Drought-Induced miRNA Expression Correlated with Heavy Metal, Phenolic Acid, and Protein and Nitrogen Levels in Five Chickpea Genotypes.

Drought is a prime stress, drastically affecting plant growth, development, and yield. Plants have evolved various physiological, molecular, and biochemical mechanisms to cope with drought. Investigating specific biochemical pathways related to drought tolerance mechanisms of plants through biotechnology approaches is one of the quickest and most effective strategies for enhancing crop production. Among them, microRNAs (miRNAs) are the principal post-transcriptional regulators of gene expression in plants during plant growth under biotic and abiotic stresses. In this study, five different chickpea genotypes (Inci, Hasan bey, Arda, Seçkin, and Diyar 95) were grown under normal and drought stress. We recorded the expression levels of microRNAs in these genotypes and found differential expression (miRNA396, miR408, miRNA414, miRNA528, and miRNA1533) under contrasting conditions. Results revealed that miRNA414 and miRNA528 considerably increased in all genotypes under drought stress, and expression levels of miRNA418, miRNA1533, and miRNA396 (except for the Seçkin genotype) were found to be higher under the watered conditions. These genotypes were also investigated for heavy metal, phenolic acid, protein, and nitrogen concentrations under normal and drought stress conditions. The Arda genotype showed a significant increase in nitrogen (5.46%) and protein contents (28.3%), while protein contents were decreased in the Hasan bey and Seçkin genotypes subjected to drought stress. In the case of metals, iron was the most abundant element in all genotypes (Inci = 15.4 ppm, Hasan bey = 29.6 ppm, Seçkin = 37.8 ppm, Arda = 26.3 ppm, and Diyar 95 = 40.8 ppm) under normal conditions. Interestingly, these results were related to miRNA expression in the chickpea genotypes and hint at the regulation of multiple pathways under drought conditions. Overall, the present study will help us to understand the miRNA-mediated regulation of various pathways in chickpea genotypes.

Characterization of Two-Component System gene (TCS) in melatonin-treated common bean under salt and drought stress.

The two-component system (TCS) generally consists of three elements, namely the histidine kinase (HK), response regulator (RR), and histidine phosphotransfer (HP) gene families. This study aimed to assess the expression of TCS genes in P. vulgaris leaf tissue under salt and drought stress and perform a genome-wide analysis of TCS gene family members using bioinformatics methods. This study identified 67 PvTCS genes, including 10 PvHP, 38 PvRR, and 19 PvHK, in the bean genome. PvHK2 had the maximum number of amino acids with 1261, whilst PvHP8 had the lowest number with 87. In addition, their theoretical isoelectric points were between 4.56 (PvHP8) and 9.15 (PvPRR10). The majority of PvTCS genes are unstable. Phylogenetic analysis of TCS genes in A. thaliana, G. max, and bean found that PvTCS genes had close phylogenetic relationships with the genes of other plants. Segmental and tandem duplicate gene pairs were detected among the TCS genes and TCS genes have been subjected to purifying selection pressure in the evolutionary process. Furthermore, the TCS gene family, which has an important role in abiotic stress and hormonal responses in plants, was characterized for the first time in beans, and its expression of TCS genes in bean leaves under salt and drought stress was established using RNAseq and qRT-PCR analyses. The findings of this study will aid future functional and genomic studies by providing essential information about the members of the TCS gene family in beans. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01406-5.

Genome skimming approach reveals the gene arrangements in the chloroplast genomes of the highly endangered Crocus L. species: Crocus istanbulensis (B.Mathew) Ruksans.

Crocus istanbulensis (B.Mathew) Ruksans is one of the most endangered Crocus species in the world and has an extremely limited distribution range in Istanbul. Our recent field work indicates that no more than one hundred individuals remain in the wild. In the present study, we used genome skimming to determine the complete chloroplast (cp) genome sequences of six C. istanbulensis individuals collected from the locus classicus. The cp genome of C. istanbulensis has 151,199 base pairs (bp), with a large single-copy (LSC) (81,197 bp), small single copy (SSC) (17,524 bp) and two inverted repeat (IR) regions of 26,236 bp each. The cp genome contains 132 genes, of which 86 are protein-coding (PCGs), 8 are rRNA and 38 are tRNA genes. Most of the repeats are found in intergenic spacers of Crocus species. Mononucleotide repeats were most abundant, accounting for over 80% of total repeats. The cp genome contained four palindrome repeats and one forward repeat. Comparative analyses among other Iridaceae species identified one inversion in the terminal positions of LSC region and three different gene (psbA, rps3 and rpl22) arrangements in C. istanbulensis that were not reported previously. To measure selective pressure in the exons of chloroplast coding sequences, we performed a sequence analysis of plastome-encoded genes. A total of seven genes (accD, rpoC2, psbK, rps12, ccsA, clpP and ycf2) were detected under positive selection in the cp genome. Alignment-free sequence comparison showed an extremely low sequence diversity across naturally occurring C. istanbulensis specimens. All six sequenced individuals shared the same cp haplotype. In summary, this study will aid further research on the molecular evolution and development of ex situ conservation strategies of C. istanbulensis.

Bryonia multiflora Extract Induces Autophagy via Regulating Long Non-coding RNAs in Breast Cancer Cells.

Bryonia multiflora, one of the species of Bryonia L. (Cucurbitaceae) genus, is a perennial, dioecious, herbaceous plant with rhizome-shaped roots. Bryonia species have anti-inflammatory, antimicrobial, cytotoxic, antioxidant, etc., activities and their components consume antitumoral effects. Purpose of the study to investigate the effect of Bryonia Multiflora extract (BMST) on breast cancer cells. Our results revealed that MCF-7 and MDA-MB-231 cells underwent significant morphological changes leading to cell rounding. No significant changes were observed in the cell viability by MTT. Acridine orange staining of our cells gave rise to think that BMST might lead our cells to autophagy. Therefore, possible molecular mechanisms underlying morphological changes such as autophagy (LC-3B, Beclin, AMBRA1) and apoptosis (Bcl-2) were evaluated on mRNA and protein levels. BMST treated MCF-7 and MDA-MB-231 cells had increased levels of autophagy markers whereas decreased levels of Bcl-2. p21 levels were also found to be increased in both cells. Analysis of lncRNA expressions has shown that BMST treatment led to changes in the expression levels of several lncRNAs playing roles in autophagy. The current study has shown that BMST induces autophagy in MCF-7 and MDA-MB-231 cells via regulating the lncRNAs revealing that BMST could be a promising therapeutic agent.

The Protective Effects of Silymarin on Thioacetamide-Induced Liver Damage: Measurement of miR-122, miR-192, and miR-194 Levels.

This study aims to investigate the protective effects of silymarin (Sm) in thioacetamide (TAA)-related liver damage. What makes this study special is that it attempts to determine the expression of changes in the liver at the level of gene expression. Routine liver damage markers were compared with changes in the levels of microRNA (miRNA) known as new biomarkers. With this in mind, we divided the rats into four groups including control, TAA, Sm + TAA (50 + 50 mg/kg), and Sm + TAA (100 + 50 mg/kg). Blood and tissue samples belonging to the rats were collected in consideration of morphological, immunohistochemistry, miRNAs levels, and biochemical evaluations. Our study results showed that miR-122, miR-192, and miR-194 levels had decreased in the experimental groups given TAA, whereas miR-122, miR-192, and miR-194 levels had increased in the doses of Sm + TAA-given group. Therefore, Sm treatment undertaken before exposure to the toxin successfully altered its effects upon the study animals.

Genetic characterization and virulence of Fusarium spp. isolated from chickpea.

Fusarium wilt causing yield losses in chickpea is one of the most important diseases, and occurs due to some fungi especially F. oxysporum and F. solani. Molecular studies are important in revealing the genetic characterization and virulence of the pathogen. In this study ten Fusarium genotypes isolated from chickpea plants showing symptoms of wilting were used. The ITS region was amplified by using the thermal cycler and genetic similarities and differences among species were revealed by performing sequence analysis of ITS region. Samples except for two of Fusarium genotypes; N1, N2, N3, N4, N5, N7, N9 and N10 genotypes showed a close relative to F. oxysporum. However, the N8 genotyped was found to be similar to F. solani, and interestingly the N6 genotype showed an equal relationship with F. solani and F. oxysporum so, it could not be fully identified. According to the phylogenetic tree, F. oxysporum, N2, N4 and N10 were determined in a separate group and F. oxysporum N5, N7 and N9 formed separate another group which showed a close relationship to each other. The severity of disease caused by isolates on ILC-482 chickpea variety varied between 1.25 and 3.50. The close relationship was found between F. oxysporum, N2, N4, N5, N7, N9 and N10 isolates was confirmed the result of the pathogenicity test of same isolates. The results revealed that N7 isolate prevented the emergence of plants by causing disease on chickpea seeds and it also indicated the high virulence.

Antioxidant capacity and phylogenetic analysis of twenty native grape cultivars in Siirt province, Turkey.

The quality of grape cultivars not only depends on the grape cultivar but also is influenced by the molecular concepts and agro-climatic factors. For this purpose, four different grape cultivars were collected from five different locations in Siirt province (Turkey). Totally twenty different grape cultivars were investigated. In the present study, the antioxidant activity (total phenolic, flavonoid, proanthocyanidin content, DPPH and FRAP activity) in seeds were indicated and phylogenetic analysis (cpDNA;trnL-F region) of twenty native grape cultivars were investigated to construct their phylogenetic tree. According to reported data on antioxidant activity and content of phytochemicals, all cultivars exhibited different values from each other, but Rutik and Gadüv cultivars were found as significantly higher in comparison to others. According to bioinformatics analysis, twenty grape cultivars were distributed into six different major groups. Rutik and Sevkeye cultivars exhibit significant distinction from other grape cultivars. The phylogenetic analysis was also associated and supported with the results of obtained data from bioactivity. The bioactivity and phylogenetic analysis were firstly identified and quantified in these grape cultivars, however, with regard to obtained data from the current study, the grape cultivars grown in Siirt province were indicated significant and valuable results and as a result, these cultivars have to be evaluated before extinction.

Transcriptome - Scale characterization of salt responsive bean TCP transcription factors.

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) proteins are important regulators of growth and developmental processes including branching, floral organ morphogenesis and leaf growth as well as stress response. This study identified 27 TCP genes of Phaseolus vulgaris (common bean), which were divided into three clusters based on phylogenetic relationship. In addition, this study showed that some of TCP genes such as Pvul-TCP-4 and Pvul-TCP-15 located on chromosomes 3 and 7, Pvul-TCP-7 and Pvul-TCP-20 located on chromosome 7 and 9, were segmentally duplicated. On the other hand, a total of 20 Pvul-TCP genes have predicted to be targeted by microRNAs (miRNA). Most of the miRNA-target genes were Pvul-TCP-1, -11, -13 and -27, which were targeted by 13, 17, 22 and 13 plant miRNAs, respectively. miR319 was one of the highly represented regulatory miRNAs to target TCP transcripts. Promoter region analysis of TCP genes resulted that the GT-1 motif, which was related to salt stress, was found in 14 different Pvul-TCP genes. Expression profiling of 10 Pvul-TCP genes based on RNA-sequencing data further confirmed with quantitative real-time RT-PCR measurements identified that Pvul-TCP genes under salt stress are expressed in a cultivar- and tissue-specific manner.

Genome-wide analysis of Phaseolus vulgaris C2C2-YABBY transcription factors under salt stress conditions.

The aim of this study was to identify and characterize the C2C2-YABBY family of genes by a genome-wide scale in common bean. Various in silico approaches were used for the study and the results were confirmed through common molecular biology techniques. Quantitative real-time PCR (qPCR) analysis was performed for identified putative PvulYABBY genes in leaf and root tissues of two common bean cultivars, namely Yakutiye and Zulbiye under salt stress condition. Eight candidate PvulYABBY proteins were discovered and the length of these proteins ranged from 173 to 256 amino acids. The isoelectric points (pIs) of YABBY proteins were between 5.18 and 9.34 and ranged from acidic to alkaline, and the molecular weight of PvulYABBYs were between 18978.4 and 28916.8 Da. Three segmentally duplicated gene couples among the identified eight PvulYABBY genes were detected. These segmentally duplicated gene couples were PvulYABBY-1/PvulYABBY-3, PvulYABBY-5/PvulYABBY-7 and PvulYABBY-6/PvulYABBY-8. The predicted number of exons among the PvulYABBY genes varied from 6 to 8 exons. Additionally, all genes found included introns within ORFs. PvulYABBY-2, -4, -5 and -7 genes were targeted by miRNAs of five plant species and a total of five miRNA families (miR5660, miR1157, miR5769, miR5286 and miR8120) were detected. According to RNA-seq analysis, all genes were up- or down-regulated except for PvulYABBY-1 and PvulYABBY-6 after salt stress treatment in leaf and root tissues of common bean. According to the qPCR analysis, six out of eight genes were expressed in the leaves but only four out of eight genes were expressed in the roots and these genes exhibited tissue- and cultivar-specific expression patterns.

Genome-wide identification of salinity responsive HSP70s in common bean.

The present study is aimed to identify and characterize HSP70 (PvHSP70) genes in two different common bean cultivars under salt stress. For this purpose various in silico methods such as RNAseq data and qRT-PCR analysis were used. A total of 24 candidate PvHSP70 gene were identified. Except for chromosome 4 and 7, these candidate PvHSP70 genes were distributed on the remaining chromosomes. While the lowest number of PvHSP70 genes was determined on chromosomes 1, 3, 5, 7, 9, 10 and 11 (one HSP70 gene), the highest number of PvHSP70s was on chromosomes 6 and 8 (seven HSP70 genes each). Three genes; PvHSP70-5, -9, and -10 were found to have no-introns. In addition, four tandemly and six segmentally duplicated gene couples were detected. A total of 13 PvHSP70 genes were targeted by miRNAs of 44 plant species and the most targeted genes were PvHSP70-5 and -23. The expression profile of PvHSP70 genes based on publicly available RNA-seq data was identified and salt treated leaf tissue was found to have more gene expression levels compared to the root. qRT-PCR analysis showed that the transcript concentrations of upregulated PvHSP70 genes in leaves of Zulbiye (sensitive) were mostly higher than those of Yakutiye (resistant). The present study revealed that PvHSP70 genes might play an important role in salt stress response for common bean cultivars and variability between cultivars also suggests that these genes could be used as functional markers for salt tolerance in common bean.

Transcriptome analysis of wheat inoculated with Fusarium graminearum.

Plants are frequently exposed to microorganisms like fungi, bacteria, and viruses that cause biotic stresses. Fusarium head blight (FHB) is an economically risky wheat disease, which occurs upon Fusarium graminearum (Fg) infection. Moderately susceptible (cv. "Mizrak 98") and susceptible (cv. "Gun 91") winter type bread wheat cultivars were subjected to transcriptional profiling after exposure to Fg infection. To examine the early response to the pathogen in wheat, we measured gene expression alterations in mock and pathogen inoculated root crown of moderately susceptible (MS) and susceptible cultivars at 12 hours after inoculation (hai) using 12X135K microarray chip. The transcriptome analyses revealed that out of 39,179 transcripts, 3668 genes in microarray were significantly regulated at least in one time comparison. The majority of differentially regulated transcripts were associated with disease response and the gene expression mechanism. When the cultivars were compared, a number of transcripts and expression alterations varied within the cultivars. Especially membrane related transcripts were detected as differentially expressed. Moreover, diverse transcription factors showed significant fold change values among the cultivars. This study presented new insights to understand the early response of selected cultivars to the Fg at 12 hai. Through the KEGG analysis, we observed that the most altered transcripts were associated with starch and sucrose metabolism and gluconeogenesis pathways.

Genome-wide fungal stress responsive miRNA expression in wheat.

MicroRNAs (miRNAs) are small non-coding class of RNAs. They were identified in many plants with their diverse regulatory roles in several cellular and metabolic processes. A number of miRNAs were involved in biotic and abiotic stress responses. Here, fungal stress responsive wheat miRNAs were analyzed by using miRNA-microarray strategy. Two different fungi (Fusarium culmorum and Bipolaris sorokiniana) were inoculated on resistant and sensitive wheat cultivars. A total of 87 differentially regulated miRNAs were detected in the 8 × 15 K array including all of the available plant miRNAs. Using bioinformatics tools, the target transcripts of responsive miRNAs were predicted, and related biological processes and mechanisms were assessed. A number of the miRNAs such as miR2592s, miR869.1, miR169b were highly differentially regulated showing more than 200-fold change upon fungal-inoculation. Some of the miRNAs were identified as fungal-inoculation responsive for the first time. The analyses showed that some of the differentially regulated miRNAs targeted resistance-related genes such as LRR, glucuronosyl transferase, peroxidase and Pto kinase. The comparison of the two miRNA-microarray analyses indicated that fungal-responsive wheat miRNAs were differentially regulated in pathogen- and cultivar-specific manners.

Diverse expression pattern of wheat transcription factors against abiotic stresses in wheat species.

Abiotic stress including drought and salinity affects quality and yield of wheat varieties used for the production of both bread and pasta flour. bZIP, MBF1, WRKY, MYB and NAC transcription factor (TF) genes are the largest transcriptional regulators which are involved in growth, development, physiological processes, and biotic/abiotic stress responses in plants. Identification of expression profiling of these TFs plays a crucial role to understand the response of different wheat species against severe environmental changes. In the current study, expression analysis of TaWLIP19 (wheat version of bZIP), TaMBF1, TaWRKY10, TaMYB33 and TaNAC69 genes was examined under drought and salinity stress conditions in Triticum aestivum cv. (Yuregir-89), Triticum turgidum cv. (Kiziltan-91), and Triticum monococcum (Siyez). After drought stress application, all five selected genes in Kiziltan-91 were induced. However, TaMBF1 and TaWLIP19 were the only downregulated genes in Yuregir-89 and Siyez, respectively. Except TaMYB33 in Siyez, expression level of the remaining genes increased under salt stress condition in all Triticum species. For determination of drought response to selected TF members, publicly available RNA-seq data were also analyzed in this study. TaMBF1, TaWLIP19 and TaNAC69 transcripts were detected through in silico analysis. This comprehensive gene expression analysis provides valuable information for understanding the roles of these TFs under abiotic stresses in modern wheat cultivars and ancient einkorn wheat. In addition, selected TFs might be used for determination of drought or salinity-tolerant and susceptible cultivars for molecular breeding studies.

Nutrition metabolism plays an important role in the alternate bearing of the olive tree (Olea europaea L.).

The olive tree (Olea europaea L.) is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year. Microarray based gene expression analysis using RNA from olive samples (on-off years leaves and ripe-unripe fruits) are particularly useful to understand the molecular mechanisms influencing the periodicity in the olive tree. Thus, we carried out genome wide transcriptome analyses involving different organs and temporal stages of the olive tree using the NimbleGen Array containing 136,628 oligonucleotide probe sets. Cluster analyses of the genes showed that cDNAs originated from different organs could be sorted into separate groups. The nutritional control had a particularly remarkable impact on the alternate bearing of olive, as shown by the differential expression of transcripts under different temporal phases and organs. Additionally, hormonal control and flowering processes also played important roles in this phenomenon. Our analyses provide further insights into the transcript changes between "on year" and "off year" leaves along with the changes from unrpipe to ripe fruits, which shed light on the molecular mechanisms underlying the olive tree alternate bearing. These findings have important implications for the breeding and agriculture of the olive tree and other crops showing periodicity. To our knowledge, this is the first study reporting the development and use of an olive array to document the gene expression profiling associated with the alternate bearing in olive tree.