Leaf movement and photosynthetic plasticity of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) alleviate stress under different light and water conditions

Leaf morphological, physiological and biochemical characteristics of Robinia pseudoacacia L. seedlings were studied under different stress conditions. The plants were subjected to drought and shade stress for one month. Leaf inclination, chlorophyll fluorescence and chlorophyll content were measured at the first day (short-term stress) and at the end of the stress period (long-term stress) and in the recovery period. Leaf inclination was affected mainly by light; a low level of irradiance caused leaves to be arranged horizontally. Diurnal rhythmicity was lost after the long-term stress, but resumed, in part, in the recovery period. Drought stress caused leaves to tilt more obviously and decreased damage to the photosystem. Sun avoiding movement in a single leaf and sun tracking movement in the whole plant coexisted. Significant physiological changes occurred under different conditions of light. Increased energy dissipation and light capture were the main responses to high and low level of irradiance, respectively, and these were reflected by changes of chlorophyll fluorescence and chlorophyll content. Phenotypic plasticity in the leaflet enhanced the protective response to stress. These adaptive mechanisms may explain better survival of R. pseudoacacia seedlings in the understory, especially during the drought periods, and made it to be the preponderant reforestation species in Shandong Province of China.     

Analysis of interactions between the invasive tree-of-heaven (<Emphasis Type="Italic">Ailanthus altissima</Emphasis>) and the native black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>)

Invasive exotic plants can persist and successfully spread within ecosystems and negatively affect the recruitment of native species. The exotic invasive Ailanthus altissima and the native Robinia pseudoacacia are frequently found in disturbed sites and exhibit similar growth and reproductive characteristics, yet each has distinct functional roles such as allelopathy and nitrogen fixation, respectively. A four-month full additive series in the greenhouse was used to analyze the intraspecific and interspecific interference between these two species. In the greenhouse experiment, the inverse of the mean total biomass (g) response per plant for each species was regressed on the density of each species and revealed that the performance of the plants was significantly reduced by interspecific interference and not by intraspecific interference (p < 0.05). Other biomass traits such as root dry weight, shoot dry weight, stem dry weight, and leaf dry weight were also negatively affected by interspecific interference. Competition indices such as Relative Yield Total and Relative Crowding Coefficient suggested that A. altissima was the better competitor in mixed plantings. Ailanthus altissima consistently produced a larger above ground and below ground relative yield while R. pseudoacacia generated a larger aboveground relative yield in high density mixed species pots.     

Investigations on influencing plant-associated bacteria in tissue cultures of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.)

During tissue culture of black locust (Robinia pseudoacacia L.), serious problems with plant-associated bacteria led to a reduction of propagation potential in several clones. Four dominant strains of plant-associated bacteria could be isolated and were assigned to the genera Acidovorax, Dyella, Microbacterium and Sphingomonas. Out of five essential oils tested, thyme and lemongrass oil at a concentration of 0.03% each and 0.015% of both oils in combination clearly inhibited the growth of these bacteria strains on bacteriologic medium. There were no significant differences in total bacterial population density when penicillin, thyme and lemongrass oil or thyme plus lemongrass oil were added to the plant propagation media. The use of lemongrass oil changed the proportion of dominant bacterial strains.     

Methyl jasmonate regulated diploid and tetraploid black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) tolerance to salt stress

Methyl jasmonate (MeJA) is an essential and promising plant growth regulation factor that can improve plant development and growth. Here, we explored the mechanism by which MeJA regulates the tolerance of black locust (Robinia pseudoacacia L.) to salt stress. In this study, diploid and tetraploid R. pseudoacacia were subjected to three treatments: 500 mM NaCl; 100 μM MeJA; and 500 mM NaCl and 100 μM MeJA, and the changes in plant growth, endogenous MeJA levels and the anti-oxidative metabolism of leaves were investigated. The results showed that salt stress significantly inhibited plant growth and induced the accumulation of Na⁺ and Cl^? ions, malondialdehyde (MDA) content and reactive oxygen species. However, these adverse effects could be alleviated by applying MeJA, which was followed by a marked increase in the activities of antioxidant enzymes. In addition, some genes encoding several antioxidant enzymes were also up-regulated. Simultaneously, the endogenous MeJA content in MeJA-treated plants was lower than in salt-treated plants. It is noteworthy that tetraploids always possessed higher salt tolerance and obtained greater positive effects from MeJA than diploids. These results suggested that MeJA might play a protective role in defense responses, enabling diploid and tetraploid black locust, especially tetraploid, to better tolerate the adverse effects of salt stress.     

Characterization of Phages Virulent for <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> Rhizobia

Three lytic phages (ΦRP1, ΦRP2, and ΦRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100–200 phage particles per one infected cell) and rise periods (90–120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4–93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.     

Cryopreservation of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Water entry for the black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) seeds observed by dedicated micro-magnetic resonance imaging

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

Plant regeneration in <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> from cell suspension cultures

A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without agar. Single cells were isolated after 3 d and the optimum cell density was 1–3 × 10⁴ cells per cm³ of the liquid MS medium. Plating efficiency was 29.6 % and callus formed within 4 weeks was subcultured and transferred to solid MS medium supplemented with 0.6 mg dm⁻³ benzyladenine (BA) along with 0.05 mg dm⁻³ α-naphthalene-1-acetic acid (NAA) for the induction of adventitious bud primordia. The shoots developed were isolated and re-cultured on MS medium containing 0.6 mg dm⁻³ BA. These microshoots after dipping in 1–2 cm³ of 10 mg dm⁻³ indole-3-butyric acid (IBA) for 24 h in dark were cultured on half strength solid MS medium supplemented with 0.05 % charcoal and showed 80–82 % rooting within 4 weeks.     

<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> in Poland and Japan is nodulated by <Emphasis Type="Italic">Mesorhizobium amorphae</Emphasis> strains

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91–98% and 90–98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51–75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61–64 mol% across M. amorphae strains.     

Photosynthetic characteristics and nitrogen allocation in the black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) grown in a FACE system

Key message

The black locust is adapted to elevated [CO ₂ ] through changes in nitrogen allocation characteristics in leaves.

Abstract

The black locust (Robinia pseudoacacia L.) is an invasive woody legume within Japan. This prolific species has a high photosynthetic rate and growth rate, and undergoes symbiosis with N₂-fixing micro-organisms. To determine the effect of elevated CO₂ concentration [CO₂] on its photosynthetic characteristics, we studied the chlorophyll (Chl) and leaf nitrogen (N) content, and the leaf structure and N allocation patterns in the leaves and acetylene reduction activity after four growing seasons, in R. pseudoacacia. Our specimens were grown at ambient [CO₂] (370 μmol mol^?1) and at elevated [CO₂] (500 μmol mol^?1), using a free air CO₂ enrichment (FACE) system. Net photosynthetic rate at growth [CO₂] (A _growth) and acetylene reduction activity were significantly higher, but maximum carboxylation rate of RuBisCo (V _cmax), maximum rate of electron transport driving RUBP regeneration (J _max), net photosynthetic rate under enhanced CO₂ concentration and light saturation (A _max), the N concentration in leaf, and in leaf mass per unit area (LMA) and ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo) content were significantly lower grown at elevated [CO₂] than at ambient [CO₂]. We also found that RuBisCo/N were less at elevated [CO₂], whereas Chl/N increased significantly. Allocation characteristics from N in leaves to photosynthetic proteins, N_L (Light-harvesting complex: LHC, photosystem I and II: PSI and PSII) and other proteins also changed. When R. pseudoacacia was grown at elevated [CO₂], the N allocation to RuBisCo (N_R) decreased to a greater extent but N_L and N remaining increased relative to specimens grown at ambient [CO₂]. We suggest that N remobilization from RuBisCo is more efficient than from proteins of electron transport (N_E), and from N_L. These physiological responses of the black locust are significant as being an adaptation strategy to global environmental changes.

     

Environmental assessment of black locust (<Emphasis Type="BoldItalic">Robinia pseudoacacia</Emphasis> L.)-based ethanol as potential transport fuel

Purpose  

Lignocellulosic ethanol has received special research interest, driven by concerns over high fuel prices, security of energy supplies, global climate change as well as the search of opportunities for rural economic development. A well-to-wheel analysis was conducted for ethanol obtained from black locust (Robinia pseudoacacia L.) by means of the life cycle assessment (LCA) methodology. This study assesses the environmental profile of using ethanol in mixtures E10 and E85 as transport fuel in comparison with conventional gasoline (CG). In addition, the best model of black locust cultivation was analysed under an environmental point of view.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Higher phenotypic plasticity does not confer higher salt resistance to <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> than <Emphasis Type="Italic">Amorpha fruticosa</Emphasis>

A greenhouse experiment was conducted in which two leguminous species commonly used in the Yellow River Delta for vegetation restoration, Robinia pseudoacacia and Amorpha fruticosa, were subjected to five salt treatments: 0, 50, 100, 150, and 200 mmol L^?1. We aimed to determine which of the two species would be better suited for growth in a saline environment, and whether the acclimation capacity to salinity resulted from an inherently higher phenotypic plasticity. The results showed that salinity affected most growth and biomass parameters but had no effects on most leaf traits and physiological parameters of the two species. Height, relative growth rate of crown area, root biomass, and leaf mass ratio of R. pseudoacacia were reduced by higher salinity, while A. fruticosa was not affected. Chlorophyll a-to-chlorophyll b ratio and total antioxidative capacity of A. fruticosa increased with higher salinity, whereas those of R. pseudoacacia remained unchanged. Root mass ratio and vitamin C concentration of both species were not affected by salinity, whereas vitamin C concentration of A. fruticosa was higher than that of R. pseudoacacia. The root-to-shoot ratio of A. fruticosa was higher than that of R. pseudoacacia in most salt treatments. Of all leaf traits, only leaf area differed between treatments. R. pseudoacacia generally exhibited a greater plasticity than A. fruticosa in response to salinity, but A. fruticosa was more resistant to the higher salinities than R. pseudoacacia, and was thus a better candidate for vegetation restoration in saline areas.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Aquaporin gene expression and physiological responses of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L. to the mycorrhizal fungus <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> and drought stress

The influence of arbuscular mycorrhiza (AM) and drought stress on aquaporin (AQP) gene expression, water status, and photosynthesis was investigated in black locust (Robinia pseudoacacia L.). Seedlings were grown in potted soil inoculated without or with the AM fungus Rhizophagus irregularis, under well-watered and drought stress conditions. Six full-length AQP complementary DNAs (cDNAs) were isolated from Robinia pseudoacacia, named RpTIP1;1, RpTIP1;3, RpTIP2;1, RpPIP1;1, RpPIP1;3, and RpPIP2;1. A phylogenetic analysis of deduced amino acid sequences demonstrated that putative proteins coded by these RpAQP genes belong to the water channel protein family. Expression analysis revealed higher RpPIP expression in roots while RpTIP expression was higher in leaves, except for RpTIP1;3. AM symbiosis regulated host plant AQPs, and the expression of RpAQP genes in mycorrhizal plants depended on soil water condition and plant tissue. Positive effects were observed for plant physiological parameters in AM plants, which had higher dry mass and lower water saturation deficit and electrolyte leakage than non-AM plants. Rhizophagus irregularis inoculation also slightly increased leaf net photosynthetic rate and stomatal conductance under well-watered and drought stress conditions. These findings suggest that AM symbiosis can enhance the drought tolerance in Robinia pseudoacacia plants by regulating the expression of RpAQP genes, and by improving plant biomass, tissue water status, and leaf photosynthesis in host seedlings.     

High somatic instability of a microsatellite locus in a clonal tree,<Emphasis Type="Italic"> Robinia pseudoacacia</Emphasis>

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13–71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.Communicated by H.F. Linskens     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.