The combined effects of elevated atmospheric CO2 and cadmium exposure on flavonoids in the leaves of Robinia pseudoacacia L. seedlings.

Flavonoids participate in several plant processes such as growth and physiological protection in adverse environments. In this study, we investigated the combined effects of eCO2 and cadmium (Cd)-contaminated soils on the total flavonoid and monomer contents in the leaves of Robinia pseudoacacia L. seedlings. Elevated CO2, Cd, and eCO2+ Cd increased the total flavonoids in the leaves relative to the control, and eCO2 mostly increased (p < 0.05) the total flavonoid content under Cd exposure. Elevated CO2 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 eCO2 on day 135. The responses of total chlorophyll, total soluble sugars, starch, C, N, S, and the C/N ratio in the leaves to eCO2 significantly affected the synthesis of total flavonoids and monomers under Cd exposure. Overall, rutin was more sensitive to eCO2+ Cd than the other flavonoids. Cadmium, CO2, and time had significant interactive effects on the synthesis of flavonoids in the leaves of R. pseudoacacia L. seedlings. Elevated CO2 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 eCO2 could improve the phytoremediation of Cd-contaminated soils.

Effects of sodium sulfide application on the growth of Robinia pseudoacacia, heavy metal immobilization, and soil microbial activity in Pb-Zn polluted soil.

Sodium sulfide (Na2S) is usually used as an amendment in industrial sewage treatment. To evaluate the effects of Na2S 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. Na2S application increased the shoot biomass under severe and mild contamination. In soil, Na2S 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, Na2S 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 Na2S application. Increased phosphatase activity and increased available phosphorous content may promote the uptake of phosphorus in R. pseudoacacia. Moreover, Na2S application is beneficial to the diversity of AM fungi under mild and severe pollution. Overall, Na2S 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, Na2S is suitable for use in Pb-Zn remediation to ameliorate environmental heavy metal pollution.

Increased nitrogen supply promoted the growth of non-N-fixing woody legume species but not the growth of N-fixing Robinia pseudoacacia.

Nitrogen (N) is an essential macronutrient for plant development and growth, and the deposition of N has increased in recent decades. Legumes that fix N can also provide N for nearby species. However, N in soil inhibits N fixation. We tested the effects of N fertilisation on one N-fixing (Robinia pseudoacacia) and two non-N-fixing (Sophora japonica and Senna surattensis) woody legume species, which were subjected to five different N levels (0, 1.5, 2.9, 5.9 and 11.4 mg N per plant day-1) under greenhouse conditions. The growth of the two non-N-fixing species was promoted by N supply, while that of R. pseudoacacia was unaffected. Among the three species, R. pseudoacacia had the largest specific leaf area and chlorophyll concentration, S. japonica had the largest root-to-shoot ratio and main root-to-lateral root ratio, and S. surattensis had the largest leaf N and phosphorus concentrations. The N-fixing species was mostly unaffected by N supply. The growth, leaf chlorophyll concentration, and leaf number in the non-N-fixing species were promoted by N supply. The N-fixing species showed better growth in low-N environments, while under increased N deposition, its growth was similar to that of the non-N-fixing species.

Three years of exposure to lead and elevated CO2 affects lead accumulation and leaf defenses in Robinia pseudoacacia L. seedlings.

Few studies have explored the long-term effects of elevated atmospheric CO2 combined with lead (Pb) contamination on plants. The objective of this study was to examine the effects of 3 years of elevated CO2 (700 ±â€¯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 CO2 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 CO2 than under ambient CO2, but seedling height and width increased under elevated CO2 relative to ambient CO2. Elevated CO2 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 CO2 + Pb relative to Pb alone. Elevated CO2 stimulated the production of soluble sugars, proline, flavonoids, saponins, and phenolics in plants exposed to Pb stress. Ove rall, long-term elevation of CO2 increased Pb-induced oxidative damage in seedlings, but enhanced the phytoextraction of Pb from contaminated soils.

Leaf defense system of Robinia pseudoacacia L. seedlings exposed to 3years of elevated atmospheric CO2 and Cd-contaminated soils.

Short-term exposure to elevated CO2 increases cadmium (Cd) uptake in some plant species (wheat, poplars, and willows), which triggers an increase in antioxidative system activity to deal with additional reactive oxygen species that are generated. Here, we examined leaf defenses in Robinia pseudoacacia L. seedlings exposed to elevated CO2+Cd for 3years. Three years of elevated CO2 decreased Cd uptake into leaves and the Cd content in soils and increased the pH of rhizosphere soil relative to ambient CO2. In plants exposed to Cd stress, leaf chlorophyll content was greater under elevated CO2 than under ambient CO2. Superoxide dismutase, peroxidase, and catalase activity increased, glutathione content increased, and malondialdehyde and phytochelatins contents decreased under elevated CO2+Cd relative to Cd alone. Proline, soluble sugars, flavonoids, saponins, and phenolic acids contents were greater under elevated CO2+Cd than under Cd alone, and condensed tannin content was lower. Overall, long-term elevation of CO2 enhanced the leaf defense system of R. pseudoacacia exposed to Cd by stimulating antioxidant enzyme activity, osmotic adjustment, and the production of glutathione, flavonoids and phenolic acids. Future research should focus on understanding the mechanisms involved in the decrease in Cd uptake into leaves and Cd content in soils and the increase in rhizosphere soil pH under long-term exposure to elevated CO2.

Plant growth in amended molybdenum mine waste rock.

This greenhouse study examined the use of organic and inorganic soil amendments in waste rock material from the former Questa Molybdenum Mine in northern New Mexico to promote beneficial soil properties. Waste rock material was amended with 11 soil amendment treatments that included municipal composted biosolids, Biosol®, inorganic fertilizer, and two controls (pure waste rock and sand). Elymus trachycaulus and Robinia neomexicana growth performance and plant chemistry were assessed across all treatments over a period of 99 and 141 days, respectively. Even though waste rock material had more than 200 times the molybdenum concentration of native soils, adverse effects were not observed for either species. The two main limiting factors in this study were soil nutritional status and soil water retention. The biosolid amendment was found to provide the greatest buffer against these limiting factors due to significant increases in both nutrition and soil water retention. As a result, both species responded with the highest levels of biomass production and the least amount of required water demands. Use of organic amendments such as biosolids, even though short lived in the soil, may provide plants the necessary growth stimulus to become more resilient to the harsh conditions found on many mine reclamation sites.

De novo sequencing and comparative transcriptome analysis of adventitious root development induced by exogenous indole-3-butyric acid in cuttings of tetraploid black locust.

BACKGROUND: Indole-3-butyric acid (IBA) is applied to the cuttings of various plant species to induce formation of adventitious roots (ARs) in commercial settings. Tetraploid black locust is an attractive ornamental tree that is drought resistant, sand tolerant, can prevent sand erosion and has various commercial uses. To further elucidate the mechanisms of AR formation, we used Illumina sequencing to analyze transcriptome dynamics and differential gene expression at four developmental stages in control (CK) and IBA-treated groups. RESULTS: The short reads were assembled into 127,038 unitranscripts and 101,209 unigenes, with average lengths of 986 and 852 bp. In total, 10,181 and 14,924 differentially expressed genes (DEGs) were detected in the CK and IBA-treated groups, respectively. Comparison of the four consecutive developmental stages showed that 282 and 260 DEGs were shared between IBA-treated and CK, suggesting that IBA treatment increased the number of DEGs. We observed 1,721 up-regulated and 849 down-regulated genes in CI vs. II, 849 up-regulated and 836 down-regulated genes in CC vs. IC, 881 up-regulated and 631 down-regulated genes in CRP vs. IRP, and 5,626 up-regulated and 4,932 down-regulated genes in CAR vs. IAR, of which 25 up-regulated DEGs were common to four pairs, and these DEGs were significantly up-regulated at AR. These results suggest that substantial changes in gene expression are associated with adventitious rooting. GO functional category analysis indicated that IBA significantly up- or down-regulated processes associated with regulation of transcription, transcription of DNA dependent, integral to membrane and ATP binding during the development process. KEGG pathway enrichment indicated that glycolysis/gluconeogenesis, cysteine and methionine metabolism, photosynthesis, nucleotide sugar metabolism, and lysosome were the pathways most highly regulated by IBA. We identified a number of differentially regulated unigenes, including 12 methionine-related genes and 12 ethylene-related genes, associated with the KEGG pathway cysteine and methionine metabolism. The GO enrichment, pathway mapping, and gene expression profile analyses revealed molecular traits for root induction and initiation. CONCLUSION: Our study presents a global view of the transcriptomic profiles of tetraploid black locust cuttings in response to IBA treatment and provides new insights into the fundamental mechanisms associated with auxin-induced adventitious rooting.

Elevated CO2 affects secondary metabolites in Robinia pseudoacacia L. seedlings in Cd- and Pb-contaminated soils.

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.

Copper tolerance mechanisms of Mesorhizobium amorphae and its role in aiding phytostabilization by Robinia pseudoacacia in copper contaminated soil.

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.

Molecular cloning, characterization and expression analysis of the SAMS gene during adventitious root development in IBA-induced tetraploid black locust.

S-Adenosylmethionine synthetase (SAMS) catalyzes the synthesis of S-adenosylmethionine (SAM), a precursor for ethylene and polyamine biosynthesis. Here, we report the isolation of the 1498 bp full-length cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) SAMS (TrbSAMS), which contains an open reading frame of 1179 bp encoding 392 amino acids. The amino acid sequence of TrbSAMS has more than 94% sequence identity to SAMSs from other plants, with a closer phylogenetic relationship to SAMSs from legumes than to SAMS from other plants. The TrbSAMS monomer consists of N-terminal, central, and C-terminal domains. Subcellular localization analysis revealed that the TrbSAMS protein localizes mainly to in the cell membrane and cytoplasm of onion epidermal cells and Arabidopsis mesophyll cell protoplasts. Indole-3-butyric acid (IBA)-treated cuttings showed higher levels of TrbSAMS transcript than untreated control cuttings during root primordium and adventitious root formation. TrbSAMS and its downstream genes showed differential expression in shoots, leaves, bark, and roots, with the highest expression observed in bark. IBA-treated cuttings also showed higher SAMS activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbSAMS might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings.

Effects of soil water and nitrogen availability on photosynthesis and water use efficiency of Robinia pseudoacacia seedlings.

The efficient use of water and nitrogen (N) to promote growth and increase yield of fruit trees and crops is well studied. However, little is known about their effects on woody plants growing in arid and semiarid areas with limited water and N availability. To examine the effects of water and N supply on early growth and water use efficiency (WUE) of trees on dry soils, one-year-old seedlings of Robinia pseudoacacia were exposed to three soil water contents (non-limiting, medium drought, and severe drought) as well as to low and high N levels, for four months. Photosynthetic parameters, leaf instantaneous WUE (WUEi) and whole tree WUE (WUEb) were determined. Results showed that, independent of N levels, increasing soil water content enhanced the tree transpiration rate (Tr), stomatal conductance (Gs), intercellular CO2 concentration (Ci), maximum net assimilation rate (Amax), apparent quantum yield (AQY), the range of photosynthetically active radiation (PAR) due to both reduced light compensation point and enhanced light saturation point, and dark respiration rate (Rd), resulting in a higher net photosynthetic rate (Pn) and a significantly increased whole tree biomass. Consequently, WUEi and WUEb were reduced at low N, whereas WUEi was enhanced at high N levels. Irrespective of soil water availability, N supply enhanced Pn in association with an increase of Gs and Ci and a decrease of the stomatal limitation value (Ls), while Tr remained unchanged. Biomass and WUEi increased under non-limiting water conditions and medium drought, as well as WUEb under all water conditions; but under severe drought, WUEi and biomass were not affected by N application. In conclusion, increasing soil water availability improves photosynthetic capacity and biomass accumulation under low and high N levels, but its effects on WUE vary with soil N levels. N supply increased Pn and WUE, but under severe drought, N supply did not enhance WUEi and biomass.

Using the pollen viability and morphology for fluoride pollution biomonitoring.

The methods using plants for biomonitoring of air and soil quality are simple, cheap, and fast and can supplement the classical physicochemical methods. In this study, biological pollen characterization of some collected legume species from an aluminum smelter area in Iran (IRALCO) was carried out to determine the actual value of pollen as a bioindicator of the effects of soil and atmospheric pollution. Young buds and flowers of six legumes (Cercis siliquastrum L., Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam, Trifolium repens L., and Sophora alopecuroides L.) in polluted and control plants were removed and compared. Studies of light and electron microscopic preparation showed some abnormalities during pollen development in affect of fluoride pollution. The viability of pollen grains estimated by staining with acetocarmine shows sharp differences in smearing advanced pollen grains from abnormal ones. Except M. officinalis, the pollen grains of C. siliquastrum, M. sativa, R. pseudoacacia, T. repens, and S. alopecuroides in polluted areas showed light, partial, or no staining with acetocarmine, whereas almost all of the control ones clearly stained. Observation of the pollen grains by light microscopy and scanning electron microscopy showed the significant effect of fluoride on shapes and sizes of pollen grains. The stimulation and inhibition of these pollen characteristics depend on the pollen species as well as on the pollutant and its concentration. Therefore, pollen grains provide essential information on biological impact of pollutants and they are good candidates for biomonitoring the atmospheric and edaphic pollutions.

Long-term impact of municipal sewage irrigation on treated soil and black locust trees in a semi-arid suburban area of Iran.

The effects of municipal sewage irrigation on the soil and black locust (Robinia pseudoacacia L.) tree were studied. For this purpose, two artificial black locust stands under irrigation of municipal sewage and well water were selected in south of Tehran, Iran. Data were collected using technique of systematic random sampling with 4 replicates in each stand. It was found that the growth of black locust tree, as indicated by diameter at breast height, total height, crown length, average crown diameter, basal area and volume, in sewage irrigation stand was much higher than that of well water irrigation stand (P < 0.01). Plant analysis indicated that concentrations of leaf nutrients (N, P, K, Ca, Mg, Na, Fe, Mn, Cu and Zn) were greater in sewage-irrigated trees, without toxicity to the minerals of tree leaf, than those of well waterirrigated trees, and positively correlated with their respective value in soil. Ni, Cr and Pb were not detected in leaf samples. Application of sewage resulted in a 1.5-fold increase in the concentrations of soil nutrients, Ni, Cr and Pb. Among these minerals only Pb and Ni in some soil samples exceeded the toxicity limit. The increase in pH, electrical conductivity (EC) and organic carbon of soil was also observed in sewage irrigation. Results confirm that besides the use as irrigation water, municipal sewages are also a potential source of plant nutrients. However, significant accumulation of heavy metals such as Pb and Ni in soil needs to be monitored.

An effective method for axillary bud culture and RAPD analysis of cloned plants in tetraploid black locust.

An efficient micropropagation technique by axillary bud multiplication was established for cloning tetraploid black locust tree (Robinia pseudoacacia L.). The result showed that the optimal medium for shoot multiplication and elongation was Murashige and Skoog (MS) medium supplemented with 0.5 mg/l 6-benzylaminopurine in combination with 0.5 mg/l kinetin and 0.1 mg/l 1-naphthaleneacetic acid. The best medium for rooting was half-strength MS medium with 0.25 mg/l indole-3-butyric acid. In the present report, we examined the genetic fidelity of the micropropagated plants by the randomly amplified polymorphic DNA (RAPD) method with 25 primers. The cloned plants of tetraploid black locust showed complete stability.