Meta-analysis of transcriptomic profiles in Dunaliella tertiolecta reveals molecular pathway responses to different abiotic stresses.

Microalgae are photosynthetic organisms and a potential source of sustainable metabolite production. However, different stress conditions might affect the production of various metabolites. In this study, a meta-analysis of RNA-seq experiments in Dunaliella tertiolecta was evaluated to compare metabolite biosynthesis pathways in response to abiotic stress conditions such as high light, nitrogen deficiency and high salinity. Results showed downregulation of light reaction, photorespiration, tetrapyrrole and lipid-related pathways occurred under salt stress. Nitrogen deficiency mostly induced the microalgal responses of light reaction and photorespiration metabolism. Phosphoenol pyruvate carboxylase, phosphoglucose isomerase, bisphosphoglycerate mutase and glucose-6-phosphate-1-dehydrogenase (involved in central carbon metabolism) were commonly upregulated under salt, light and nitrogen stresses. Interestingly, the results indicated that the meta-genes (modules of genes strongly correlated) were located in a hub of stress-specific protein-protein interaction (PPI) network. Module enrichment of meta-genes PPI networks highlighted the cross-talk between photosynthesis, fatty acids, starch and sucrose metabolism under multiple stress conditions. Moreover, it was observed that the coordinated expression of the tetrapyrrole intermediated with meta-genes was involved in starch biosynthesis. Our results also showed that the pathways of vitamin B6 metabolism, methane metabolism, ribosome biogenesis and folate biosynthesis responded specifically to different stress factors. Since the results of this study revealed the main pathways underlying the abiotic stress, they might be applied in optimised metabolite production by the microalga Dunaliella in future studies. PRISMA check list was also included in the study.

Identification of Novel QTLs Associated with Frost Tolerance in Winter Wheat (Triticum aestivum L.).

Low temperature (cold) and freezing stress is a major problem during winter wheat growth. Low temperature tolerance (LT) is an important agronomic trait in winter wheat and determines the plants' ability to cope with below-freezing temperatures; thus, the development of cold-tolerant cultivars has become a major goal of breeding in various regions of the world. In this study, we sought to identify quantitative trait loci (QTL) using molecular markers related to freezing tolerance in winter. Thirty-four polymorphic markers among 425 SSR markers were obtained for the population, including 180 inbred lines of F12 generation wheat, derived from crosses (Norstar × Zagros) after testing with parents. LT50 is used as an effective selection criterion for identifying frost-tolerance genotypes. The progeny of individual F12 plants were used to evaluate LT50. Several QTLs related to wheat yield, including heading time period, 1000-seed weight, and number of surviving plants after overwintering, were identified. Single-marker analysis illustrated that four SSR markers with a total of 25% phenotypic variance determination were linked to LT50. Related QTLs were located on chromosomes 4A, 2B, and 3B. Common QTLs identified in two cropping seasons based on agronomical traits were two QTLs for heading time period, one QTL for 1000-seed weight, and six QTLs for number of surviving plants after overwintering. The four markers identified linked to LT50 significantly affected both LT50 and yield-related traits simultaneously. This is the first report to identify a major-effect QTL related to frost tolerance on chromosome 4A by the marker XGWM160. It is possible that some QTLs are closely related to pleiotropic effects that control two or more traits simultaneously, and this feature can be used as a factor to select frost-resistant lines in plant breeding programs.

Molecular insights into the genetic diversity and population structure of Artemisia annua L. as revealed by insertional polymorphisms.

The knowledge about the level of genetic diversity and population structure in natural populations of Artemisia annua L. is a primary step in breeding programs for development of new cultivars with higher artemisinin level and better quality of secondary metabolites composition. We used PCR-based "retrotransposon-microsatellite amplified polymorphisms" (REMAPs) to study insertional polymorphism in A. annua genome to assess genetic variability and population structure in a collection of 118 accessions collected from north and northwest of Iran. Twenty-five primer combinations of 10 retrotransposon and seven ISSR primers amplified a total of 693 clear and unambiguous fragments in the studied accessions. The average number of bands, polymorphic bands, polymorphism, effective number of alleles, Shannon's information index and expected heterozygosity were 27.72, 24.76, 88.14%, 1.47, 0.42 and 0.28, respectively. The analysis of molecular variance revealed high genetic variation present within sampled geographical regions. Distance-based cluster analysis assigned the studied accessions into four clusters according to their geographical origin, which were also confirmed by principal coordinate analysis. In model-based Bayesian clustering, the maximum value of Δk was obtained when the collection of 118 assayed A. annua accessions assigned into two subgroups (K = 2). The results showed the high genetic variation in the collection of Iranian sweet wormwood which revealed by REMAP markers indicating the reliability and efficiency of this marker system for analysis of genetic diversity and population structure of A. annua. Supplementary Information: The online version contains supplementary material available at 10.1007/s40415-022-00860-x.

High genetic diversity in Aegilops tauschii Coss. accessions from North Iran as revealed by IRAP and REMAP markers.

BACKGROUND: Aegilops tauschii Coss. as a donor of wheat D genome has an important role in wheat breeding programs. Genetic and phylogeographic diversity of 79 Ae. tauschii accessions collected from north and northwest of Iran were analyzed based on retroelement insertional polymorphisms using inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers. RESULTS: In total, 306 and 151 polymorphic bands were amplified in IRAP and REMAP analyses, respectively. As a result, a high level of polymorphism was observed among the studied accessions as revealed by an average of 25.5 bands per primer/primer combination and mean PIC value of 0.47 in IRAP and an average of 25.16 bands per primer combination and mean PIC value of 0.47 in REMAP. Genetic relationships of the accessions were analyzed using distance- and model-based cluster analyses. CONCLUSION: The result showed that genetic distance did not seem to be related to geographic distribution, and the accessions could be divided into three groups, which was further supported by principal coordinate analysis. These results on genetic diversity and population structure of Ae. tauschii in Iran should provide important knowledge on genetic resources and their applications in wheat breeding programs.

The influence of breeding history, origin and growth type on population structure of barley as revealed by SSR markers.

Natural and mass selection during domestication and cultivation favored particular traits of interest in barley. In the present study, population structure, and genetic relationships among 144 accessions of barley landraces and breeding materials from various countries were studied using a set of 77 and 72 EST-SSR and gSSR markers, respectively distributed on seven chromosomes of barley. In total, 262 and 429 alleles were amplified in 77 EST-SSRs and 72 gSSR loci, respectively. Out of which, 185 private/group-specific alleles were identified in the landraces compared with 14 in "cultivar and advanced breeding lines", indicating the possibility to introgress favorite alleles from landraces into breeding materials. Comparative analysis of genetic variation among breeding materials, Iranian landraces, and exotic landraces revealed higher genetic diversity in Iranian landraces compared with others. A total of 37, 15, and 14 private/group-specific alleles were identified in Iranian landraces, exotic landraces, and breeding materials, respectively. The most likely groups for 144 barley genotypes were three as inferred using model- and distance-based clustering as well as principal coordinate analysis which assigned the landraces and breeding materials into separate groups. The distribution of alleles was found to be correlated with population structure, domestication history and eco-geographical factors. The high allelic richness in the studied set of barley genotype provides insights into the available diversity and allows the construction of core groups based on maximizing allelic diversity for use in barley breeding programs.

Apoptotic Effect of Saccharomyces cerevisiae on Human Colon Cancer SW480 Cells by Regulation of Akt/NF-ĸB Signaling Pathway.

Drug resistance is one of the major problems, which causes recurrence of cancers. Therefore, complementary treatments are needed to improve the impacts of chemotherapy agents. The effect of probiotics as cancer-preventing agents through involvement in the activation of apoptotic pathways has been established. The present study sought to investigate how the heat-killed form of Saccharomyces cerevisiae (as a probiotic) could affect the Akt/NF-kB-induced apoptosis in colon cancer cells, the SW480 cell line. The cytotoxic effects of heat-killed yeast (HKY) and 5-fluorouracil (5-FU, as a positive control drug) were assayed using the MTT method. Morphological changes followed by apoptosis were examined using DAPI staining. The transcription and translation level of apoptosis genes were explored with qRT-PCR and western blotting. The data were analyzed using GraphPad Prism V6.0 Software. The results showed that HKY could induce apoptosis in colon cancer cell line through downregulation of p-Akt1, Rel A, Bcl-XL, pro-caspase 3, and pro-caspase 9 expressions, and upregulation of BAX, cleaved caspase-3, and cleaved caspase-9. Besides, Akt protein expression was not affected. It is noticeable that HKY had a better modulating effect on BAX expression compared with 5-FU. It was able to modulate Akt/NF-kB signaling pathway followed by the apoptotic cascade.

Genome-wide identification and co-expression network analysis of nuclear factor-Y in barley revealed potential functions in salt stress.

Nuclear factor-Ys (NF-Ys) were previously shown to have important regulatory impacts in different developmental and physiological process. However, in barley the function of the NF-Y genes at system levels is not well known. To identify barley NF-Ys, Arabidopsis and wheat NF-Y protein sequences were retrieved and the BLAST program along with the hidden Markov model were used. Multiple sequence alignments of identified NF-Ys were constructed using ClustalW. Expression patterns of the NF-Ys at different physiological and developmental conditions were also surveyed based on microarray datasets in public databases and subsequently co-expression network were constructed. Validation of in silico expression analysis was performed by real-time qPCR under salt stress condition. In total, 23 barley NF-Ys (8 NF-YA, 11 NF-YB and 4 NF-YC) were identified. Based on the sequence homology, the subunits of the NF-Y complex were divided into three to five groups. Structural analysis highlighted the conserved domains of HvNF-YA, HvNF-YB and HvNF-YC. Co-expression network analysis indicated the potential functions of HvNF-Ys in photosynthesis, starch biosynthesis and osmotic stress tolerance. The results of qRT-PCR also confirmed the HvNF-Ys roles in adaptation responses of barley to salt stress. We identified some potential candidate genes which could be used for improvements of cereals tolerance to salinity stress.

Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase.

PURPOSE: Recently discovered Anabaena variabilis phenylalanine ammonia lyase (AvPAL) proved to be a good candidate for enzyme replacement therapy of phenylketonuria. Outstanding stability properties of a mutant version of this enzyme, produced already in our laboratory, have led us to the idea of culture conditions optimization for soluble expression of this therapeutically valuable enzyme in E. coli. METHODS: In the present study, the gene encoding mutant version of AvPAL was cloned into the pET28a expression vector. Different concentrations of IPTG, induction period, growth temperature, shaking speed, as well as different types of culture media were examined with respect to the amount of recombinant protein produced and specific activity of the enzyme. RESULTS: Based upon our findings, maximum amount of active mutant enzyme was attained by addition of 0.5 mM IPTG at 150 rpm to the TB culture media. The yield of active enzyme at cluture tempreature of 25 °C and induction period of 18 hour was the highest. CONCLUSION: The results of this study indicated that the yield of mutant AvPAL production in E. coli can be affected mainly by culture temperature and inducer concentration.

Engineering and kinetic stabilization of the therapeutic enzyme Anabeana variabilis phenylalanine ammonia lyase.

Anabeana variabilis phenylalanine ammonia lyase has just recently been discovered and introduced in clinical trials of phenylketonuria enzyme replacement therapy for its outstanding kinetic properties. In the present study, kinetic stabilization of this therapeutically important enzyme has been explored by introduction of a disulfide bond into the structure. Site-directed mutagenesis was performed with quick-change PCR method. Recombinant wild-type and mutated enzymes were expressed in Escherichia coli, and his-tagged proteins were affinity purified. Formation of disulfide bond was confirmed by Ellman's method, and then chemical unfolding, kinetic behavior, and thermal inactivation of mutated enzyme were compared with the wild type. Based on our results, the Q292C mutation resulted in a significant improvement in kinetic stability and resistance against chemical unfolding of the enzyme while kinetic parameters and pH profile of enzyme activity were remained unaffected. The results of the present study provided an insight towards designing phenylalanine ammonia lyases with higher stability.