Endophytic Bacillus and Pseudomonas spp. Modulate Apple Shoot Growth, Cellular Redox Balance, and Protein Expression Under in Vitro Conditions

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant–endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant–endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families

Peer reviewe

Cloning and expression of transmembrane domain segments of Arabidopsis thaliana RBOH D enzyme

Production of reactive oxygen species (ROS) is important cell signaling component involved in plant response to stress. NADPH-oxidase, also known as respiratory burst oxidase homologue (RBOH) in plants, is a superoxide producing NOX family protein that is located in plasma membrane and contributes to ROS generation outside the plasma membrane in the apoplast. A. thaliana has ten RBOH genes (RBOH A-J). RBOH D is expressed in all plant tissues and takes part in regulation of response to pathogen and abiotic stress. Plant RBOH proteins show unique structural and regulation features that include calcium binding and different cytosolic component role in regulation of the enzyme activity. Meanwhile, structure of small N-terminal cytosolic domain of plant RBOH was studied so far, further structural and functional studies of plant RBOH homologues could help to understand the role of these enzymes in cell signaling. Heterologous expression and purification of transmembrane proteins is challenging due to their hydrophobic membrane domain. However, valuable information about transmembrane protein structure and function may be obtained by expression and characterization of separate membrane domain fragments. Aim of this study was to identify, clone and optimize the production of segments of A. thaliana RBOH D protein membrane domain in E. coli expression system. To identify transmembrane domain of RBOH D, bioinformatics analysis of amino acid sequence hydrophobicity profile, transmembrane protein domain modeling and assessment of previously predicted structures of other NOX family proteins, such as NOX2, was used. Six transmembrane helices were identified and the third and fifth helices each contained two conserved His residues, which are considered to coordinate the two hemes involved in superoxide production. [...]Gamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro filialas Sodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Influence of different exogenous saccharose and proline of strawberrymicroshoots in vitro

LAMMC Sodininkystės ir daržininkystės institute, Sodo augalų genetikos ir biotechnologijos skyriuje, buvo tiriamas įvairių braškių veislių atsparumas šalčiui po 14 dienų grūdinimo ir šaldymo in vitro –10 o C temperatūroje. Buvo įvertintas braškių atsparumas šalčiui, priklausomai nuo sacharozės, rafinozės ir prolino priedų, įdėtų į maitinamąją terpę (MS). T.Murashige’as ir F. Skoogas (1962) maitinamąją terpę papildė 3 ir 6% sacharozės, 5 % sacharozės + 1 % rafinozės, 6 % sacharozės + 0,001 % prolino priedais. Pašaldžius buvo vertinamas išgyvenusių mikroūglių skaičius (%) ir pažeidimas šalčiu (balais). Išryškėjo tiesioginė priklausomybė tarp grūdinimo trukmės, sacharozės, rafinozės ir prolino kiekio maitinamojoje terpėje ir mikroūglių būklės. Analizuojant cukrų priedų maitinamojoje terpėje įtaką jų kaupimuisi braškių mikroūgliuose, MS terpė buvo papildyta įvairiu kiekiu sacharozės (0,5%, 2% ir 6%). Nustatyta, kad tirpių angliavandenių kiekis braškių mikroūgliuose, auginant juos MS terpėje, pasižyminčioje skirtinga sacharozės koncentracija, didėja nepriklausomai nuo veislėsWinter and cold hardiness remain among most important traits for strawberry and other fruit crops. Biotechnological, in vitro methods make it possible to investigate separate factors of those complex traits in detail and give recommendations for improving cold hardiness by genetic engineering, marker assisted selection or breeding. The study was performed at Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry Effect of sucrose, raffinose and proline additions to growing media for cold acclimation efficiency, duration and cold hardiness were investigated in vitro. Plants of strawberry cultivars ‘Melody’, ‘Venta’ were planted on MS media with addition of different variants of 3%, 6% sucrose, 5% sucrose +1 % raffinose and 6% sucrose + 0.01% proline. After one week plants were transferred for acclimation at +4°C temperature for 14 days, frozen at -10°C temperature. Survival rate and cold injury score (0-5) were evaluated 3 weeks after freezing. It was shown direct correlation between acclimation duration, sucrose, raffinose concentration and cold hardiness of investigated strawberry plants. It was found that the soluble carbohydrate content of strawberries microshoots, growing them in MS medium with different sucrose concentrations increased regardless of the varietyBotanikos sodasSodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Production of recombinant Fibronectin tipe III 9-10 domain in bacterial expression system

Fibronectin (Fn) is a glycoprotein that plays important roles in cell adhesion, growth, differentiation and migration by mediating a wide variety of cellular interactions with the extracellular matrix (ECM). Fn usually exists as a dimer composed of two nearly identical 220~250 kDa subunits where each monomer is composed of homologous repeats of three prototypical domains: type I, type II and type III. Fn interacts with many integrins such as α3β1, α5β1, α8β1, αvβ1, αIIββ3, αvβ3, αvβ5, and αvβ6. In previous studies, the specific integrin-recognition sequences involved in cell adhesion have been identified. The best known of these – Arg-Gly-Asp (RGD) sequence – is located in the central cell-binding domain - FnIII10. It is the most important recognition site that can interact with about half of all known integrins. Another important sequence which acts in synergy with the RGD site is - Pro-His-Ser-Arg-Asn (the ‘synergy site’ PHSRN) found in Fn repeat III9, that promotes specific α5β1 integrin binding. Because of the ECM binding properties Fn is a perfect protein for nanofabrication and integrin-mediated immobilization of cells into synthetic scaffolds. Heterologous expressions of large eucaryotic proteins, such as Fn, in bacterial expression system is complicated. Therefore the aim of this study was to establish a recombinant protein production system for the "cell-binding domain" of Fn protein - including FnIII9-10 fragment and to assess the effect of His-tag position on the recombinant peptide purification efficiency. For this purpose, a sequence of the FnIII9-10 fragment was cloned to pLATE bacterial expression vector using a ligation independent cloning system. This vector includes bacteriophage T7 promoter that ensures high yields of expressed proteins. Two constructs including either amino- or carboxy- terminal 6xHis-tag were developed.[...]Biochemijos katedraGamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasVilniaus universitetasVytauto Didžiojo universiteta

Bacterial endophytes in agricultural crops and their role in stress tolerance: a review

Abstract Bacterial endophytes are a class of endosymbiotic microorganisms widespread among plants that colonize intercellular and intracellular spaces of all plant compartments and do not cause plant disease or significant morphological changes. Plant and endophytic bacteria association includes vast diversity of bacterial taxa and plant hosts and in this review we present an overview of taxonomic composition of endophytes identified in common agricultural crops. Further, during the last decade, new aspects of the microbial diversity have emerged with application of new metagenomic analysis methods in studies of bacterial endophytes. Endophytic bacteria community structure is influenced by plant genotype, abiotic and biotic factors such as environment conditions, microbe -microbe interactions and plant -microbe interactions. Agricultural practices, such as soil tillage, irrigation, use of pesticides and fertilizers have a major effect on function and structure of soil and endophytic microbial populations. Therefore, the use of agricultural practices that maintain natural diversity of plant endophytic bacteria is becoming an important element of sustainable agriculture that could ensure plant productivity and quality of agricultural production. The diverse endophytic microbial communities play integral and unique role in the functioning of agroecosystems. Endophytic bacteria have been shown to have several beneficial effects on their host plant, including growth promoting activity, modulation of plant metabolism and phytohormone signalling that leads to adaptation to environmental abiotic or biotic stress. Use of endophytic bacteria presents a special interest for development of agricultural applications that ensure improved crop performance under cold, draught or contaminated soil stress conditions or enhanced disease resistance

Microbial antagonism of putative bacterial endophytes from apple (Malus × domestica Borkh)

It is presumed that all plants are inhabited by diverse species of microorganisms known as endophytes. Information about composition and activity of endophytic microbiome of woody plants is mostly concerned with bacterial and fungal populations of plant rhizosphere. Meanwhile only fragmented knowledge is present about endophytes that reside in phyllosphere of cultivated tree plants such as domestic apple (Malus x domestica Borkh.). In this study, thirty-eight putative endophytic bacterial isolates were cultured from buds of apple. Analysis of 16 S rRNA gene sequence revealed presence of Curtobacterium, Pantoea and six Pseudomonas species among the isolates. Characterization of microbial growth suppresing properties demonstrated that 17, 2, 3 and 4 of the isolates displayed antagonistic activity against Micrococcus luteus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa, respectively. None of the culture filtrates was inhibiting growth of all four bacteria or Salmonella typhimurium. Ability to produce siderophores was characteristic to 27 of the isolates, and 8 isolates were producers of hydrogen cyanide. However, these traits only weekly correlated to the microbial growth inhibiting properties of sterile culture filtrates prepared from the isolates. Characterization of capability to cross-inhibit growth among the isolates revealed a complex picture of potential interactions that are present within the population of endophytic bacteria of apple phyllosphere. In addition, it was determined that three of endophytic strains were able to inhibit growth of pathogen of apple Venturia inaequalis, suggesting potential for biocontrol applicationBiochemijos katedraLietuvos agrarinių ir miškų mokslų centrasVytauto Didžiojo universiteta

Biochemical and Physiological Plant Processes Affected by Seed Treatment with Non-Thermal Plasma

Among the innovative technologies being elaborated for sustainable agriculture, one of the most rapidly developing fields relies on the positive effects of non-thermal plasma (NTP) treatment on the agronomic performance of plants. A large number of recent publications have indicated that NTP effects are far more persistent and complex than it was supposed before. Knowledge of the molecular basis and the resulting outcomes of seed treatment with NTP is rapidly accumulating and requires to be analyzed and presented in a systematic way. This review focuses on the biochemical and physiological processes in seeds and plants affected by seed treatment with NTP and the resulting impact on plant metabolism, growth, adaptability and productivity. Wide-scale changes evolving at the epigenomic, transcriptomic, proteomic and metabolic levels are triggered by seed irradiation with NTP and contribute to changes in germination, early seedling growth, phytohormone amounts, metabolic and defense enzyme activity, secondary metabolism, photosynthesis, adaptability to biotic and abiotic stress, microbiome composition, and increased plant fitness, productivity and growth on a longer time scale. This review highlights the importance of these novel findings, as well as unresolved issues that remain to be investigated

Hemagglutinin from Echinacea purpurea L. root separation and identification

[no. VNS17/045]Lectins or hemagglutinins are non-immune origin glycoproteins, which can bind carbohydrate structures in specific and reversible manner. They have big potential for their therapeutical applications such as immunomodulatory, anticancer antibacterial and other activities. Hemagglutinins from Echinarea purpurea L. (Moench) roots haven’t been investigated. The aim of the experiment: To purify and identify hemagglutinins from Echinarea purpurea L. (Moench) roots. Experiment tasks: 1. Purify hemagglutinins from purple coneflower roots; 2. Identify hemagglutinating glycoproteins. Materials and methods: 1. Affinity column with immobilized D-(+)-mannose ligands was equilibrated and unbound proteins were washed out with phosphate buffer saline (PBS) pH 7,4. Hemagglutinins were eluted out of the column with 0.2 M lactose solution in PBS. Following hemagglutinin fraction was collected and checked for hemagglutinating activity. 2. Hemagglutinating active fraction was separated by polyacrylamide gel electrophoresis and immunoblotted for glycosylated proteins. 3. Glycosylated protein bands were identified by liquid chromatography tandem mass spectrometry by sunflower genome database search. Results: 1. Hemagglutinating active proteins from purple coneflower root were separated from non-active proteins and collected into one fraction. 2. Lysin motive (LysM) peptidoglycan binding domain was identified after database search with identity score equal 182.0. Conclusions: 1. Affinity chromatography method was suitable for separation of hemagglutinating active proteins from purple coneflower root crude protein extract. 2. Identified glycoprotein – LysM domain was responsible for the hemagglutinating activityBotanikos sodasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta

Estraction conditions of proteins from Echinacea Purpurea L. (Moench) roots and leaves

The purple coneflower (Echinacea purpurea L. (Moench)) has been known and used in traditional medicine for decades, but the interest of this plant pharmacological properties isn't decreasing. Previous studies showed hemagglutinating activity of ethanolic extracts from purple coneflower roots and leaves. Nevertheless, information about purple coneflower proteins and their hemagglutinating properties are still not defined. Aim of experiment: To determine the best extraction conditions with highest protein yield and highest hemagglutinating activity. Experiment tasks: 1. Extraction of proteins from coneflower roots and leaves. 2. Determination of extraction conditions for the highest protein yield. 3. Determination of extraction conditions for the highest hemagglutinating activity. Materials and methods: 1. Proteins were extracted out of coneflower leaves (ratio of herb material and extraction buffer - 1:10) and roots (ratio of herb material and extraction buffer - 1:6) in different conditions: A. At 20±2°C for 2 hours in 3 buffers with different pH (Written below); B. At 4±1°C temperature for 2 hours with 5% of protese inhibitor cocktail (Halt™, Thermo Scientific). C: At 4±1°C for 2 hours with 1% of polivynyl polypirolidone, 0,5% of dithiotrethiol and 0,5% of ethylenediaminetetraacetic acid. After extraction proteins were precipitated with 13.3% trichloracetic acid in acetone and 0.2% of β-mercaptoethanol. 2. Protein amount was measured by Bradford assay. 3. Determination of hemagglutinating activity: protein fractions were poured on suspension with 2% trypsin treated rabbit erythrocytes and incubated for 30 min at 20±2°C temperature. Buffers used for extraction: Acetic buffer pH=5.0, Phosphate buffered saline (PBS) pH=7.4, Tris-HCI buffer pH=8.0. [...]Botanikos sodasLietuvos agrarinių ir miškų mokslų centrasLietuvos sveikatos mokslų universitetas. Medicinos akademijaVytauto Didžiojo universiteta