Genome-wide investigation and expression analysis of AP2-ERF gene family in salt tolerant common bean

Apetala2-ethylene-responsive element binding factor (AP2-ERF) superfamily with common AP2-DNA binding domain have developmentally and physiologically important roles in plants. Since common bean genome project has been completed recently, it is possible to identify all of the AP2-ERF genes in the common bean genome. In this study, a comprehensive genome-wide in silico analysis identified 180 AP2-ERF superfamily genes in common bean (Phaseolus vulgaris). Based on the amino acid alignment and phylogenetic analyses, superfamily members were classified into four subfamilies: DREB (54), ERF (95), AP2 (27) and RAV (3), as well as one soloist. The physical and chemical characteristics of amino acids, interaction between AP2-ERF proteins, cis elements of promoter region of AP2-ERF genes and phylogenetic trees were predicted and analyzed. Additionally, expression levels of AP2-ERF genes were evaluated by in silico and qRT-PCR analyses. In silico micro-RNA target transcript analyses identified nearly all PvAP2-ERF genes as targets of by 44 different plant species’ miRNAs were identified in this study. The most abundant target genes were PvAP2/ERF-20-25-62-78-113-173. miR156, miR172 and miR838 were the most important miRNAs found in targeting and BLAST analyses. Interactome analysis revealed that the transcription factor PvAP2-ERF78, an ortholog of Arabidopsis At2G28550, was potentially interacted with at least 15 proteins, indicating that it was very important in transcriptional regulation. Here we present the first study to identify and characterize the AP2-ERF transcription factors in common bean using whole-genome analysis, and the findings may serve as a references for future functional research on the transcription factors in common bean

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean (Phaseolus vulgaris)

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks  <  1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean (Phaseolus vulgaris)

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks  <  1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume

Genome editing for healthy crops: traits, tools and impacts

Crop cultivars in commercial use have often been selected because they show high levels of resistance to pathogens. However, widespread cultivation of these crops for many years in the environments favorable to a pathogen requires durable forms of resistance to maintain “healthy crops”. Breeding of new varieties tolerant/resistant to biotic stresses by incorporating genetic components related to durable resistance, developing new breeding methods and new active molecules, and improving the Integrated Pest Management strategies have been of great value, but their effectiveness is being challenged by the newly emerging diseases and the rapid change of pathogens due to climatic changes. Genome editing has provided new tools and methods to characterize defense-related genes in crops and improve crop resilience to disease pathogens providing improved food security and future sustainable agricultural systems. In this review, we discuss the principal traits, tools and impacts of utilizing genome editing techniques for achieving of durable resilience and a “healthy plants” concept

Kışlık buğdaylarda (Kızıltan-91 and Bezostaja-01) doku kültürünün, rejenerasyonun ve agrobakteriuma dayalı transformasyon parametrelerinin optimizasyonu

The objective of this study was to optimize tissue culture and regeneration parameters of immature inflorescence culture of Triticum aestivum cv. Bezostaja- 01 and Triticum durum cv. Kızıltan-91. The effects of callus age and vernalisation time of explants on regeneration success were evaluated. For determination of optimum vernalisation time of immature inflorescence, plants subjected to 4°C for 1, 2, 3, 4, and 5 weeks, respectively. Tillers containing immature inflorescences were collected at the same time. Percentage of inflorescence formed tillers over total explants were reached the highest value, 79 %, at 4 weeks cold treated Kızıltan cultivar and, 73 %, at 5 weeks cold treated Bezostaja cultivar. Isolated immature inflorescences were put onto 2mg /L 2,4-dichlorophenoxyacetic acid and picloram containing callus induction medium for Kızıltan and Bezostaja cultures, respectively. Callus induction rate were found to be 100 % for Kızıltan and Bezostaja. These explants were taken to regeneration after 6, 9, 12 and 15 weeks of dark incubation period. The regeneration capacities of calli were determined as shooting percentage and data were collected after 4, 8, 12, and 15 week regeneration period. The highest shooting percentage of 69 %, were obtained from 6 weeks old calli produced from 4 weeks vernalised explants in Kızıltan cultures at the end of 15 weeks regeneration period. However, shooting percentage was 57.2 % for 9 weeks old calli while it decreases to 37.6 % in 12 weeks old calli and 44.2 % in 15 weeks old calli at the end of 15 weeks regeneration period. This showed that prolonged dark incubation period decreased regeneration capacity of the callus. However, there was no significant difference in regeneration capacities of calli produced from Bezostaja immature inflorescence and the highest shooting percentage was obtained from 9 weeks old calli producedM.S. - Master of Scienc

TaNHX1 ve TaSTR genleri kullanarak tuza dirençli bitkilerin geliştirilmesi.

Soil salinity negatively affects agricultural production in Turkey by decreasing the yield and quality. Direct introduction of stress related genes by genetic engineering is one of the most rapid approaches to develop stress tolerant crops. In this study, TaNHX1 gene was isolated from bread wheat and three different local wheat cultivars were transformed with overexpression vectors containing TaNHX1 gene by using Agrobacterium-mediated and particle bombardment gene transfer techniques. Immature embryo and inflorescence of Triticum durum cv. Kızıltan-91 and Triticum aestivum cv. Yüreğir-89 and mature embryo of Triticum durum cv. Mirzabey-2000 were used as an explant. In this manner, totally 8960 and 5650 explants were used during particle bombardment and Agrobacterium-mediated transformation, respectively. Moreover, leaves of Nicotiana tabacum cv. Petit Havana were transformed by TaSTR gene to develop salt resistant transgenic tobacco plants by using Agrobacterium-mediated transformation. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Expression of TaSTRG in tobacco was evaluated by physiological and biochemical analysis, such as germination test, root length and MDA analysis. In addition to the nuclear transformation, chloroplast transformation of tobacco was performed with Xyl10B gene responsible for the synthesis of hyperthermostable xylanase enzyme. Stable integration of transgenes and homoplasmy were confirmed with PCR and Southern blotting.Ph.D. - Doctoral Progra

Development of resistant sunflower lines to broomrape using CRISPR-CAS9

Sunflower is one of the most important oil crops in the world that become a strategic plant due to the increased demand for its oil in recent years. Sunflower has low climate demand that enables it to grow in many regions of Europe. The biggest problem in sunflower cultivation is the presence of parasitic plants called broomrape (O. cumana). The seeds of these non-photosynthetic parasitic plants germinate with the secretion of Sesquiterpene Lactones (STL) from the roots of the sunflower. After attachment to the roots of the sunflower, it absorbs the water and all the nutrients from the host. Just one broomrape plant can produce millions of tiny seeds that can survive more than 15 years in the soil and can contaminate many fields in a region. Classical herbicides and mechanical techniques are not effective on these parasitic plants, since it already causes great damage to the plant when it rises above the ground. Many sunflower lines resistant to broomrape have been developed in last decades. However, the resistance of these lines was broken by emergence of new virulent broomrape strains. In recent years, secretion of Sesquiterpene Lactones (STLs) from sunflower roots has been found to trigger the germination of broomrape seeds. The genes encoding the enzymes (HaGAS, HaGAO, HaG8H, HaCOS) functional in STL biosynthesis in sunflower have been well characterized in recent years. In the light of all these information, genes of the enzymes that catalyze the production STLS was aimed to knockout with CRISPR/Cas9 technique in the project

Enhanced salt tolerance of transgenic tobacco expressing a wheat salt tolerance gene

Soil salinity is one of the most important limiting factors of agricultural productivity in the world. The Triticum aestivum salt tolerance-related gene (TaSTRG) possesses a functional response to salt and drought stress conditions. A variety of stress factors, such as salt, drought, abscisic acid, and cold, may induce the expression of TaSTRG in wheat. In this study, the TaSTRG gene was transferred to tobacco via Agrobacterium-mediated transformation. Overexpression of TaSTRG in transgenic tobacco plants indicated higher salt tolerance and mediated more vigorous growth than in wild-type plants. Under salt stress conditions, the transgenic tobacco plants had higher germination and survival rates and longer root length than the control plants. Under salt treatments (200-250 mM), TaSTRG-overexpressing tobacco plants accumulated a higher amount of proline and had significantly lower malondialdehyde content than wild-type plants. Furthermore, transgene inheritance followed Mendelian laws, indicating the stability of TaSTRG in transgenic tobacco plants. These results indicated that the wheat TaSTRG gene plays an important role in responding to salt stress

Development of Highly Efficient Resistance to <i>Beet Curly Top Iran Virus</i> (<i>Becurtovirus</i>) in Sugar Beet (<i>B. vulgaris</i>) via CRISPR/Cas9 System

Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10–55%), disease symptoms and biomass reduction (13–45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53–88%), especially for those targeting BCTIV’s movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet

Antioxidative and physiological responses of two sunflower (Helianthus annuus) cultivars under PEG-mediated drought stress

Drought stress is one of the most important yield-reducing factors in crop production. Sunflower, an oilseed crop, is severely affected by abiotic stress. In this study, 2 sunflower cultivars (Musala and Aydin) were evaluated in terms of various biochemical and physiological responses under 2 different polyethylene glycol-mediated drought stress conditions. Stress-determining parameters such as malondialdehyde (MDA), hydrogen peroxide (H2O2), and proline contents were determined. Both cultivars showed similar responses at osmotic potentials of both -0.4 and -0.8 MPa. Aydin was less affected than Musala under these stress conditions. MDA, H2O2, and proline levels were similar at both -0.4 and -0.8 MPa osmotic potentials in the 2 different cultivars. The 2 cultivars differed significantly in ascorbate peroxidase and catalase enzyme activities, which were more prominent in Aydin for both stress levels. However, glutathione reductase activity did not appear to be an essential part of the antioxidative system in either of the cultivars. Engineering antioxidative enzyme levels might provide a potential mechanism to cope with drought stress in sunflower