Growth enhancement, metabolic profile improvement, and DXR and TPS2 gene expression changes in Thymus vulgaris L. by cyanobacterial inoculation.

BACKGROUND: In recent decades cyanobacterial species have attracted research attention as potential sources of new biostimulants. In this study, the biostimulant effects of five cyanobacterial suspensions on the growth and essential oil composition of Thymus vulgaris L. were evaluated. The expression of key genes involved in the biosynthesis of thymol and carvacrol, such as DXR and TPS2, were investigated. RESULTS: A pot culture experiment revealed that cyanobacterial application significantly improved T. vulgaris L. growth indices, including plant height, dry and fresh weight, leaf and flower number, leaf area, and photosynthetic pigment content. Total phenol and flavonoid content in inoculated plants also showed a significant increase compared with the control. Anabaena torulosa ISB213 inoculation significantly increased root and shoot biomass by about 65.38% and 92.98% compared with the control, respectively. Nostoc calcicola ISB215 inoculation resulted in the highest amount of essential oil accumulation (18.08 ± 0.62) in T. vulgaris leaves, by about 72.19% compared with the control (10.5 ± 0.50%). Interestingly, the amount of limonene in the Nostoc ellipsosporum ISB217 treatment (1.67%) increased significantly compared with the control and other treatments. The highest expression rates of DXR and TPS2 genes were observed in the treatment of N. ellipsosporum ISB217, with 5.92-fold and 5.22-fold increases over the control, respectively. CONCLUSION: This research revealed the potential of the cyanobacteria that were studied as promising biostimulants to increase the production of biomass and secondary metabolites of T. vulgaris L., which could be a suitable alternative to chemical fertilizers. © 2024 Society of Chemical Industry.

Sesame (Sesamum indicum L.) response to drought stress: susceptible and tolerant genotypes exhibit different physiological, biochemical, and molecular response patterns.

Drought is one of the main environmental stresses affecting the quality and quantity of sesame production worldwide. The present study was conducted to investigate the effect of drought stress and subsequent re-watering on physiological, biochemical, and molecular responses of two contrasted sesame genotypes (susceptible vs. tolerant). Results showed that plant growth, photosynthetic rate, stomatal conductance, transpiration rate, and relative water content were negatively affected in both genotypes during water deficit. Both genotypes accumulated more soluble sugars, free amino acids, and proline and exhibited an increased enzyme activity for peroxidase, catalase, superoxide dismutase, and pyruvate dehydrogenase in response to drought damages including increased lipid peroxidation and membrane disruption. However, the tolerant genotype revealed a more extended root system and a more efficient photosynthetic apparatus. It also accumulated more soluble sugars (152%), free amino acids (48%), proline (75%), and antioxidant enzymes while showing lower electrolyte leakage (26%), lipid peroxidation (31%), and starch (35%) content, compared to the susceptible genotype at severe drought. Moreover, drought-related genes such as MnSOD1, MnSOD2, and PDHA-M were more expressed in the tolerant genotype, which encode manganese-dependent superoxide dismutase and the alpha subunit of pyruvate dehydrogenase, respectively. Upon re-watering, tolerant genotype recovered to almost normal levels of photosynthesis, carboxylation efficiency, lipid peroxidation, and electrolyte leakage, while susceptible genotype still suffered critical issues. Overall, these results suggest that a developed root system and an efficient photosynthetic apparatus along with the timely and effective accumulation of protective compounds enabled the tolerant sesame to withstand stress and successfully return to a normal growth state after drought relief. The findings of this study can be used as promising criteria for evaluating genotypes under drought stress in future sesame breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01372-y.

Biochemical and Transcriptional Responses in Cold-Acclimated and Non-Acclimated Contrasting Camelina Biotypes under Freezing Stress.

Cold-acclimated and non-acclimated contrasting Camelina (Camelina sativa L.) biotypes were investigated for changes in stress-associated biomarkers, including antioxidant enzyme activity, lipid peroxidation, protein, and proline content. In addition, a well-known freezing tolerance pathway participant known as C-repeat/DRE-binding factors (CBFs), an inducer of CBF expression (ICE1), and a cold-regulated (COR6.6) genes of the ICE-CBF-COR pathway were studied at the transcriptional level on the doubled-haploid (DH) lines. Freezing stress had significant effects on all studied parameters. The cold-acclimated DH34 (a freezing-tolerant line) showed an overall better performance under freezing stress than non-acclimated plants. The non-cold-acclimated DH08 (a frost-sensitive line) showed the highest electrolyte leakage after freezing stress. The highest activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) was also detected in non-acclimated plants, whereas the cold-acclimated plants showed lower enzyme activities upon stress treatment. Cold acclimation had a significantly positive effect on the total protein and proline content of stressed plants. The qRT-PCR analysis revealed significant differences in the expression and cold-inducibility of CsCBF1-3, CsICE1, and CsCOR6.6 genes among the samples of different treatments. The highest expression of all CBF genes was recorded in the non-acclimated frost-tolerant biotype after freezing stress. Interestingly a significantly higher expression of COR6.6 was detected in cold-acclimated samples of both frost-sensitive and -tolerant biotypes after freezing stress. The presented results provide more insights into freezing tolerance mechanisms in the Camelina plant from both a biochemical point of view and the expression of the associated genes.

Antimicrobial activity, environmental sensitivity, mechanism of action, and food application of αs165-181 peptide.

αs165-181 is a peptide derived from αs2-casein of ovine milk. Herein, we report the antimicrobial activity and mechanism, and food application of the peptide. αs165-181 showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Bacillus cereus, and Salmonella enterica serovar Enteritidis in a dose-dependent manner. The minimum inhibitory concentration of the peptide was 3.9 mg/ml for E. coli and 7.8 mg/ml for the other bacteria. The peptide did not show antimicrobial activity against Lactobacillus plantarum up to 3.9 mg/ml concentration. The minimum bactericidal concentration of αs165-181 peptide was 7.8 mg/ml for E. coli, S. aureus, L. monocytogenes, and B. cereus. The peptide was sensitive to monovalent and divalent cations, pH, and high temperatures. Transmission electron microscopy, cytoplasmic ß-galactosidase leakage, and DNA electrophoresis analyses showed that αs165-181 peptide affects bacteria by damaging cell membrane and binding to the genomic DNA. When αs165-181 peptide was applied to minced beef or UHT cream, the antimicrobial activity (7.8 mg/g) was almost the same as or even better than nisin (0.5 mg/g). This study helps understand the antimicrobial mode of action of αs165-181 peptide and develop strategies for application in food products.

Recombinant pebulin protein, a type 2 ribosome-inactivating protein isolated from dwarf elder (Sambucus ebulus L.) shows anticancer and antifungal activities in vitro.

In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.

Genetic analysis of freezing tolerance in camelina [Camelina sativa (L.) Crantz] by diallel cross of winter and spring biotypes.

MAIN CONCLUSION: Camelina biotypes had different responses to freezing stress, which was mainly inherited by additive gene effects and can be reliably used in breeding programs and for a better understanding of freezing tolerance mechanisms in camelina plants. Camelina [Camelina sativa (L.) Crantz] is a frost-tolerant oilseed plant that is cultivated as an autumn crop in semi-arid regions. However, camelina establishment in these areas is limited by low temperatures in winter that results in decreased seed yield. In the present study, genetic basis of freezing tolerance (FT) in spring and winter biotypes of camelina was analyzed at seedling stage using a diallel cross experiment. The parents consisted of two winter doubled haploid (DH) lines with high (DH34 and DH31), two spring lines with medium (DH19 and DH26), and two spring lines with low FT (DH08 and DH91). For this purpose, the parents along with F1 entries were subjected to freezing stress and survival percentage, electrolyte leakage, and lethal temperature for 50% mortality (LT50) of the lines were measured. Results showed that although both additive and non-additive effects of the genes determine the FT, further analyses indicated that it was mainly controlled by the additive effects. Therefore, selection-based methods may be more efficient for improving FT in camelina genotypes. The results of specific combining ability (SCA) and heterosis analysis among various DH lines suggested that more tolerant cultivars of camelina could be developed by targeted crossings. When a tolerant winter line and a susceptible spring line were crossed, their progenies showed a higher FT compared with the progenies of a cross between two susceptible spring lines indicating FT is controlled by additive effects of the genes in camelina plants. These findings provided new insight into the genetic basis of freezing-related traits in camelina and could be used for more sophisticated breeding programs.

Molecular cloning and in-depth bioinformatics analysis of type II ribosome-inactivating protein isolated from Sambucus ebulus.

Plant ribosome-inactivating proteins (RIPs) are N-glycosidases which inhibit protein synthesis through depurination of the ribosomal RNA sequence. Type II RIPs are heterodimer proteins which can bind to cell surfaces. The cytotoxicity of these RIPs is different. Sambucus spp. are a rich source of RIP proteins with different properties. In the present study, a type II RIP was isolated from S. ebulus plant that grows widely in the north of Iran, and different bioinformatics tools were used for the evaluation of physicochemical, functional and 3D protein characteristics. The results showed significant differences among isolated RIP and other Sambucus RIP proteins. The study of these differences can not only expand our insight into the functioning mechanisms of plant RIPs but also provide information about a novel RIP protein with potential biological applications.

Recombinant production of a bioactive peptide from spotless smooth-hound (Mustelus griseus) muscle and characterization of its antioxidant activity.

Bioactive peptides are short amino acid sequences with desirable health effects which are derived from animals, plants, and marine sources. In this study, recombinant production of a bioactive peptide (GIISHR) from spotless smooth-hound (Mustelus griseus) muscle and its antioxidant properties is discussed. A gene composed of 12 tandem copies of the peptide sequence was cloned in pET-28a and expressed as a His-tagged polypeptide in Escherichia coli. The recombinant polypeptide was then purified by Ni-NTA affinity chromatography, cleaved by Trypsin and purified by ultrafiltration. DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) and hydroxyl radical scavenging activity assays, ferric reducing antioxidant power (FRAP) assay and ß-carotene bleaching test were used to characterize the antioxidant activity of the GIISHR. Liquid chromatography-mass spectrometry analysis revealed 60% purity for released bioactive peptide. Production yield was estimated as 60-80 mg GIISHR active peptide per 1 L bacterial culture. Antioxidant activity assays indicated that the antioxidant activity was increased with increase in peptide concentration. Though the DPPH radical scavenging activity, FRAP and ß-carotene bleaching power of the peptide were lower than those of the synthetic antioxidant tert-butylhydroquinone (TBHQ), the ABTS and hydroxyl radical scavenging activities of the peptide (at a concentration of 20 mg/mL) were similar to those of TBHQ (at a concentration of 0.1 mg/mL). The findings of the present study may be helpful in development of a process for production of the bioactive antioxidant peptides and its application in food industry.

Increased cell wall thickness of endodermis and protoxylem in Aeluropus littoralis roots under salinity: The role of LAC4 and PER64 genes.

Enhanced cell wall lignification is one of the major salinity tolerance strategies in the roots of halophytes. A deep insight into the exact root developmental system in halophytes may be of great importance for understanding plant salt tolerance mechanisms. In this work the developmental and anatomical changes in the roots of halophyte Aeluropus littoralis along with expression patterns of two genes encoding for cell wall laccase (LAC4) and peroxidase (PER64) were investigated. The plants were treated with 0, 300 and 600mM NaCl and root samples were collected 3, 6 and 9days after treatment (DAT). Upon salinity treatment, root diameter and parenchyma thickness were increased significantly in the tip and middle segments compared to upper zones, but the change trend was reversed with the time. It was interestingly revealed that protoxylem was the tissue of target for lignification at root tips, while the highest lignification rates were observed in metaxylem and endodermis in upper segments. Compared to endodermis, protoxylem is restrictively involved in early stages of salt stress in root tips as an efficient barrier against Na+ flow. Gene expression analysis revealed that LAC4 expression was higher in root tips resulting in enhanced protoxylem lignification while PER64 expression was higher in more differentiated zones leading to endodermis thickening. The overall results of this study reveal the crucial role of LAC4 as an important gene in specialized protoxylem lignification in undifferentiated root tips leading to enhanced tolerance in early stages of salt stress.

Comparison between the effects of potassium phosphite and chitosan on changes in the concentration of Cucurbitacin E and on antibacterial property of Cucumis sativus.

BACKGROUND: Cucurbitacins are mostly found in the members of the family Cucurbitaceae and are responsible for the bitter taste of cucumber. Pharmacological activities such as anti-bacterial and anti-tumor effects have been attributed to these structurally divers triterpens. The aim of this study was to investigate the effect of potassium phosphite (KPhi) and chitosan on Cucurbitacin E (CuE) concentration in different tissues of Cucumis sativus. The antibacterial effect of plant ethanolic extracts was also examined against E.coli PTCC 1399 and Pseudomonas aeruginosa PTCC 1430 bacterial strains. METHODS: After emergence of secondary leaves, cucumber plants were divided into 4 groups (each group consisted of 6 pots and each pot contained one plant) and different treatments performed as follows: group1. Leaves were sprayed with distilled water (Control), group 2. The leaves were solely treated with potassium phosphite (KPhi), group 3. Leaves were solely sprayed with chitosan (Chitosan), group 4. Leaves were treated with KPhi and chitosan (KPhi + chitosan). The KPhi (2 g L-1) and chitosan (0.2 g L-1) were applied twice every 12 h for one day. Fruits, roots and leaves were harvested 24 h later. The ethanolic extract of plant organs was used for determination of CuE concentration using HPLC approach. The antimicrobial activity was evaluated by the agar well diffusion method. The experiments were arranged in a completely randomized design (CRD) and performed in six biological replications for each treatment. Analysis of variance was performed by one-way ANOVA and Dunnette multiple comparison using SPSS. RESULTS: The highest level of CuE was recorded in fruit (2.2 g L-1) of plants under concomitant applications of KPhi and chitosan. Result of antibacterial activity evaluation showed that under concomitant treatments of KPhi and chitosan, fruit extract exhibited the highest potential for activity against E. coli PTCC 1399 (with mean zone of inhibition equal to 36 mm) and Pseudomonas aeruginosa PTCC 1430 (with mean zone of inhibition equal to 33 mm). CONCLUSIONS: KPhi and chitosan can induce production of CuE compound and increase antibacterial potential of cucumber plant extract. The application of KPhi and chitosan may be considered as promising prospect in the biotechnological production of CuE.

Essential oil composition of sweet basil (Ocimum basilicum L.) in symbiotic relationship with Piriformospora indica and paclobutrazol application under salt stress.

Essential oil content and oil composition of paclobutrazol treated sweet basil (Ocimum basilicum L.) plant inoculated with Piriformospora indica under salt stress were investigated by GC-MS. The results show a slight increase in essential oil content when basil plants subjected to moderate salinity stress (3 dS m-1 of NaCl). It decreased signifiicantly with increasing salinity level to 9 dS m-1. The findings revealed that leaf area, above ground and leaf dry weights, essential oil content and yield were significantly affected by P. indica inoculation, however paclobutrazol application significantly influenced essential oil yield but not content. Fungal symbiosis as well as paclobutrazol application ameliorated the negative effects of salinity on dry matter and essential oil yield. The main constituents found in the volatile oil of O. basilicum in control treatment were Geranial (26.03%), Neral (24.88%) and Estragole (24.78%). The compounds concentrations showed some differences in P. indica and paclobutrazol treatments. The results demonstrate that micorrhiza-like fungi concomitantly increase essential oil production and biomass in sweet basil, a medicinal herb rich in commercially valuable essential oils.

First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2.

Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus.

Chitinolytic and antifungal activity of a Bacillus pumilus chitinase expressed in Arabidopsis.

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin-glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.