Investigation of pectolytic and PR genes expression, quality and phytochemical contents in organic and non-organic table grapes at harvest and during storage.

The demand for organic table grapes is increasing worldwide. However, comprehensive information of quality parameters and phytochemical compounds in organically grown fruit remain unclear. Furthermore, table grapes are perishable and postharvest quality retention and waste prevention is very important. In this study we have compared the differences between organic and non-organic table grapes in terms of phytochemical compounds and quality parameters as well as the changes in the expression levels of pathogen related and lytic genes during storage. Organic fruit showed higher levels of phenolics, flavonoids, caffeic acid, hydrogen peroxide, protein content, antioxidant and anti-stress enzymes and total antioxidant activities at harvest and during storage. Although, the expression levels of polygalactronases, pectin methyl esterase, chitinase and glucanase genes was lower in organically grown table grapes at harvest, but the expression of all these genes was significantly increased during cold storage. After 60 days of cold storage the expression levels of pectin methyl esterase, chitinase and glucanase genes was significantly higher than the conventionally grown grape berries in organic ones. The highest expression of polygalacturonase was recorded in organic samples after 30 days of storage. There was no significant difference between the two types of table grapes for decay extension and tissue deterioration rate. The results of this study indicate that due to higher levels of phytochemicals and antioxidant compounds the organic table grapes have a higher nutritional quality. Furthermore, the increase in PR and pectolytic genes expression levels is enough for decreasing the fruit susceptibility to decay pathogens and enhancing the postharvest life of organic grapes.

Inductive effect of titanium dioxide nanoparticles on the anticancer compounds production and expression of rosmarinic acid biosynthesis genes in Dracocephalum kotschyi transformed roots.

Methoxylated flavonoids, mainly xanthomicrol and cirsimaritin that can be extracted from Dracocephalum kotschyi Boiss, have anticancer, antispasmodic and antiplatelet effects. The production of these valuable pharmaceutical compounds is one of the major goals of biotechnology studies. In this work, induced transformed roots were influenced by various concentrations of titanium dioxide nanoparticles (TiO2 NPs) at 24 or 48 h exposure time. The effects of TiO2 NPs were assessed on growth rate, activity of antioxidant enzymes, total phenol and flavonoid content (TPC and TFC) and rosmarinic acid (RA) and some flavonoids accumulation. The gene expression level of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) genes were assessed by real time PCR analysis. The transformed roots biomass was substantially increased in elicited roots in comparison with the control. The TPC, TFC and antioxidant enzymes activitywere affected by TiO2 NPs concentration and exposure time. Valuable flavonoids with anticancer characteristics along with xanthomicrol, cirsimaritin and isokaempferide exhibited an increase (70, 34.28 and 7.81-fold, respectively) versus the control. The maximum content of RA (530.5 µg g-1 FW), which was 4.30 times as great as that of control was detected in samples treated with TiO2 NPs (50 mg L-1) 24 h after elicitation. Real-time PCR analysis revealed a considerable increase in pal and ras expression rate engaged by TiO2 NPs levels and exposure time. Overall D. kotschyi transformed roots elicitation by TiO2 NPs led to a massive increment in the production of valuable anticancer flavonoids such as xanthomicrol, cirsimaritin and RA as polyphenol.

Exogenous application of 24-epibrassinosteroid mitigates NaCl toxicity in flax by modifying free amino acids profile and antioxidant defence system.

In the present study, we investigated the ameliorative effects of 24-epibrassinosteroid (24-epiBL) on antioxidant response and ion homeostasis in two NaCl-stressed Linum usitatissimum L. (flax) cultivars differing in salt tolerance. The content and profile of amino acids were also studied in the tolerant cultivar. Salt stress differently altered the activity of antioxidant enzymes, phenol and flavonoid contents, total antioxidant capacity and ion homeostasis in both cultivars, whereas H2O2 and malondialdehyde (MDA) contents were induced only in the TN-97-95 cultivar. Free amino acid concentrations showed variable patterns under salinity conditions compared with the control plants. 24-epiBL decreased the soluble protein content in NaCl-treated plants and also decreased stimulatory effects of salinity on the production and accumulation of phenol and flavonoid contents and antioxidant capacity with altered ion (Na+, K+, and Cl-) contents. The 24-epiBL reduced the chlorophylls (a, b) and carotenoid contents in salt-treated TN-97-95 cultivar while enhanced the activity of antioxidant enzymes and declined the H2O2 content and lipid peroxidation in both NaCl-stressed cultivars. The profile and content of amino acids were significantly changed by 24-epiBL application under salinity treatment. In summary, our findings demonstrate that 24-epiBL seed priming mitigates the deleterious effects of salt stress in flax plants.

Cold stress changes antioxidant defense system, phenylpropanoid contents and expression of genes involved in their biosynthesis in Ocimum basilicum L.

Environmental stresses might alter the activity of antioxidant defense system and both quantity and quality of the essential oil constituents in aromatic plants. In the current study, a greenhouse experiment was designed to assess the influence of cold stress on total phenolic (TPC) and flavonoid contents (TFC), DPPH radical scavenging, antioxidant and phenylalanine ammonia-lyase (PAL) enzymes activity and content of phenylpropanoid compounds in Ocimum basilicum L. The genes expression levels of chavicol O-methyl transferase (CVOMT), cinnamate 4-hydroxylase (C4H), eugenol synthase 1 (EGS1) and eugenol O-methyl transferase (EOMT) were also investigated. Results revealed the highest TPC, TFC and DPPH at 4 °C for 12 h. Positive significant correlation was observed between TFC and DPPH, as well as TPC and PAL enzyme activity. The highest activity of superoxide dismutase and guaiacol peroxidase was recorded in 4 °C for 48 h, while this treatment caused the highest reduction in the activities of ascorbate peroxidase and catalase. In plants exposed to 10 °C for 48 h, the contents of methyleugenol and methylchavicol was positively associated with the expression levels of EGS1 and EOMT. A positive correlation was also found between C4H expression and eugenol, methyleugenol and methylchavicol contents under 4 °C for 12 h.

Iron oxide nanoparticles: a novel elicitor to enhance anticancer flavonoid production and gene expression in Dracocephalum kotschyi hairy-root cultures.

BACKGROUND: Dracocephalum kotschyi Boiss. is a valuable source of rosmarinic acid (RA) and methoxylated hydroxyflavones (such as xanthomicrol and cirsimaritin) with antioxidative and antiplatelet effects and with antiproliferative potential against various cancer cells. The extensive application of nanotechnology in hairy root cultures is a new sustainable production platform for producing these active constituents. In the present study, hairy roots derived from 4-week-old leaves and Agrobacterium rhizogenes strain ATCC15834 were used to investigate the impact of various concentrations of iron oxide nanoparticles (Fe NPs) in two elicitation time exposures (24 and 48 h) on growth, antioxidant enzyme activity, total phenolic and flavonoid content (TPC and TFC), and some polyphenols. Gene expression levels of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) were also analyzed. RESULTS: Iron nanoparticles enhanced biomass accumulation in hairy roots. The treatment time and Fe NP dosage largely improved the activity of antioxidant enzymes, TPC and TFC. The highest RA (1194 µg g-1 FW) content (9.7-fold), compared to controls, was detected with 24 h of exposure to 75 mg L-1 Fe NP, which was consistent with the expression of pal and ras genes under the influence of elicitation. The xanthomicrol, cirsimaritin, and isokaempferide content was increased 11.87, 3.85, and 2.27-fold, respectively. CONCLUSION: Stimulation of D. kotschyi hairy roots by Fe NPs led to a significant increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and xanthomicrol. © 2019 Society of Chemical Industry.

24-Epibrassinolide alters DNA cytosine methylation of Linum usitatissimum L. under salinity stress.

Salinity is a common environmental challenge limiting worldwide agricultural crop yield. Plants employ epigenetic regulatory strategies, such as DNA methylation which relatively allows rapid adaptation to new conditions in response to environmental stresses. Brassinosteroids (BRs) are a novel group of phytohormones recognized as transcription and translation regulators which are able to mitigate the impact of environmental stresses on the plants. In the current investigation, the influence of salinity and 24-epibrassinolide (24-epiBL) was investigated on the extent and pattern of cytosine DNA methylation using methylation-sensitive amplified polymorphisms (MSAP) technique in flax. Upon NaCl (150 mM) exposure, total methylation of CCGG sequences was decreased in comparison to control plants, while 24-epiBL (10-8 M) induced total methylation under salinity stress. Sequencing and analysis of six randomly selected MSAP fragments detected genes involved in various biological and molecular processes such as vitamine B1 biosynthesis, protein targeting and localization, post-translational modification and gene regulation. In conclusion, 24-epiBL seed priming could play critical role in regulation of cellular and biological processes in response to salt stress by epigenetic modification and induction of methylation.

The impact of cold stress on genes expression pattern of mono- and sesquiterpene biosynthesis and their contents in Ocimum basilicum L.

Basil (Ocimum basilicum L.) contains valuable monoterpene and sesquiterpene compounds used for medical purposes. Environmental stresses are suggested to change the essential oil composition in medicinal plants. In the current investigation, an experiment was arranged in greenhouse to study the effect of cold stress on genes expression patterns of linalool synthase (LIS), ß-myrcene synthase (MYS), γ-cadinene synthase (CDS), germacrene D synthase (GDS) and geraniol synthase (GES) which is involved in monoterpenes and sesquiterpenes biosynthesis in O. basilicum. The monoterpenes and sesquiterpenes composition and content were also investigated. Plants were exposed to temperatures 22 (control), 4 and 10 °C at 6 to 8 leaf stage for 12, 24 and 48 h. The genes expression levels were determined by real time PCR in plant leaves. Essential oil was extracted at flowering stage by distillation using Clevenger apparatus and its compounds were identified using gas chromatography/mass spectrometry (GC-MS). The results revealed that the LIS expression increasingly occurred to 4.86 fold at 10 °C after 12 h while that of GES reached to 5.7 fold at 10 °C after 48 h. Temperature 4 °C for 12 h increased the expression levels of MYS and GDS genes to 41.5 and 14.2 fold, respectively, while the expression level of CDS increased to 25.5 fold at 4 °C for 48 h. Significant differences (P ≤ 0.01) were observed among treatments with respect to all compounds except α-pinene and camphene. The maximum proportion of geraniol and γ-cadinene were observed at 4 °C for 24 h, while the maximum proportion of germacrene D and α-bergamotene obtained at 10 °C for 12 h. The highest proportion of 1, 8-cineole was achieved at 4 °C for 48 h. Positive associations between germacrene D content and GDS expression (r = 0.8, P ≤ 0.05) and γ-cadinene and CDS expression (r = 0.78, P ≤ 0.05) proposed that the content of terpenoid compounds in basil can be enhanced through increasing the expression levels of genes involved in their biosynthesis.

Genetic Mapping of Quantitative Trait Loci for Yield-Affecting Traits in a Barley Doubled Haploid Population Derived from Clipper × Sahara 3771.

Many traits play essential roles in determining crop yield. Wide variation for morphological traits exists in Hordeum vulgare L., but the genetic basis of this morphological variation is largely unknown. To understand genetic basis controlling morphological traits affecting yield, a barley doubled haploid population (146 individuals) derived from Clipper × Sahara 3771 was used to map chromosome regions underlying days to awn appearance, plant height, fertile spike number, flag leaf length, spike length, harvest index, seed number per plant, thousands kernel weight, and grain yield. Twenty-seven QTLs for nine traits were mapped to the barley genome that described 3-69% of phenotypic variations; and some genomic regions harbor a given QTL for more than one trait. Out of 27 QTLs identified, 19 QTLs were novel. Chromosomal regions on 1H, 2H, 4H, and 6H associated with seed grain yield, and chromosome regions on 2H and 6H had major effects on grain yield (GY). One major QTL for seed number per plant was flanked by marker VRS1-KSUF15 on chromosome 2H. This QTL was also associated with GY. Some loci controlling thousands kernel weight (TKW), fertile spike number (FSN), and GY were the same. The major grain yield QTL detected on linkage PSR167 co-localized with TAM10. Two major QTLs controlling TKW and FSN were also mapped at this locus. Eight QTLs on chromosomes 1H, 2H, 3H, 4H, 5H, 6H, and 7H consistently affected spike characteristics. One major QTL (ANIONT1A-TACMD) on 4H affected both spike length (SL) and spike number explained 9 and 5% of the variation of SL and FSN, respectively. In conclusion, this study could cast some light on the genetic basis of the studied pivotal traits. Moreover, fine mapping of the identified major effect markers may facilitate the application of molecular markers in barley breeding programs.

Preliminary evidence for associations between molecular markers and quantitative traits in a set of bread wheat (Triticum aestivum L.) cultivars and breeding lines.

The identification of polymorphic markers associated with various quantitative traits allows us to test their performance for the exploitation of the extensive quantitative variation maintained in gene banks. In the current study, a set of 97 wheat germplasm accessions including 48 cultivars and 49 breeding lines were evaluated for 18 agronomic traits. The accessions were also genotyped with 23 ISSR, nine IRAP and 20 REMAP markers, generating a total of 658 clear and scorable bands, 86% of which were polymorphic. Both neighbor-joining dendrogram and Bayesian analysis of clustering of individuals revealed that the accessions could be divided into four genetically distinct groups, indicating the presence of a population structure in current wheat germplasm. Associations between molecular markers and 18 agronomic traits were analyzed using the mixed linear model (MLM) approach. A total of 94 loci were found to be significantly associated with agronomic traits (P≤0.01). The highest number of bands significantly associated with the 18 traits varied from 11 for number of spikelets spike-1 (NSS) to two for grain yield in row (GRY). Loci ISSR16-9 and REMAP13-10 were associated with three different traits. The results of the current study provide useful information about the performance of retrotransposon-based and ISSR molecular markers that could be helpful in selecting potentially elite gene bank samples for wheat-breeding programs.

The effect of drought stress on the expression of key genes involved in the biosynthesis of phenylpropanoids and essential oil components in basil (Ocimum basilicum L.).

Basil (Ocimum basilicum L.), a medicinal plant of the Lamiaceae family, is used in traditional medicine; its essential oil is a rich source of phenylpropanoids. Methylchavicol and methyleugenol are the most important constituents of basil essential oil. Drought stress is proposed to enhance the essential oil composition and expression levels of the genes involved in its biosynthesis. In the current investigation, an experiment based on a completely randomized design (CRD) with three replications was conducted in the greenhouse to study the effect of drought stress on the expression level of four genes involved in the phenylpropanoid biosynthesis pathway in O. basilicum c.v. Keshkeni luvelou. The genes studied were chavicol O-methyl transferase (CVOMT), eugenol O-methyl transferase (EOMT), cinnamate 4-hydroxylase (C4H), 4-coumarate coA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD). The effect of drought stress on the essential oil compounds and their relationship with the expression levels of the studied genes were also investigated. Plants were subjected to levels of 100%, 75%, and 50% of field capacity (FC) at the 6-8 leaf stage. Essential oil compounds were identified by gas chromatography/mass spectrometry (GC-MS) at flowering stage and the levels of gene expression were determind by real time PCR in plant leaves at the same stage. Results showed that drought stress increased the amount of methylchavicol, methyleugenol, ß-Myrcene and α-bergamotene. The maximum amount of these compounds was observed at 50% FC. Real-time PCR analysis revealed that severe drought stress (50% FC) increased the expression level of CVOMT and EOMT by about 6.46 and 46.33 times, respectively, whereas those of CAD relatively remained unchanged. The expression level of 4CL and C4H reduced under drought stress conditions. Our results also demonstrated that changes in the expression levels of CVOMT and EOMT are significantly correlated with methylchavicol (r = 0.94, P ≤ 0.05) and methyleugenol (r = 0.98, P ≤ 0.05) content. Thus, drought stress probably increases the methylchavicol and methyleugenol content, in part, through increasing the expression levels of CVOMT and EOMT.

Development of IRAP- and REMAP-derived SCAR markers for marker-assisted selection of the stripe rust resistance gene Yr15 derived from wild emmer wheat.

KEY MESSAGE: Yr15 provides broad resistance to stripe rust, an important wheat disease. REMAP- and IRAP-derived co-dominant SCAR markers were developed and localize Yr15 to a 1.2 cM interval. They are reliable across many cultivars. Stripe rust [Pucinia striiformis f.sp. tritici (Pst)] is one of the most important fungal diseases of wheat, found on all continents and in over 60 countries. Wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of durum wheat, is a valuable source of novel stripe rust resistance genes for wheat breeding. T. dicoccoides accession G25 carries Yr15 on chromosome 1BS. Yr15 confers resistance to virtually all tested Pst isolates; it is effective in durum and bread wheat introgressions and their derivatives. Retrotransposons generate polymorphic insertions, which can be scored as Mendelian markers using techniques such as REMAP and IRAP. Six REMAP- and IRAP-derived SCAR markers were mapped using 1,256 F2 plants derived from crosses of the susceptible T. durum accession D447 (DW1) with its resistant BC3F9 and BC3F10 (B9 and B10) near isogenic lines, which carried Yr15 introgressed from G25. The nearest markers segregated 0.1 cM proximally and 1.1 cM distally to Yr15. These markers were also mapped and validated at the same position in another 500 independent F2 plants derived from crosses of B9 and B10 with the susceptible cultivar Langdon (LDN). SC2700 and SC790, defining Yr15 on an interval of 1.2 cM, were found to be reliable and robust co-dominant markers in a wide range of wheat lines and cultivars with and without Yr15. These markers are useful tags in marker-assisted wheat breeding programs that aim to incorporate Yr15 into elite wheat lines and cultivars for durable and broad-spectrum resistance to stripe rust.

Retrotransposon insertional polymorphism in Iranian bread wheat cultivars and breeding lines revealed by IRAP and REMAP markers.

Inter-retrotransposon amplified polymorphisms (IRAPs) and retrotransposon-microsatellite amplified polymorphisms (REMAPs) were used to detect retrotransposon integration events and genetic diversity in 101 Iranian bread wheat (Triticum aestivum L.) cultivars and breeding lines. The 9 IRAP primers amplified 128 loci, and 20 REMAP primers amplified 263 loci. Percentage of polymorphic loci, average expected heterozygosity, number of effective alleles, and Shannon's information index for the REMAP markers were slightly higher than those for the IRAP markers. The same estimated parameters calculated for native and nonnative retrotransposons were not considerably different. A Mantel test between IRAP and REMAP cophenetic matrices evidenced no significant correlation. Cluster analysis based on the Dice similarity coefficient and complete linkage algorithm using IRAP+REMAP loci identified five groups among the genotypes studied that could be applied as crossing parents in T. aestivum breeding programs.