseudoacacia in open fields and in the root growth chamber were 0.24 and 0.39 mm, respectively. The resistance to water flow at the soil-root interface in both environments increased with decreasing soil water potential. Stepwise regression analysis demonstrated that soil water potential and soil temperature were the best predictors of variation in the root-soil air gap. A combination of soil water potential, soil temperature, root-air water potential difference and soil-root water potential difference best predicted the resistance to water flow at the soil-root interface., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The mouth cavity hosts many types of anaerobic bacteria, including Streptococcus mutans and Porphyromonas gingivalis, which cause periodontal inflammatory diseases and dental caries. The present study was conducted to evaluate the antibacterial potential of extracts of Robinia pseudoacacia and its different fractions, as well as some of its natural compounds against oral pathogens and a nonpathogenic reference bacteria, Escherichia coli. The antibacterial activity of the crude extract and the solvent fractions (hexane, chloroform, ethyl acetate and butanol) of R. pseudoacacia were evaluated against S. mutans, P. gingivalis and E. coli DH5α by standard micro-assay procedure using conventional sterile polystyrene microplates. The results showed that the crude extract was more active against P. gingivalis (100% growth inhibition) than against S. mutans (73% growth inhibition) at 1.8 mg/mL. The chloroform and hexane fractions were active against P. gingivalis, with 91 and 97% growth inhibition, respectively, at 0.2 mg/mL. None of seven natural compounds found in R. pseudoacacia exerted an antibacterial effect on P. gingivalis; however, fisetin and myricetin at 8 µg/mL inhibited the growth of S. mutans by 81% and 86%, respectively. The crude extract of R. pseudoacacia possesses bioactive compounds that could completely control the growth of P. gingivalis. The antibiotic activities of the hexane and chloroform fractions suggest that the active compounds are hydrophobic in nature. The results indicate the effectiveness of the plant in clinical applications for the treatment of dental plaque and periodontal inflammatory diseases and its potential use as disinfectant for various surgical and orthodontic appliances.

Knowledge about the long-term influences of climate change on the amount of potential carbon (C) sequestration in forest ecosystems, including age-related dynamics, remains unclear. This study used two similar age-sequences of black locust forests (Robinia pseudoacacia L.) in the semi-arid and semi-humid zones of China's Loess Plateau to assess the variation in C stocks and age-related dynamics. Our results demonstrated that black locust forests of the semi-humid zone stored significantly more C than did forests in the semi-arid zone, across the chronosequence (p < 0.001). The C carrying capacity of the plantations was measured at 166.4 Mg C ha-1 (1 Mg = 106 g) in the semi-humid zone, while the semi-arid zone had a capacity of only 79.4 Mg C ha-1. Soil organic C (SOC) increased continuously with stand age in the semi-arid zone (R2 = 0.84, p = 0.010). However, in the semi-humid zone, SOC declined sharply by 47.8% after the initial stage (5 to 10 y). The C stock in trees increased continuously with stand age in the semi-humid zone (R2 = 0.83, p = 0.011), yet in the semi-arid zone, it decreased dramatically from 43.0 Mg C ha-1 to 28.4 Mg C ha-1 during the old forest stage (38 to 56 y). The shift from being a net C sink to a net C source occurred at the initial stage in the semi-humid zone versus at the old forest stage in the semi-arid zone after reforestation. Surprisingly, with the exception of the initial and later stages (55 y), the patterns of C allocation among trees, soils, understory and litter were not statistically different between the two climate zones. Our results suggest that climate factors can alter the potential amount and age-related dynamics of forest C sequestration.

Juglans regia, Robinia pseudoacacia, Eucalyptus sp. and Populus sp. plantations, suffering from Mn and Cu toxicity, were limed in order to reduce Cu and Mn solubility in soil. The purposes of the present work were: i) to study the changes in soil chemical properties after the addition of CaCO3, ii) to investigate the influence of liming on the reduction of Mn and Cu toxicity. After the addition of CaCO3 (three applications, during three successive years), pH and CaCO3 content were significantly increased, while organic C and N were significantly reduced. Exchangeable Ca concentrations have been slightly, or significantly, increased, while those of Mg have been decreased; in addition, ratios Ca/Mg and C/N have been significantly increased after liming. Impressive reductions of DTPA extractable Cu and Mn concentrations (more than 10 times in most cases) were recorded. It was also found that trees without Mn and Cu toxicity symptoms (healthy tress) before liming did not have, in many cases, significantly greater leaf Mn, Cu and Fe concentrations, than trees after soil liming (all the trees were healthy). This probably happened because excess Mn and Cu quantities had been accumulated into their root system. Finally, leaf Mn, Cu and Zn concentrations of trees suffering from toxicity were significantly decreased after soil liming, while leaf Fe concentrations, in all the plant species studied, were increased., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The legume-rhizobium symbiosis has been proposed as an important system for phytoremediation of heavy metal contaminated soils due to its beneficial activity of symbiotic nitrogen fixation. However, little is known about metal resistant mechanism of rhizobia and the role of metal resistance determinants in phytoremediation. In this study, copper resistance mechanisms were investigated for a multiple metal resistant plant growth promoting rhizobium, Mesorhizobium amorphae 186. Three categories of determinants involved in copper resistance were identified through transposon mutagenesis, including genes encoding a P-type ATPase (CopA), hypothetical proteins, and other proteins (a GTP-binding protein and a ribosomal protein). Among these determinants, copA played the dominant role in copper homeostasis of M. amorphae 186. Mutagenesis of a hypothetical gene lipA in mutant MlipA exhibited pleiotropic phenotypes including sensitivity to copper, blocked symbiotic capacity and inhibited growth. In addition, the expression of cusB encoding part of an RND-type efflux system was induced by copper. To explore the possible role of copper resistance mechanism in phytoremediation of copper contaminated soil, the symbiotic nodulation and nitrogen fixation abilities were compared using a wild-type strain, a copA-defective mutant, and a lipA-defective mutant. Results showed that a copA deletion did not affect the symbiotic capacity of rhizobia under uncontaminated condition, but the protective role of copA in symbiotic processes at high copper concentration is likely concentration-dependent. In contrast, inoculation of a lipA-defective strain led to significant decreases in the functional nodule numbers, total N content, plant biomass and leghemoglobin expression level of Robinia pseudoacacia even under conditions of uncontaminated soil. Moreover, plants inoculated with lipA-defective strain accumulated much less copper than both the wild-type strain and the copA-defective strain, suggesting an important role of a healthy symbiotic relationship between legume and rhizobia in phytostabilization.

The study is a part of research project on using short rotation plantations in biomass production. The aim of this investigation was to determine the accumulation characteristics of Robinia pseudoacacia leaves as a biomonitor of heavy-metal pollution in the industrial regions in Bulgaria. The study was carried out in 25-year-old plantations located in close vicinity of industrial area Devnia, Eastern Bulgaria. Devnia is a zone, highly contaminated by cement factories, nitrogen fertilizers and polyvinylchloride factories. Controls were similarly aged plantations on a background area 15 km away from the emission sources. The concentration of nutrients and heavy metals in the leaves of damaged and control plants were investigated. Decreased levels of total nitrogen (6%), total phosphorous (11%), potassium (36%) and magnesium (3%) were detected in September for polluted trees as compared to control trees. Lead content (30.7 mg kg(-1)) was 1.38 times higher as compared to control, whereas accumulation of zinc (19.0 mg kg2) about 1.37 times more than control. An excessive accumulation of copper in the leaves collected from the polluted area (17.2 mg kg(-1)) was 2.15 times higher than control. The concentrations of Pb, Zn and Cu in the contaminated soil under black locust plantation were 38.2, 77.4 and 101.3 mg kg(-1), respectively. Our results showed that the leaves of R. pseudoacacia accumulated Pb, Zn and Cu in parallelity with their increase in the contaminated soil. R. pseudoacacia may be considered as a good biomonitor of soil pollution especially with Cu, and at lower extent for Pb and Zn in the industrial region of Devnia.

The propagation of hard-branch cuttings of tetraploid Robinia pseudoacacia (black locust) is restricted by the low rooting rate; however, etiolated juvenile tetraploid black locust branches result in a significantly higher rooting rate of cuttings compared with non-etiolated juvenile tetraploid branches. To identify proteins that influence the juvenile tetraploid branch rooting process, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectra (MALDI-TOF/TOF-MS) were used to analyze proteomic differences in the phloem of tetraploid R. pseudoacacia etiolated and non-etiolated juvenile branches during different cutting periods. A total of 58 protein spots differed in expression level, and 16 protein spots were only expressed in etiolated branches or non-etiolated ones. A total of 40 highly expressed protein spots were identified by mass spectrometry, 14 of which were accurately retrieved. They include nucleoglucoprotein metabolic proteins, signaling proteins, lignin synthesis proteins and phyllochlorin. These results help to reveal the mechanism of juvenile tetraploid R. pseudoacacia etiolated branch rooting and provide a valuable reference for the improvement of tetraploid R. pseudoacacia cutting techniques.

The 9-, 17-, 30- and 37-year-old Robinia pseudoacacia plantations in the loess hilly region were investigated to study the dynamics and allocation patterns of carbon and nitrogen storage. The results showed that the ranges of carbon and nitrogen contents were 435.9-493.4 g x kg(-1) and 6.8-21.0 g x kg(-1) in the arbor layer, 396.3-459.2 g x kg(-1) and 14.2-23.5 g x kg(-1) in the herb and litter layer, and 2.7-10.7 g x kg(-1) and 0.2-0.7 g x kg(-1) in the soil layer, respectively. The branch was the major carbon and nitrogen pool in the arbor layer, accounting for 46.9%-63.3% and 39.3%-57.8%, respectively. The maximum storage values were 30.1 and 1.8 Mg x hm(-2) for carbon and nitrogen, respectively, in the 0-20 cm soil layer in the 37-year-old R. pseudoacacia plantation. The total carbon and nitrogen storage in the R. pseudoacacia plantation ecosystem increased with increasing forest age, and the maximum values were 127.9 Mg x hm(-2) and 6512.8 kg x hm(-2) for carbon and nitrogen storage, respectively, in the 37-year-old R. pseudoacacia plantation. Soil layer was the major carbon and nitrogen pool of R. pseudoacacia plantation ecosystem, accounting for 63.3%-83.3% and 80.3%-91.4%, respectively.

Tetraploid black locust (Robinia pseudoacacia L.) is adaptable to salt stress. Here, we compared morphological, physiological, ultrastructural, and proteomic traits of leaves in tetraploid black locust and its diploid relatives under salt stress. The results showed that diploid (2×) plants suffered from greater negative effects than those of tetraploid (4×) plants. After salt treatment, plant growth was inhibited, photosynthesis was reduced, reactive oxygen species, malondialdehyde content, and relative electrolyte leakage increased, and defense-related enzyme activities decreased in 2× compared to those in 4×. In addition, salt stress resulted in distorted chloroplasts, swollen thylakoid membranes, accumulation of plastoglobules, and increased starch grains in 2× compared to those in 4×. However, 4× developed diverse responses under salt stress. A comparative proteomic analysis revealed that 41 and 37 proteins were differentially expressed in 2× and 4×, respectively. These proteins were mainly involved in photosynthesis, stress and defense, energy, metabolism, transcription/translation, and transportation. Distinct patterns of protein changes between 2× and 4× were analyzed. Collectively, our results suggest that the plants showed significantly different responses to salt stress based on ploidy level of the plant. The 4× possessed a better salt protection mechanism than that of 2×, suggesting salt tolerance in the polyploid plant.

Emasculation and bagging of flowers, which are widely used in the controlled pollination of monoclinous plants, may induce premature senescence, flower abscission and low fruit set. To determine the mechanism responsible for these phenomena, levels of abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA), ethylene, soluble sugars, reducing sugars and free amino acids in black locust (Robinia pseudoacacia) flowers subjected to different treatments were quantified at different developmental stages. The phytohormones and assimilates were also quantified in untreated flowers to investigate the presence of discernible patterns. The levels of ethylene and ABA in emasculated and bagged (EB) flowers increased prematurely compared with those of untreated flowers, whereas the content of reducing sugars in EB flowers decreased compared with that of untreated flowers. These results indicated that the premature increase in ethylene and ABA synthesis, and the decrease in reducing sugars content, in EB flowers may cause flower abscission and result in low fruit set, which may be relevant for assimilate applications and future research on the regulation of controlled pollinations with exogenous phytohormones.

Using indigenous tree species to transform large area pure plantations has been an effective close-to-nature forest management mode in China islands. By using the biomass allometric equation and combined with the investigation data from sampling plots, the carbon storage of the dominant species Pinus thunbergii and Robinia pseudoacacia in the plantations on the Nanchangshan Island of Miaodao Archipelago in Changdao County of Shandong Province was estimated. The average carbon storage in the arbor layer of P. thunbergii and R. pseudoacacia plantations was 56.81 and 37.26 t x hm(-2), respectively, being higher than the average carbon stock (27.62 t x hm(-2)) of tree plantations in Shandong Province. Slope aspect and stand density were the important environmental and biological factors affecting the tree's carbon stock in the Island, respectively. There was no significant correlation between the average cumulative rate of biomass in the arbor layer of P. thunbergi plantation and the stand age. The P. thunbergii with good carbon sequestration function could be an ideal tree species on Nanchangshan Island.

This study aimed to identify drought-mediated differences in amino nitrogen (N) composition and content of xylem and phloem in trees having different symbiotic N(2)-fixing bacteria. Under controlled water availability, 1-year-old seedlings of Robinia pseudoacacia (nodules with Rhizobium), Hippophae rhamnoides (symbiosis with Frankia) and Buddleja alternifolia (no such root symbiosis) were exposed to control, medium drought and severe drought, corresponding soil water content of 70-75%, 45-50% and 30-35% of field capacity, respectively. Composition and content of amino compounds in xylem sap and phloem exudates were analysed as a measure of N nutrition. Drought strongly reduced biomass accumulation in all species, but amino N content in xylem and phloem remained unaffected only in R. pseudoacacia. In H. rhamnoides and B. alternifolia, amino N in phloem remained constant, but increased in xylem of both species in response to drought. There were differences in composition of amino compounds in xylem and phloem of the three species in response to drought. Proline concentrations in long-distance transport pathways of all three species were very low, below the limit of detection in phloem of H. rhamnoides and in phloem and xylem of B. alternifolia. Apparently, drought-mediated changes in N composition were much more connected with species-specific changes in C:N ratios. Irrespective of soil water content, the two species with root symbioses did not show similar features for the different types of symbiosis, neither in N composition nor in N content. There was no immediate correlation between symbiotic N fixation and drought-mediated changes in amino N in the transport pathways., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Here, we describe the draft genome sequence of Mesorhizobium amorphae strain CCNWGS0123, isolated from nodules of Robinia pseudoacacia growing on zinc-lead mine tailings. A large number of metal(loid) resistance genes, as well as genes reported to promote plant growth, were identified, presenting a great future potential for aiding phytoremediation in metal(loid)-contaminated soil.

Taking the pot-cultured seedlings of four leguminous tree species (Albizia julibrissin, Robinia pseudoacacia, Sophora japonica, and Gleditsia sinensis) as test materials, this paper studied their growth indices, critical salt concentration (C50), and K+ and Na+ allocation under different levels of NaCl stress, aimed to understand the difference of test tree species in salt tolerance. NaCl stress inhibited the seedling growth of the tree species. Under NaCl stress, the dry matter accumulation decreased, while the root/shoot ratio increased, especially for A. julibrissin and G. sinensis. Quadratic regression analysis showed that the C50 of A. julibrissin, R. pseudoacacia, S. japonica, and G. sinensis was 3.0 per thousand, 5.0 per thousand, 4.5 per thousand, and 3.9 per thousand, respectively, i.e., the salt tolerance of the four tree species was in the order of R. pseudoacacia > S. japonica > G. sinensis > A. julibrissin. In the root, stem, and leaf of the four tree species seedlings, the Na+ content increased with the increase of NaCl stress, while the K+ content (except in the root of A. julibrissin) decreased after an initial increase, resulting in a larger difference in the K+/Na+ ratio in the organs. Under the same NaCl stress, the allocation of Na+ in different organs of the four tree species seedlings decreased in the order of root>stem>leaf, while that of K+ differed with tree species and NaCl stress, and leaf was the main storage organ for K+. The K+/Na+ ratio in different organs decreased in the sequence of leaf>stem>root. R. pseudoacacia under NaCl stress accumulated more K+ and less Na+ in stem and leaf, and had higher K+/Na+ ratio in all organs and higher dry mass, being assessed to be more salt-tolerant. In contrast, A. julibrissin under high NaCl stress accumulated more Na+ in stem and leaf, and had a lower K+/Na+ ratio in all organs and lower dry mass, being evaluated to be lesser salt-tolerant. The K+ accumulation in seedling stem and leaf and the Na+ retention in seedling root could be the main reasons for the salt tolerance of leguminous tree species under NaCl stress.

A field experiment was conducted to study the leaf N, P, and chlorophyll contents, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters in pure Ulmus pumila forest, pure Robinia pseudoacacia forest, and U. pumila-R. pseudoacacia mixed forests [1:1 (1B1C), 1:2 (1B2C), and 2:1 (2B1C)] in different growth periods. From May to September, the plant leaf N and P contents in pure and mixed forests all presented a decreasing trend. By the end of growth period, the leaf N content of U. pumila and the P content of R. pseudoacacia in 1B2C were obviously higher than those in pure forests. In the mixed forests, the chlorophyll content of U. pumila was obviously higher than that of R. pseudoacacia, and the chlorophyll content of U. pumila in 1B2C reached the highest in July. The photosynthetic rate (Pn) of U. pumila and R. pseudoacacia in mixed forests was higher than that in pure forests, and the Pn of R. pseudoacacia in 1B2C reached the highest (18.54 micromol x m(-2) x s(-1)) in July. The transpiration rate (Tr) and stomatal conductance (Gs) of R. pseudoacacia in mixed forests were higher than those in pure forests, and the Tr and Gs in mixed forests were in the order of 1B2C>1B1C>2B1C. In September, the quantum yield of PSII electron transport (phi(PS II)) of U. pumila in mixed forests was obviously higher than that in pure forest. The photochemical quenching coefficients (q(P)) of U. pumila and R. pseudoacacia in pure and mixed forests had no significant difference, but the non-photochemical quenching coefficient (NPQ) of the two tree species in 1B2C was significantly lower than that in corresponding pure forests. It was suggested that mixed planting U. pumila and R. pseudoacacia could significantly improve the leaf nutrient contents and photosynthetic capacity of the two tree species, and the optimum mixed ratio of U. pumila and R. pseudoacacia was 1:2.