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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.)

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).)

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.)

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.)

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.)

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.

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.)

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.)

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 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.)

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 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.

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.

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.

A lectin has been identified in black locust (Robinia pseudoacacia) bark that shares approximately 50% sequence identity with plant class V chitinases but is essentially devoid of chitinase activity. Specificity studies indicated that the black locust chitinase-related agglutinin (RobpsCRA) preferentially binds to high-mannose N-glycans comprising the proximal pentasaccharide core structure. Closely related orthologs of RobpsCRA could be identified in the legumes Glycine max, Medicago truncatula, and Lotus japonicus but in no other plant species, suggesting that this novel lectin family most probably evolved in an ancient legume species or possibly an earlier ancestor. This identification of RobpsCRA not only illustrates neofunctionalization in plants, but also provides firm evidence that plants are capable of developing a sugar-binding domain from an existing structural scaffold with a different activity and accordingly sheds new light on the molecular evolution of plant lectins.

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.