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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.

A key process in forest management planning is the estimation of tree volume and, more specifically, merchantable volume. The ability to predict the cumulative stem volume relative to any upper stem diameter on standing trees or stands is essential for forest inventories and the management of forest resources. In the 1980s, the Hellenic Public Power Corporation (HPPC) started the rehabilitation of lignite post-mining areas in Greece by planting mainly black locust ( Robinia pseudoacacia, L.). Today, these plantations occupy an area of approximately 2570 ha, but the stem volume has not yet been estimated. Therefore, we aimed to estimate the over- and under-bark stem volume using taper function models for 30 destructively sampled trees. Of the nineteen calibrated fixed-effects models, Kozak’s (2004) equation performed best for both the over-bark and under-bark datasets, followed by Lee’s (2003) and Muhairwe’s (1999) equations. Two fixed effect models were compared with fitted coefficients from Poland and the United States confirming that the local model fits were better suited, as the foreign model coefficients caused an increase in root mean square error (RMSE) for stem diameter predictions of 13% and 218%, respectively. The addition of random effects on a single-stem basis for two coefficients of Kozak’s (2004) equation improved the model fit significantly at 86% of the over-bark fixed effect RMSE and 69% for the under-bark model. Integrated taper functions were found to slightly outperform three volume equations for predictions of single stem volume over and under bark. Ultimately it was shown that these models can be used to precisely predict stem diameters and total stem volume for the population average as well as for specific trees of the black locust plantations in the study area.

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.

Key message High P increased the tolerance of R. pseudoacacia roots to salt stress. Salt is an important abiotic factor that restricts plant growth and development in soil. An appropriate concentration of P can increase plant tolerance to salt stress. We investigated the physiological and transcriptional regulatory effects of high P (HP) or low P (LP) on the response of R. pseudoacacia roots to salt stress . A pot experiment was carried out to grow R. pseudoacacia seedlings in vermiculite media supplemented with 0 mM, 150 mM or 300 mM NaCl under HP or LP conditions. The root dry weight and concentrations of free proline, P, ions, and phytohormones were measured, and the transcription of the genes was analyzed under NaCl stress under HP or LP conditions. The results revealed that R. pseudoacacia responds to NaCl stress by regulating the absorption and utilization of P and the levels of free proline, phytohormones and Na + , K + , Ca 2+ , and Mg 2+ as well as changing the expression levels of key genes. Compared with those under the LP condition, the roots of the R. pseudoacacia under the HP condition presented greater P concentrations, lower JA concentrations, and more stable K + levels when subjected to NaCl stress, which increased their tolerance to NaCl stress. Moreover, genes involved in the cell wall, root growth, root architecture regulation, biomass accumulation, stress response, osmotic regulation and ion balance maintenance were upregulated under NaCl stress under HP conditions. In addition, NaCl stress impairs N metabolism under LP conditions. Our findings provide new insights into the response of woody plants to salt stress under different P conditions and contribute to the development of scientific afforestation in saline–alkali areas.

Hydrogen sulphide (H2S), a gaseous signalling molecule, plays a multifaceted role in plant physiology by enhancing adaptability to environmental stresses. However, the regulatory mechanism of symbiotic nitrogen (N) fixation by H2S in indeterminate nodules of woody legumes remains unclear. In this study, we investigated the mechanism by which H2S promotes nodulation and N fixation in the woody legume Robinia pseudoacacia . Exogenous H2S significantly enhanced rhizobium infection, nodule formation and nitrogenase activity, demonstrating its positive role in the symbiotic process. Transcriptomic analysis of roots and nodules revealed that H2S signalling modulates auxin metabolism, particularly through the regulation of indole‐3‐acetic acid (IAA) homeostasis. H2S was found to promote free IAA accumulation and reduce IAA conjugation (IAA‐Asp and IAA‐Glu). Further investigation revealed that H2S directly targets GH3.1, a key IAA‐amido synthetase responsible for IAA conjugation. Specifically, H2S mediated persulfidation at Cys304 of GH3.1, inhibiting its enzymatic activity and preventing IAA inactivation. This modification was confirmed by LC–MS/MS, UPLC‐ESI‐MS/MS and site‐directed mutagenesis. This post‐translational modification maintained active IAA levels, facilitating early nodule development. These findings highlight the active role of H2S in regulating IAA homeostasis, thereby enhancing indeterminate nodule formation and N fixation through persulfidation of the Cys304 residue of GH3.1 in R. pseudoacacia . H2S persulfidates GH3.1 at Cys304, reducing its activity, sustaining IAA levels, triggering H2S‐IAA feedback, enhancing nodulation gene expression, boosting nodule formation, nitrogen fixation and Robinia pseudoacacia growth.

Potassium (K) in plants participates in a variety of physiological processes and is kept at a higher concentration than its soluble form in soil. Potassium solubilizing bacteria (KSB) release K from minerals. Arbuscular mycorrhizal (AM) fungi facilitate bacterial movement along their extraradical hyphae and improve plant K status. However, the interaction of KSB and AM fungi is rarely reported. This study aimed to isolate KSB and evaluate their interaction with AM fungi in promoting plant K uptake and growth. Soil was sampled from the rhizosphere of Robinia pseudoacacia in the southern area of the Loess Plateau, where soil available K is lower than plant demand. KSB from soil was isolated using a select medium in which K-feldspar is the only K source. KSB that showed an obvious dissolving circle and relatively high K solubilizing efficiency (over 20%) were isolated and identified. A pot experiment was conducted in a randomized design to evaluate the effect of KSB and AM fungi and their interaction. Four out of 12 isolated strains that showed high potassium solubilizing efficiency were from the genus Pseudomonas. Inoculation of KSB promoted the growth and K content of R. pseudoacacia. KSB showed a variety of (increase, decrease, and not-obvious) influences on the colonization status of R. irregularis. Co-inoculation of R. irregularis and KSB promoted plant growth, K content of the plant, and the available K in the growth substrate. This study provided a basis for the utilization of KSB and AM fungi as biofertilizers in the Loess Plateau.

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.

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.

Main Conclusion Permanent glandular trichomes of Robinia viscosa var. hartwigii produce viscous secretion containing several secondary metabolites, as lipids, mucilage, flavonoids, proteins and alkaloids. Robinia viscosa var. hartwigii (Hartweg’s locust) is an ornamental tree with high apicultural value. It can be planted in urban greenery and in degraded areas. The shoots, leaves, and inflorescences of this plant are equipped with numerous persistent glandular trichomes producing sticky secretion. The distribution, origin, development, morphology, anatomy, and ultrastructure of glandular trichomes of Hartweg's locust flowers as well as the localisation and composition of their secretory products were investigated for the first time. To this end, light, scanning, and transmission electron microscopy combined with histochemical and fluorescence techniques were used. The massive glandular trichomes differing in the distribution, length, and stage of development were built of a multicellular and multiseriate stalk and a multicellular head. The secretory cells in the stalk and head had large nuclei with nucleoli, numerous chloroplasts with thylakoids and starch grains, mitochondria, endoplasmic reticulum profiles, Golgi apparatus, vesicles, and multivesicular bodies. Many vacuoles contained phenolic compounds dissolved or forming various condensed deposits. The secretion components were transported through symplast elements, and the granulocrine and eccrine modes of nectar secretion were observed. The secretion was accumulated in the subcuticular space at the trichome apex and released through a pore in the cuticle. Histochemical and fluorescence assays showed that the trichomes and secretion contained lipophilic and polyphenol compounds, polysaccharides, proteins, and alkaloids. We suggest that these metabolites may serve an important function in protection of plants against biotic stress conditions and may also be a source of phytopharmaceuticals in the future.

Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45 days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.