Functional analysis of the 1-aminocyclopropane-1-carboxylate deaminase gene of the biocontrol fungus Trichoderma asperellum ACCC30536

2016 ◽  
Vol 96 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Fuli Zhang ◽  
Zhihua Liu ◽  
Mijiti Gulijimila ◽  
Yucheng Wang ◽  
Haijuan Fan ◽  
...  
Keyword(s):  
Salt Stress ◽  
Transgenic Plants ◽  
Salt Content ◽  
Membrane Integrity ◽  
Acc Deaminase ◽  
Blue Staining ◽  
Trichoderma Asperellum ◽  
Reading Frame ◽  
Populus Davidiana ◽  
Ethylene Content

1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) cleaves ACC, the immediate precursor of the ethylene, decreasing the level of ethylene and inhibition of plant growth resulted by environmental stresses. Here, TaACCD was cloned from the biocontrol agent Trichoderma asperellum ACCC30536. Its open reading frame was 1047 bp long encoding a 37 kD protein of 348 aa, and a pI of 5.77. Phylogenetic analysis demonstrated this protein to be closely related to ACCD from T. asperellum T203 (ACX94231). Transformation of Populus davidiana × P. bolleana with TaACCD, increased salinity tolerance of transgenic plants Pdb-ACCD3 and Pdb-ACCD5. Transgenic plants could survive at salinity of 200 mM NaCl, whereas untransformed control poplar Pdb-NT could withstand salinity to 150 mM NaCl. Transformed plants accumulated higher amounts of chlorophyll compared to Pdb-NT plants. Accumulation of reactive oxygen species (ROS) was regulated by TaACCD under salt stress, as shown from higher superoxide dismutase (SOD) and peroxidase (POD) activities, as well as NBT and DAB staining. Evans blue staining showed that TaACCD maintained membrane integrity in Populus under salt stress conditions. Additionally, TaACCD expression decreased ethylene content of transgenic plants compared to nontransgenic plants, but salt content in plant leaves didnt show obvious difference under same salt concentration. To the best of our knowledge, the current study is the first demonstration that the TaACCD gene from T. asperellum ACCC30536 can enhance tolerance of Populus to salt stress.

scholarly journals Alleviation of Salt Stress in Wheat Seedlings via Multifunctional Bacillus aryabhattai PM34: An In-Vitro Study

2021 ◽  
Vol 13 (14) ◽  
pp. 8030
Author(s):  
Shehzad Mehmood ◽  
Amir Abdullah Khan ◽  
Fuchen Shi ◽  
Muhammad Tahir ◽  
Tariq Sultan ◽  
...  
Keyword(s):  
Salt Stress ◽  
Plant Growth ◽  
Stress Tolerance ◽  
Bacterial Strain ◽  
Acc Deaminase ◽  
Wheat Growth ◽  
Wheat Seedlings ◽  
Pgp Traits ◽  
Bacillus Aryabhattai

Plant growth-promoting rhizobacteria play a substantial role in plant growth and development under biotic and abiotic stress conditions. However, understanding about the functional role of rhizobacterial strains for wheat growth under salt stress remains largely unknown. Here we investigated the antagonistic bacterial strain Bacillus aryabhattai PM34 inhabiting ACC deaminase and exopolysaccharide producing ability to ameliorate salinity stress in wheat seedlings under in vitro conditions. The strain PM34 was isolated from the potato rhizosphere and screened for different PGP traits comprising nitrogen fixation, potassium, zinc solubilization, indole acetic acid, siderophore, and ammonia production, along with various extracellular enzyme activities. The strain PM34 showed significant tolerance towards both abiotic stresses including salt stress (NaCl 2 M), heavy metal (nickel, 100 ppm, and cadmium, 300 ppm), heat stress (60 °C), and biotic stress through mycelial inhibition of Rhizoctonia solani (43%) and Fusarium solani (41%). The PCR detection of ituC, nifH, and acds genes coding for iturin, nitrogenase, and ACC deaminase enzyme indicated the potential of strain PM34 for plant growth promotion and stress tolerance. In the in vitro experiment, NaCl (2 M) decreased the wheat growth while the inoculation of strain PM34 enhanced the germination% (48%), root length (76%), shoot length (75%), fresh biomass (79%), and dry biomass (87%) over to un-inoculated control under 2M NaCl level. The results of experiments depicted the ability of antagonistic bacterial strain Bacillus aryabhattai PM34 to augment salt stress tolerance when inoculated to wheat plants under saline environment.

scholarly journals The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 1143-1151
Author(s):  
Gil Shalev ◽  
Avraham A Levy
Keyword(s):  
Transposable Element ◽  
Donor Site ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Blue Staining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Reading Frame ◽  
Strand Breaks ◽  
Ectopic Recombination

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby β-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5′ end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5′ end of the GUS open reading frame and had a deletion at the 3′ end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.

scholarly journals Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

2002 ◽  
Vol 68 (11) ◽  
pp. 5209-5216 ◽  
Author(s):  
Kaouther Ben Amor ◽  
Pieter Breeuwer ◽  
Patrick Verbaarschot ◽  
Frank M. Rombouts ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Salt Stress ◽  
Bile Salt ◽  
Cell Sorting ◽  
Physiological Activity ◽  
Membrane Integrity ◽  
Multiparameter Flow Cytometry ◽  
Bifidobacterium Adolescentis ◽  
Multiparametric Flow Cytometry ◽  
Plate Counts

ABSTRACT Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

scholarly journals Applications of CRISPR/Cas9 for the Treatment of Duchenne Muscular Dystrophy

2018 ◽  
Vol 8 (4) ◽  
pp. 38 ◽  
Author(s):  
Kenji Lim ◽  
Chantal Yoon ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Wide Spectrum ◽  
Membrane Integrity ◽  
Dystrophin Gene ◽  
Muscle Membrane ◽  
Reading Frame ◽  
Long Term Treatment

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein that leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.

scholarly journals A novel transcription factor-like gene SbSDR1 acts as a molecular switch and confers salt and osmotic endurance to transgenic tobacco

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Vijay Kumar Singh ◽  
Avinash Mishra ◽  
Intesaful Haque ◽  
Bhavanath Jha
Keyword(s):  
Transcription Factor ◽  
Transgenic Plants ◽  
Soluble Sugars ◽  
Membrane Integrity ◽  
Molecular Switch ◽  
Stress Conditions ◽  
Growth And Yield ◽  
Physiological Status ◽  
Growth Stages ◽  
Potential Candidate

Abstract A salt- and drought-responsive novel gene SbSDR1 is predominantly localised to the nucleus, up-regulated under abiotic stresses and is involved in the regulation of metabolic processes. SbSDR1 showed DNA-binding activity to genomic DNA, microarray analysis revealed the upregulation of host stress-responsive genes and the results suggest that SbSDR1 acts as a transcription factor. Overexpression of SbSDR1 did not affect the growth and yield of transgenic plants in non-stress conditions. Moreover, the overexpression of SbSDR1 stimulates the growth of plants and enhances their physiological status by modulating the physiology and inhibiting the accumulation of reactive oxygen species under salt and osmotic stress. Transgenic plants that overexpressed SbSDR1 had a higher relative water content, membrane integrity and concentration of proline and total soluble sugars, whereas they showed less electrolyte leakage and lipid peroxidation than wild type plants under stress conditions. In field conditions, SbSDR1 plants recovered from stress-induced injuries and could complete their life cycle. This study suggests that SbSDR1 functions as a molecular switch and contributes to salt and osmotic tolerance at different growth stages. Overall, SbSDR1 is a potential candidate to be used for engineering salt and drought tolerance in crops without adverse effects on growth and yield.

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