Isolation and Characterization of Phenylalanine Ammonia Lyase (PAL) Genes in Ferula pseudalliacea: Insights into the Phenylpropanoid Pathway.

Phenylalanine ammonia lyase (PAL) is a key enzyme regulating the biosynthesis of the compounds of the phenylpropanoid pathway. This study aimed to isolate and characterize PAL genes from Ferula pseudalliacea Rech.f. (Apiales: Apiaceae) to better understand the regulation of metabolite production. Three PAL gene isoforms (FpPAL1-3) were identified and cloned using the 3'-RACE technique and confirmed by sequencing. Bioinformatics analysis revealed important structural features, such as phosphorylation sites, physicochemical properties, and evolutionary relationships. Expression analysis by qPCR demonstrated the differential transcription profiles of each FpPAL isoform across roots, stems, leaves, flowers, and seeds. FpPAL1 showed the highest expression in stems, FpPAL2 in roots and flowers, and FpPAL3 in flowers. The presence of three isoforms of PAL in F. pseudalliacea, along with the diversity of PAL genes and their tissue-specific expression profiles, suggests that complex modes of regulation exist for phenylpropanoid biosynthesis in this important medicinal plant. The predicted interaction network revealed associations with key metabolic pathways, emphasizing the multifaceted roles of these PAL genes. In silico biochemical analyses revealed the hydrophilicity of the FpPAL isozyme; however, further analysis of substrate specificity and enzyme kinetics can clarify the specific role of each FpPAL isozyme. These comprehensive results increase the understanding of PAL genes in F. pseudalliacea, helping to characterize their contributions to secondary metabolite biosynthesis.

Phytochemical profile and antifungal activity of essential oils obtained from different Mentha longifolia L. accessions growing wild in Iran and Iraq.

BACKGROUND: Mentha longifolia L. is a perennial plant belonging to the Lamiaceae family that has a wide distribution in the world. M. longifolia has many applications in the food and pharmaceutical industries due to its terpenoid and phenolic compounds. The phytochemical profile and biological activity of plants are affected by their genetics and habitat conditions. In the present study, the content, constituents and antifungal activity of the essential oil extracted from 20 accessions of M. longifolia collected from different regions of Iran and Iraq countries were evaluated. RESULTS: The essential oil content of the accessions varied between 1.54 ± 0.09% (in the Divandarreh accession) to 5.49 ± 0.12% (in the Khabat accession). Twenty-seven compounds were identified in the essential oils of the studied accessions, which accounted for 85.5-99.61% of the essential oil. The type and amount of dominant compounds in the essential oil were different depending on the accession. Cluster analysis of accessions based on essential oil compounds grouped them into three clusters. The first cluster included Baziyan, Boukan, Sarouchavah, Taghtagh, Darbandikhan, Isiveh and Harir. The second cluster included Khabat, Kounamasi, Soni and Mahabad, and other accessions were included in the third cluster. Significant correlations were observed between the essential oil content and components with the climatic and soil conditions of the habitats. The M. longifolia essential oil indicated antifungal activity against Fusarium solani in both methods used. In all studied accessions, the fumigation method compared to the contact method was more able to control mycelia growth. In both methods, the inhibition percentage of essential oil on mycelia growth increased with an increase in essential oil concentration. Significant correlations were found between the essential oil components and the inhibition percentage of mycelium growth. CONCLUSION: The studied M. longifolia accessions showed significant differences in terms of the essential oil content and components. Differences in phytochemical profile of accessions can be due to their genetic or habitat conditions. The distance of the accessions in the cluster was not in accordance with their geographical distance, which indicates the more important role of genetic factors compared to habitat conditions in separating accessions. The antifungal activity of essential oils was strongly influenced by the essential oil quality and concentration, as well as the application method. Determining and introducing the elite accession in this study can be different depending on the breeder's aims, such as essential oil content, desired chemical composition, or antifungal activity.

Strawberry crinkle (SCV) and Strawberry mottle (SMoV) viruses affect yield and physio-biochemical responses of three strawberry cultivars under different seasonal conditions.

BACKGROUND: The current study was performed to evaluate the impact of Strawberry crinkle virus (SCV) and Strawberry mottle virus (SMoV) viruses, in single and mixed infections, on the yield and physio-biochemical traits of strawberry plants with different genetic backgrounds. Accordingly, the virus-free plantlets were produced via meristem culture and were then transplanted in a research field in different seasons. METHODS AND RESULTS: The results indicated that genetic background clearly affects the responses of the strawberry plants to the viral infection. In addition, considering the effects of changing environmental factors, different seasons had significant influences on the strawberry responses to the virus infection. The results indicated that the components indicating destructive effects were significantly higher during the summer season. Reverse transcription-polymerase chain reaction using viral specific primers revealed 345 and 461 bp fragments for SCV and SMoV viruses, respectively, where all plants were positive for studied viruses. The comparison between the influence of SCV and SMoV viruses on the host plants indicated that SCV caused more damaging effects on the strawberry fruit yield and physio-biochemical traits. Simultaneous infection with SCV and SMoV severely impacted the strawberry plants compared with the effect of each virus individually. CONCLUSION: It is recommended to keep the temperature of the greenhouses producing strawberry at the cooler levels to prevent the spreading of the aphid-transmitted viruses.

Exploring genetic variations in threatened medicinal orchids using start codon targeted (SCoT) polymorphism and marker-association with seed morphometric traits.

We aimed to study the genetic diversity and population structure of eight Iranian terrestrial orchid species, including Anacamptis coriophora (L.) R. M. Bateman, Pridgeon and M. W. Chase, Dactylorhiza umbrosa (Kar. & Kir.) Nevski, Himantoglossum affine (Boiss.) Schltr., Orchis collina Banks and Solander, Orchis mascula (L.) L., Orchis simia Lam., Ophrys schulzei Bornm. and Fleischm., and Ophrys straussii H. Fleischm. and Bornm. using start target codon markers (SCoT) and finding markers associated with seed morphometric traits. A total of 254 reproducible SCoT fragments were generated, of which 248 fragments were polymorphic (average polymorphism of 96.18%). The SCoT markers showed a narrow range of polymorphism information content (PIC) varied from 0.397 for S9 primer to 0.499 for S11 and S20 primers. Based on the population analysis results, the Orchis simia accessions collected from Paveh region (Os.P) represented the lowest observed number of alleles (Na) (1.13) and effective number of alleles (Ne) (1.09). At the same time, the highest Na (1.29) and Ne (1.18) values were obtained in O. schulzei collected from Javanrood (Oyst.JA). Shannon's information index (I) was ranged from 0.03 for D. umbrosa accessions collected from Marivan (Du.M population) to 0.263 for Ha.Ja population (H. affine accessions collected from Javanrood). The UPGMA dendrogram obtained with the Jaccard similarity coefficient (r = 0.97295) divided 97 studied terrestrial orchid accessions into eight groups mainly based on species type and geographical origin. Based on the Bayesian statistical index, the highest probability of the data was achieved when accessions were divided into eight groups (K = 8). Multiple association analysis (MRA) revealed significant associations between some of SCoT bands with seed morphometric traits. Our findings can be useful for germplasm characterization, conservation, and improvement of Iranian terrestrial orchid species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00978-4.

Molecular characterization of endangered Iranian terrestrial orchids using ISSR markers and association with floral and tuber-related phenotypic traits.

We aimed to study the genetic diversity, population structure, and phylogeny of Iranian orchids using inter-simple sequence repeat (ISSR) markers to find markers associated with phenotypic traits. Based on the phenotypic analysis, the inflorescence length and the flower number of studied accessions ranged from 3.92 to 27.13 cm and 5 to 50, respectively. On the other hand, the tuber length ranged from 1.80 to 9.35 cm. A total of 310 reproducible ISSR fragments with a size range of 150 to 3000 bp were amplified. ISSR primers provided an average polymorphism information content of 0.391, varied from 0.488 for UBC-876 to 0.351 for UBC-842. Os.J population showed the lowest genetic diversity (H = 0.057 and I = 0.075), while Oyst.JA population showed the highest genetic diversity (H = 0.114 and I = 0.158). At species level, the average coefficient of genetic differentiation (G ST) ranged from 0.265 for Orchis simia to 0.587 for Himantoglossum affine. Gene flow (Nm) varied from 1.38 (O. simia) to 0.756 (Anacamptis collina). The UPGMA genetic similarity dendrogram using Jaccard coefficients (r = 0.973) revealed six main clusters. Based on the Bayesian clustering method, the highest probability of the data was achieved when accessions were divided into eight groups. Floral and tuber-related phenotypic traits represented high correlations together, and they were associated with some ISSR bands based on the multiple association analysis. Altogether, ISSR markers proved to be useful for discrimination and clarification of the relationships among species and populations collected from geographically different locations. Furthermore, it could identify the polymorphism among accessions within each population and species. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12298-020-00920-0) contains supplementary material, which is available to authorized users.

Selenium mitigates cadmium toxicity by preventing oxidative stress and enhancing photosynthesis and micronutrient availability on radish (Raphanus sativus L.) cv. Cherry Belle.

We aimed to examine the effects of selenium on the tolerance of radish plants CV. Cherri Belle under cadmium phytotoxicity. The biomass accumulation was drastically decreased under Cd toxicity and the supplementary Se maintained the biomass acquisition under Cd pressure. The chlorophyll index (SPAD), PSII efficiency (Fv/Fm), and PSII quantum yield (ΦPSII) were declined in response to Cd treatment, while Se nutrition improved these variables in a dose-dependent manner. The highest H2O2 and MDA contents were observed in the plants fed with 10 mg-1 L Cd. The Cd stress resulted in a considerable decline in the activities of GPX, CAT, and APX antioxidant enzymes, while Se supplementation increased their activities in the Cd-treated plants. Based on the mineral analyses, no Cd was traced in the control plants, while the Cd concentration in both roots and leaves of the Cd-stressed radish plants increased with increasing the supplemented Cd levels. Compared with plants solely treated with 10 mg L-1 Cd, Se nutrition declined the Cd absorption in roots and in leaves. The concentration of evaluated micronutrients including Fe, Mn, Cu, and Zn tended to decrease in the Cd-imposed plants in comparison with control plants. Se nutrition of both stressed and non-stressed radish plants increased the concentrations of the studied microelements, except for Zn in which the individual use of Se led to a decrease in the Zn content. Significant positive and negative correlation values were found among the studied traits and the principle component analysis (PCA) biplot and Ward dendrogram confirmed the results of the correlation analysis. Se proved to be efficient in the alleviation of Cd-triggered deleterious effects by improving biomass acquisition, enhancing chlorophyll biosynthesis and fluorescence, and increasing micronutrient uptake in a dose-dependent manner. Furthermore, the Se alleviation mechanism under Cd stress was also connected with the activation of enzymatic antioxidative protection system as well as with decreasing Cd uptake, transport, and distribution in radish leaves. Altogether, our research strongly suggests the implementation of Se in the growth medium to enhance the tolerance of radish plants under Cd stress.

Phytochemical composition and in vitro antioxidant potential of Cynodon dactylon leaf and rhizome extracts as affected by drying methods and temperatures.

The effect of five drying methods including shade drying (SHD), solar drying (SOD), and oven drying at 30 (OD30), 40 (OD40) and 50 °C (OD50) on the phytochemical composition and antioxidant potential of C. dactylon leaf and rhizome was assessed. Among drying methods, OD50 resulted in the shortest drying time (18.3 and 12 h for rhizome and leaf, respectively), when compared with SHD and SOD. Based on GC-MS analyses, 15 and 17 constituents were identified in leaf and rhizome extracts, respectively, accounting for ~ 99% of all components. Fatty acids (palmitic acid and linoleic acid) along with their methyl esters (ethyl palmitate, ethyl linoleate and ethyl oleate) and other derivatives (dihomo-γ-linoleic acid) were the main identified constituents shortly after drying procedures; however, other components such as 5-hydroxymethylfurfural, maltol, retinol and phytol were also traced. Some of C. dactylon phytochemicals including 5-hydroxymethylfurfural and ethyl linoleate were sensitive to high drying temperatures. Besides, higher drying temperatures lead to the production or increasing the level of substances such as 2,3-dihydrobenzofuran, tricyclopentadeca-3,7-dien and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one and diacetin. Based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, the IC50 values were generally higher (significance level of 0.05) for oven-dried rhizome compared with shade-dried leaves and rhizomes that quenched more than 84% of the DPPH at the concentration of 400 mg/ml (IC50 59.12). Our findings suggest that OD30 is a versatile drying method not only to reduce drying time but also to preserve the main phytochemicals and antioxidant activity of C. dactylon during dehydration.

In vitro propagation and conservation of Satureja avromanica Maroofi-an indigenous threatened medicinal plant of Iran.

An efficient and rapid in vitro propagation system for Satureja avromanica, a rare and endangered folk medicinal plant of Iran was developed through the shoot tip and leaf disc explants. Nodal and leaf explants from wild plants were established on MS and WPM media supplemented with BA, BAP and TDZ (0, 0.1, 0.5, 1, 1.5, 2, 5 and 10 mgl(-1)) alone or by application of BA and TDZ (0, 2, 5 and 10 mgl(-1)) in combination with IBA and 2,4-D (0, 0.1, 0.5 and 1 mgl(-1)), respectively. Based on results, the highest mean shoot number (6.21) was obtained on MS medium supplemented with 2 mgl(-1) BA. Regarding the shoot elongation, MS supplemented with 2 mgl(-1) TDZ and MS containing 5 mgl(-1) BA showed the longest shoots (4.82 and 4.39 cm, respectively) after 6 weeks of culture. As a matter of fact, increasing all three tested cytokinins levels led to enhancement of explant response frequency and regenerated shoot number. On the other side, WPM medium supplemented with 0.1 mgl(-1) IBA was found suitable for rooting of regenerated shoots. RAPD molecular analysis revealed genetic stability of in vitro raised plants. In conclusion, individual application of BA, BAP and TDZ were in favor of S. avromanica direct shoot regeneration while treatment media with a combination of IBA and BA as well as 2,4-D and TDZ resulted in callogenesis in most explants. Finally, the in vitro raised plantlets were acclimatized and successfully established in the greenhouse conditions. Our developed protocol can be employed for the large-scale micropropagation and conservation of S. avromanica as a threatened medicinal plant.

A modular cloning toolbox for the generation of chloroplast transformation vectors.

Plastid transformation is a powerful tool for basic research, but also for the generation of stable genetically engineered plants producing recombinant proteins at high levels or for metabolic engineering purposes. However, due to the genetic makeup of plastids and the distinct features of the transformation process, vector design, and the use of specific genetic elements, a large set of basic transformation vectors is required, making cloning a tedious and time-consuming effort. Here, we describe the adoption of standardized modular cloning (GoldenBraid) to the design and assembly of the full spectrum of plastid transformation vectors. The modular design of genetic elements allows straightforward and time-efficient build-up of transcriptional units as well as construction of vectors targeting any homologous recombination site of choice. In a three-level assembly process, we established a vector fostering gene expression and formation of griffithsin, a potential viral entry inhibitor and HIV prophylactic, in the plastids of tobacco. Successful transformation as well as transcript and protein production could be shown. In concert with the aforesaid endeavor, a set of modules facilitating plastid transformation was generated, thus augmenting the GoldenBraid toolbox. In short, the work presented in this study enables efficient application of synthetic biology methods to plastid transformation in plants.