Tight coupling between leaf δ13 C and N content along leaf ageing in the N2 -fixing legume tree black locust (Robinia pseudoacacia L.).

N2 -fixing legumes can strongly affect ecosystem functions by supplying nitrogen (N) and improving the carbon-fixing capacity of vegetation. Still, the question of how their leaf-level N status and carbon metabolism are coordinated along leaf ageing remains unexplored. Leaf tissue carbon isotopic composition (δ13 C) provides a useful indicator of time-integrated intrinsic water use efficiency (WUEi). Here, we quantified the seasonal changes of leaf δ13 C, N content on a mass and area basis (Nmass , Narea , respectively), Δ18 O (leaf 18 O enrichment above source water, a proxy of time-integrated stomatal conductance) and morphological traits in an emblematic N2 -fixing legume tree, the black locust (Robinia pseudoacacia L.), at a subtropical site in Southwest China. We also measured xylem, soil and rainwater isotopes (δ18 O, δ2 H) to characterize tree water uptake patterns. Xylem water isotopic data reveal that black locust primarily used shallow soil water in this humid habitat. Black locust exhibited a decreasing δ13 C along leaf ageing, which was largely driven by decreasing leaf Nmass , despite roughly constant Narea . In contrast, the decreasing δ13 C along leaf ageing was largely uncoupled from parallel increases in Δ18 O and leaf thickness. Leaf N content is used as a proxy of leaf photosynthetic capacity; thus, it plays a key role in determining the seasonality in δ13 C, whereas the roles of stomatal conductance and leaf morphology are minor. Black locust leaves can effectively adjust to changing environmental conditions along leaf ageing through LMA increases and moderate stomatal conductance reduction while maintaining constant Narea to optimize photosynthesis and carbon assimilation, despite declining leaf Nmass and δ13 C.

Does increasing forest age lead to greater trade-offs in ecosystem services? A study of a Robinia pseudoacacia artificial forest on the Loess Plateau, China.

Artificial forest ecosystems offer various ecosystem services (ES) and help mitigate climate change effects. Trade-offs or synergies exist among ES in artificial forests. Although forest age influences ES and ecosystem processes, the long-term dynamics of trade-offs among ES in artificial forests and during vegetation restorations remain unclear, complicating vegetation and sustainable management. We studied a Robinia pseudoacacia plantation on the Loess Plateau, China, with a restoration time of 10-44 years. The entropy weight method was used to assess five ES (carbon sequestration, water conservation, soil conservation, understory plant diversity, and runoff and sediment reduction) and investigate how ES change with forest age. The root mean square deviation (RMSD) was used to quantify the trade-offs among ES, and redundancy analysis (RDA) analysis was used to identify the key factors influencing the ES and trade-offs. The results showed that (1) as forest age increased, ES scores initially increased and then decreased. The optimal range for ES values was observed during the middle-aged to mature stages of the forest. (2) Before reaching maturity, the planted forests primarily delivered services related to water conservation and runoff and sediment reduction. (3) In young forests, ES showed a synergistic relationship (RMSD = 0.06), whereas trade-offs occurred in forests at other ages. The largest trade-off was observed in middle-aged forests. (4) The ES pairs with the dominant trade-offs in planted forests differed at different forest age stages. The largest trade-off occurred between carbon sequestration and water conservation (RMSD = 0.28). RDA analysis showed that understory vegetation coverage had a positive correlation with all ES. The ES indicators that significantly (P < 0.001) affected the water­carbon trade-off were tree carbon storage, soil organic carbon storage, soil total nitrogen, and soil total phosphorus. Thus, the water and carbon relationship must be balanced, and the key factors affecting ES trade-offs in forest management must be regulated to support ES multifunctionality.

Co-inoculation of rhizobia and AMF improves growth, nutrient uptake, and cadmium resistance of black locust grown in sand culture.

Rhizobia and arbuscular mycorrhizal fungi (AMF) are symbiotic microorganisms important for plants grown in nutrient-deficient and heavy metal-contaminated soils. However, it remains unclear how plants respond to the coupled stress by heavy metal and nitrogen (N) deficiency under co-inoculation. Here, we investigated the synergistic effect of Mesorhizobium huakuii QD9 and Funneliformis mosseae on the response of black locust (Robinia pseudoacacia L.) grown in sand culture to cadmium (Cd) under N deficiency conditions. The results showed that single inoculation of AMF improved the growth and Cd resistance of black locust, co-inoculation improved the most. Compared to non-inoculated controls, co-inoculation mediated higher biomass and antioxidant enzyme activity, reduced oxidative stress, and promoted nodulation, mycorrhizal colonization, photosynthetic capacity, and N, P, Fe and Mg acquisition when exposed to Cd. This increase was significantly higher under N deficiency compared to N sufficiency. In addition, the uptake of Cd by co-inoculated black locust roots increased, but Cd translocation to the above-ground decreased under both N deficiency and sufficiency. Thus, in the tripartite symbiotic system, not merely metabolic processes but also Cd uptake increased under N deficiency. However, enhanced Cd detoxification in the roots and reduced allocation to the shoot likely prevent Cd toxicity and rather stimulated growth under these conditions.

Significance of phosphorus deficiency for the mitigation of mercury toxicity in the Robinia pseudoacacia L.- rhizobia symbiotic association.

Nitrogen (N2)-fixing legumes can be used for phytoremediation of toxic heavy metal Mercury (Hg) contaminated soil, but N2-fixation highly relies on phosphorus (P) availability for nodule formation and functioning. Here, we characterized the significance of P deficiency for Hg accumulation and toxicity in woody legume plants. Consequences for foliar and root traits of rhizobia inoculation, Hg exposure (+Hg) and low P (-P) supply, individually and in combination were characterized at both the metabolite and transcriptome levels in seedlings of two Robinia pseudoacacia L. provenances originating from contrasting climate and soil backgrounds, i.e., GS in northwest and the DB in northeast China. Our results reveal that depleted P mitigates the toxicity of Hg at the transcriptional level. In leaves of Robinia depleted P reduced oxidative stress and improved the utilization strategy of C, N and P nutrition; in roots depleted P regulated the expression of genes scavenging oxidative stress and promoting cell membrane synthesis. Rhizobia inoculation significantly improved the performance of both Robinia provenances under individual and combined +Hg and -P by promoting photosynthesis, increasing foliar N and P content and reducing H2O2 and MDA accumulation despite enhanced Hg uptake. DB plants developed more nodules, had higher biomass and accumulated higher Hg amounts than GS plants and thus are suggested as the high potential Robinia provenance for future phytoremediation of Hg contaminated soils with P deficiency.

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

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

Temporal profiling of physiological, histological, and transcriptomic dissection during auxin-induced adventitious root formation in tetraploid Robinia pseudoacacia micro-cuttings.

MAIN CONCLUSION: Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.

Transcriptomic and Phenotypic Analyses Reveal the Molecular Mechanism of Dwarfing in Tetraploid Robinia pseudoacacia L.

Polyploid breeding techniques aid in the cultivation of new forestry cultivars, thus expanding the suite of strategies for the improvement of arboreal traits and innovation within the field of forestry. Compared to diploid Robinia pseudoacacia L. (black locust) 'D26-5①' (2×), its dwarfed homologous tetraploid 'D26-5②' (4×) variety has better application prospects in garden vegetation guardrails and urban landscape. However, the molecular mechanism of the generation and growth of this dwarf variety is still unclear. Here, plant growth and development as well as histological differences between the diploid and its autotetraploid were investigated. Levels of endogenous hormones at three different developmental stages (20, 40, and 70 days) of 2× and homologous 4× tissue culture plantlets were assessed, and it was found that the brassinosteroid (BR) contents of the former were significantly higher than the latter. Transcriptome sequencing data analysis of 2× and homologous 4× showed that differentially expressed genes (DEGs) were significantly enriched in plant hormone synthesis and signal transduction, sugar and starch metabolism, and the plant circadian rhythm pathway, which are closely related to plant growth and development. Therefore, these biological pathways may be important regulatory pathways leading to dwarfism and slow growth in tetraploids. Additionally, utilizing weighted gene coexpression network analysis (WGCNA), we identified three crucial differentially expressed genes (DEGs)-PRR5, CYP450, and SPA1-that potentially underlie the observed ploidy variation. This study provides a new reference for the molecular mechanism of dwarfism in dwarfed autotetraploid black locusts. Collectively, our results of metabolite analysis and comparative transcriptomics confirm that plant hormone signaling and the circadian rhythm pathway result in dwarfism in black locusts.

Antagonistic effect of mercury and excess nitrogen exposure reveals provenance-specific phytoremediation potential of black locust-rhizobia symbiosis.

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 (N2)-fixing legumes can be used for phytoremediation of Hg accumulation, whereas N availability could greatly affect its N2-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%) δ15N 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.

Effects of AMF inoculation on the growth, photosynthesis and root physiological morphology of root-pruned Robinia pseudoacacia seedlings.

Root pruning hinders the absorption and utilization of nutrients and water by seedlings in the short term. Arbuscular mycorrhizal fungi (AMF) are an important source of nutrient and water for seedlings except for the root system. However, the mechanism by which AMF affect the physiological growth of seedlings after root pruning has rarely been studied. In this study, a pot experiment was conducted through a three-compartment partition system to clarify the effects of Funneliformis mosseae (F. mosseae) strain BGC XJ07A on the physiological growth of root-pruned Robinia pseudoacacia seedlings. Five root pruning treatments (zero, one-fifth, one-fourth, one-third and one-half of the taproot length were removed) were applied to noninoculated seedlings and those inoculated with F. mosseae. The results showed that the presence of F. mosseae significantly increased the shoot and root biomasses, leaf photosynthetic rate, stomatal conductance and transpiration rate. The root projected area, root surface area, average root diameter, root density, root volume and number of root tips of the inoculated seedlings were higher than those without inoculation in all root pruning treatments. The root cytokinin, gibberellins and indole-3-acetic acid concentrations, but root abscisic acid concentration, were higher than those measured in the absence of inoculation in all root pruning treatments. Moreover, the changes in the root endogenous hormone concentrations of the seedlings were closely related to the root morphological development and seedling biomass. The AMF increased the soil available nitrogen, soil available phosphorus, soil available potassium and soil organic matter concentrations compared with the noninoculated treatment. These results indicate that AMF can alleviate the adverse effects of root pruning on the physiological growth of R. pseudoacacia and soil properties, and can provide a basis for AMF application to forest cultivation and the sustainable development of forest ecosystems.

Exogenous chelating agents influence growth, physiological characteristics and cell ultrastructure of Robinia pseudoacacia seedlings under lead-cadmium stress.

Heavy metal pollution of soil, especially by lead (Pb) and cadmium (Cd), is a serious problem worldwide. The application of safe chelating agents, combined with the growing of tolerant trees, constitutes an approach for phytoremediation of heavy-metal-contaminated soil. This study aimed to determine whether the two safe chelators, tetrasodium glutamate diacetate (GLDA) and citric acid (CA), could improve the phytoremediation capacity of black locust (Robinia pseudoacacia L.) in a Pb-Cd-contaminated soil and to find the key factors affecting the biomass accumulation of stressed black locust. In Pb- and Cd-stressed black locust plants, medium- and high-concentration GLDA treatment inhibited the growth, chlorophyll synthesis and maximum photochemical efficiency (Fv/Fm), promoted the absorption of Pb and Cd ions and resulted in the shrinkage of chloroplasts and starch grains when compared with those in Pb- and Cd-stressed plants that were not treated with GLDA. The effects of CA on plant growth, ion absorption, chlorophyll content, chlorophyll fluorescence and organelle size were significantly weaker than those of GLDA. The effect of both agents on Cd absorption was greater than that on Pb absorption in all treatments. The levels of chlorophyll a and plant tissue Cd and rates of starch metabolism were identified as the key factors affecting plant biomass accumulation in GLDA and CA treatments. In the future, GLDA can be combined with functional bacteria and/or growth promoters to promote the growth of Pb- and Cd-stressed plants and to further improve the soil restoration efficiency following pollution by heavy metals. Application of CA combined with the growing of black locust plants has great potential for restoring the Cd-polluted soil. These findings also provide insights into the practical use of GLDA and CA in phytoremediation by R. pseudoacacia and the tolerant mechanisms of R. pseudoacacia to Pb-Cd-contaminated soil.

Physiological tolerance of black locust (Robinia pseudoacacia L.) and changes of rhizospheric bacterial communities in response to Cd and Pb in the contaminated soil.

Woody plants possess great potential for phytoremediation of heavy metal-contaminated soil. A pot trial was conducted to study growth, physiological response, and Cd and Pb uptake and distribution in black locust (Robinia pseudoacacia L.), as well as the rhizosphere bacterial communities in Cd and Pb co-contaminated soil. The results showed that R. pseudoacacia L. had strong physiological regulation ability in response to Cd and Pb stress in contaminated soil. The total chlorophyll, malondialdehyde (MDA), soluble protein, and sulfhydryl contents, as well as antioxidant enzymes (superoxide dismutase, peroxidase, catalase) activities in R. pseudoacacia L. leaves under the 40 mg·kg-1 Cd and 1000 mg·kg-1 Pb co-contaminated soil were slightly altered. Cd uptake in R. pseudoacacia L. roots and stems increased, while the Pb content in the shoots of R. pseudoacacia L. under the combined Cd and Pb treatments decreased in relative to that in the single Pb treatments. The bacterial α-diversity indices (e.g., Sobs, Shannon, Simpson, Ace, and Chao) of R. pseudoacacia L. rhizosphere soil under Cd and Pb stress were changed slightly relative to the CK treatment. However, Cd and Pb stress could significantly (p < 0.05) alter the rhizosphere soil microbial communities. According to heat map and LEfSe (Linear discriminant analysis Effect Size) analysis, Bacillus, Sphingomonas, Terrabacter, Roseiflexaceae, Paenibacillus, and Myxococcaceae at the genus level were notably (p < 0.05) accumulated in the Cd- and/or Pb-contaminated soil. Furthermore, the MDA content was notably (p < 0.05) negatively correlated with the relative abundances of Isosphaeraceae, Gaiellales, and Gemmatimonas. The total biomass of R. pseudoacacia L. was positively (p < 0.05) correlated with the relative abundances of Xanthobacteraceae and Vicinamibacreraceae. Network analysis showed that Cd and Pb combined stress might enhance the modularization of bacterial networks in the R. pseudoacacia L. rhizosphere soil. Thus, the assembly of the soil bacterial communities in R. pseudoacacia L. rhizosphere may improve the tolerance of plants in response to Cd and/or Pb stress.

Broad environmental adaptation of abundant microbial taxa in Robinia pseudoacacia forests during long-term vegetation restoration.

Vegetation restoration has significant impacts on ecosystems, and a comprehensive understanding of microbial environmental adaptability could facilitate coping with ecological challenges such as environmental change and biodiversity loss. Here, abundant and rare soil bacterial and fungal communities were characterized along a 15-45-year chronosequence of forest vegetation restoration in the Loess Plateau region. Phylogenetic-bin-based null model analysis (iCAMP), niche breadth index, and co-occurrence network analysis were used to assess microbial community assembly and environmental adaptation of a Robinia pseudoacacia plantation under long-term vegetation restoration. The drift process governed community assembly of abundant and rare soil fungi and bacteria. With increasing soil total phosphorus content, the relative importance of drift increased, while dispersal limitation and heterogeneous selection exhibited opposite trends for abundant and rare fungi. Rare soil fungal composition dissimilarities were dominated by species replacement processes. Abundant microbial taxa had higher ecological niche width and contribution to ecosystem multifunctionality than rare taxa. Node property values (e.g., degree and betweenness) of abundant microbial taxa were substantially higher than those of rare microbial taxa, indicating abundant species occupied a central position in the network. This study provides insights into the diversity and stability of microbial communities during vegetation restoration in Loess Plateau. The findings highlight that abundant soil fungi and bacteria have broad environmental adaptation and major implications for soil multifunctionality under long-term vegetation restoration.

Consequences of excess urea application on photosynthetic characteristics and nitrogen metabolism of Robinia pseudoacacia seedlings.

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 N2-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.

Effects of stand density on the structure of soil microbial functional groups in Robinia pseudoacacia plantations in the hilly and gully region of the Loess Plateau, China.

Elucidating the responses of soil microbial functional groups to changes in stand density is crucial for understanding the sustainability of forest development. In this study, we obtained soil samples from Robinia pseudoacacia plantations of three different stand densities (low, middle, and high densities of 750, 1125, and 1550 trees ha-1, respectively) in the hilly and gully region of the Loess Plateau, China. We sought to determine the effects of stand density on the structure of soil microbial functional groups. Stand density had no significant effects on species diversity indices of fungal trophic modes or bacterial functional groups involved in carbon (C) cycling and nitrogen (N) cycling. However, differences in stand density substantially altered the composition of fungal functional groups. In low-density plantations, saprophytic fungi were the main trophic mode, with a high relative abundance of ∼62 %, whereas the fungal communities associated with middle- and high-density plantations were dominated by other fungi with a combined trophic mode, which accounted for ∼43 % and ∼41 % of the fungal trophic modes, respectively. Furthermore, we detected increases in the relative abundance of plant pathogens, nitrifiers, and nitrous oxide-denitrifying bacteria with increasing stand density. Results of the Monte Carlo test showed that soil pH influenced the composition of soil fungal (but not bacterial) groups. These findings suggested that a high density of trees might inhibit the decomposition of recalcitrant organic material and stimulate nitrous oxide emission, consequently decreasing soil nutrient availability and stimulating soil N loss. Moreover, high-density stands might increase the potential risk for plant disease. Overall, the present study suggested that reducing stand density to coverage between 750 and 1125 trees ha-1 would increase soil nutrient availability and prevent N loss from the soil. To verify these suppositions, further research is needed to determine the links between microbial functional groups composition and soil biogeochemistry.

The Rpf107 gene, a homolog of LOR, is required for the symbiotic nodulation of Robinia pseudoacacia.

MAIN CONCLUSION: Rpf107 is involved in the infection process of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The LURP-one related (LOR) protein family plays a pivotal role in mediating plant defense responses against both biotic and abiotic stresses. However, our understanding of its function in the symbiotic interaction between legumes and rhizobia remains limited. Here, Rpf107, a homolog of LOR, was identified in Robinia pseudoacacia (black locust). The subcellular localization of Rpf107 was analyzed, and its function was investigated using RNA interference (RNAi) and overexpression techniques. The subcellular localization assay revealed that Rpf107 was mainly distributed in the plasma membrane and nucleus. Rpf107 silencing prevented rhizobial infection and hampered plant growth. The number of infected cells in the nitrogen fixation zone of the Rpf107-RNAi nodules was also noticeably lower than that in the control nodules. Notably, Rpf107 silencing resulted in bacteroid degradation and the premature aging of nodules. In contrast, the overexpression of Rpf107 delayed the senescence of nodules and prolonged the nitrogen-fixing ability of nodules. These results demonstrate that Rpf107 was involved in the infection of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The findings reveal that a member of the LOR protein family plays a role in leguminous root nodule symbiosis, which is helpful to clarify the functions of plant LOR protein family and fully understand the molecular mechanisms underlying legume-rhizobium symbiosis.

Root distribution characteristics of monoculture and mixture of Pinus tabuliformis and Robinia pseudoacacia plantation.

In this study, we analyzed the vertical distribution characteristics of root biomass density, root length density, root surface area density in monoculture and mixture of Pinus tabuliformis and Robinia pseudoacacia plantations in Caijiachuan small watershed of Jixian County, Shanxi. We examined their relationships with soil physical and chemical properties in different stand types. The results showed that the total root biomass density of P. tabuliformis and R. pseudoacacia in mixture was more than 75% higher than that in monoculture. Root system of P. tabuli-formis mainly distributed in shallow layer (0-40 cm), while that of R. pseudoacacia was deeper (40-80 cm). Fine roots were predominant in different diameter classes. Length density and surface area density of fine roots were in the order of R. pseudoacacia in mixture > P. tabuliformis in mixture > R. pseudoacacia stand > P. tabuliformis stand. Root biomass density of fine roots was in the order of P. tabuliformis in mixture ＞ R. pseudoacacia in mixture ＞ P. tabuliformis stand ＞ R. pseudoacacia stand. In vertical profile, the total root and fine root biomass, root length and root surface area density of P. tabuliformis stand, R. pseudoacacia stand, P. tabuliformis in mixture and R. pseudoacacia in mixture showed a rule of decreasing with the increases of soil depth. Under different stand types, fine root length density, root biomass density and total root length density were positively correlated with soil total nitrogen, soil organic carbon, and soil water contents. Total root surface area density was significantly positively correlated with soil organic carbon, soil water content, and soil total nitrogen. The distribution of roots in pure and mixed stands of P. tabuliformis and R. pseudoacacia showed different patterns. Compared with the pure stand, the mixed stand had higher root biomass, soil nutrient contents, and soil water content.

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We investigated tree growth in Robinia pseudoacacia plantations at Ansai in Shaanxi Province and at Ji-xian in Shanxi Province by comparing the tree-ring width, basal area increase (BAI), δ13C value, intrinsic water-use efficiency (iWUE), and stomatal regulation. We quantified the responses of tree growth and iWUE to climatic factors at each site. The tree-ring width at Ansai and Jixian decreased with stand age, whereas the BAI at Ansai increased, and that at Jixian decreased after the BAI peaked. The δ13C value and iWUE of trees at Jixian were higher than those at Ansai. The iWUE of trees at both sites was similar to the constant intercellular CO2 concentration/atmospheric CO2 concentration (Ci/Ca) scenario, indicating that the Ci of trees was elevated with increasing Ca, while the stomata remained open. The BAI at Ansai was significantly positively correlated with highest temperature in May, relative humidity in June, precipitation in August, relative humidity in September, and standardized precipitation evapotranspiration index (SPEI) in September and October of current year, but negatively correlated with temperature in June. The BAI at Jixian was significantly positively correlated with SPEI in June and July, and lowest temperature in October of current year. The iWUE of trees at Ansai was significantly positively correlated with relative humidity and precipitation in June of the current year, but negatively correlated with minimum temperature in May, relative humidity in June, and temperature and maximum temperature in July of current year. A significant positive correlation between iWUE of trees at Jixian and lowest temperature in June of current year was detected. At the annual scale, the BAI of trees at Ansai was positively correlated with precipitation and SPEI, but no significant relationship was observed for trees at Jixian. However, the iWUE of trees at both sites was significantly affected by precipitation. Path analysis showed that SPEI and minimum temperature had a direct effect on BAI and iWUE of trees at Ansai, whereas precipitation and average temperature indirectly affected BAI and iWUE through SPEI. The highest temperature had a direct effect on tree growth at Jixian, whereas precipitation, minimum temperature, and average temperature had direct effects on iWUE. These results suggested that SPEI was the main climatic factor that affected the growth of R. pseudoacacia, while Ci was an important physiological factor. Our results could provide reference for the protection and management of R. pseudoacacia plantations under climate change.

Divergent patterns and drivers of leaf functional traits of Robinia pseudoacacia and Pinus tabulaeformis plantations along a precipitation gradient in the Loess plateau, China.

Changes in precipitation patterns in arid and semi-arid regions can reshape plant functional traits and significantly affect ecosystem functions. However, the synchronous responses of leaf economical, anatomical, photosynthetic, and biochemical traits to precipitation changes and their driving factors have rarely been investigated, which hinders our understanding of plants' ecological adaptation strategies to drought tolerance in arid areas. Therefore, the leaf traits of two typical plantations (Robinia pseudoacacia, RP and Pinus tabulaeformis, PT) along the precipitation gradient in the Loess Plateau, including economical, anatomical, photosynthetic, and biochemical traits, were investigated in this study. The results show that the leaf photosynthetic traits of RP and PT increase along the precipitation gradient, whereas leaf biochemical traits decrease. The anatomical traits of PT decrease with increasing precipitation, whereas no significant variation was observed for RP. Random Forest analysis show that LNC, LDMC, Chl, and PRO are leaf traits that significantly vary with the precipitation gradient in both plantations. Correlation analysis reveals that the traits coordination of RP is better than that of PT. The LMG model was used to determine driving factors. The results suggest that MAP explains the variation of PT leaf traits better (30.38%-36.78%), whereas SCH and SPH contribute more to the variation of RP leaf traits (20.88%-41.76%). In addition, the piecewise Structural Equation Model shows that the climate and soil physical and chemical properties directly affect the selected leaf functional traits of RP, whereas only the soil chemical properties directly affect the selected leaf functional traits of PT. The results of this study contribute to the understanding of the ecological adaptation of plants to environmental gradients and highlight that correlations among leaf traits should be considered when predicting plant adaptation strategies under future global change scenarios.

Green synthesis of fluorescent carbon dots from Robinia hispida L. leaves for selective detection of Hg (II).

In this study, Robinia hispida L leaves (RH) was used as a precursor for the first time to synthesize fluorescent carbon dots (CDs) with stable blue fluorescence by a single-step hydrothermal synthesis method. Notably, the innovative approach eliminates the necessity for toxic chemicals or hazardous substances, marking a significant advancement in the field. The synthesized CDs demonstrate CDs demonstrates the predominance of spherical shapes with an average size of 11.63 ± 1.92 nm. The CDs not only exhibit an enhanced fluorescent efficiency with a relatively high quantum yield of up to 6.8%, but they also possess the potential for direct utilization in the selective determination of Hg(II) through fluorescence quenching, even without any functionalization. Under the optimized conditions at a pH of 7.0, a robust linear correlation was found to exist between the fluorescence intensity and the concentration of Hg (II) within the range of 5-17.5µM, exhibiting a detection limit (3σ) of 1.5µM. Additionally, this methodology was effectively employed to successfully detect Hg (II) ions in various aqueous samples, including tap water, spring water, drinking water, and a certified reference material (CRM-SA-C Sandy Soil C). The spike recoveries of 97.6%-101.6% with less than 2.7% variability were performed on all samples.

Adaptive response of flavonoids in Robinia pseudoacacia L. affected by the contamination of cadmium and elevated CO2 to arbuscular mycorrhizal symbiosis.

As a key component in non-enzyme resistance system, flavonoids play a crucial role in the plant growth and defenses, which are significantly affected by biotic and abiotic factors such as fungi, bacteria, viruses, heavy metals, and atmospheric CO2. Arbuscular mycorrhizal fungi (AMF) play an important role in enhancing plant tolerance to adverse environments, which can significantly affect the synthesis of flavonoids by forming mycorrhizal symbionts with plant roots. However, few studies explored the combined effects of AMF, elevated CO2, and heavy metals on flavonoids in plants. Here, we investigated the adaptive response of flavonoids accumulation in Robinia pseudoacacia L. seedlings affected by the contamination of cadmium (Cd) and elevated CO2 to arbuscular mycorrhizal symbiosis. The results showed that G. mosseae decreased (p < 0.05) Cd content in leaves by 62.2% under elevated CO2. Moreover, G. mosseae colonization led to significant decreases in robinin, quercetin, kaempferol and acacetin by 17.4%, 11.1%, 15.5% and 23.1% under elevated CO2 + Cd, respectively. Additionally, G. mosseae down-regulated (p < 0.05) expression levels of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes under elevated CO2 + Cd, and CHS and uridine diphosphate flavonoid glucosyltransferase (UFGT) activities decreased (p < 0.05). Quercetin, kaempferol and acacetin showed positive (p < 0.05) correlation with PAL and CHS genes expression and PAL, CHS, and UFGT activities. Cadmium, C/N ratio, carotenoids, leaf biomass, total chlorophyll, P, and starch in leaves and G. mosseae colonization rate in roots influenced (p < 0.05) flavonoids content. Overall, G. mosseae reduced flavonoids synthesis by down-regulating gene expression levels and activities of key enzymes under elevated CO2 + Cd. The results improved our understanding of the regulation of AMF on non-enzymatic resistance of plants grown in heavy metal-contaminated soils under increasing atmospheric CO2 scenarios.