Turgay Unver

Genome-wide identification of miRNAs responsive to drought in peach (Prunus persica) by high-throughput deep sequencing.

Peach (Prunus persica L.) is one of the most important worldwide fresh fruits. Since fruit growth largely depends on adequate water supply, drought stress is considered as the most important abiotic stress limiting fleshy fruit production and quality in peach. Plant responses to drought stress are regulated both at transcriptional and post-transcriptional level. As post-transcriptional gene regulators, miRNAs (miRNAs) are small (19–25 nucleotides in length), endogenous, non-coding RNAs. Recent studies indicate that miRNAs are involved in plant responses to drought. Therefore, Illumina deep sequencing technology was used for genome-wide identification of miRNAs and their expression profile in response to drought in peach. In this study, four sRNA libraries were constructed from leaf control (LC), leaf stress (LS), root control (RC) and root stress (RS) samples. We identified a total of 531, 471, 535 and 487 known mature miRNAs in LC, LS, RC and RS libraries, respectively. The expression level of 262 (104 up-regulated, 158 down-regulated) of the 453 miRNAs changed significantly in leaf tissue, whereas 368 (221 up-regulated, 147 down-regulated) of the 465 miRNAs had expression levels that changed significantly in root tissue upon drought stress. Additionally, a total of 197, 221, 238 and 265 novel miRNA precursor candidates were identified from LC, LS, RC and RS libraries, respectively. Target transcripts (137 for LC, 133 for LS, 148 for RC and 153 for RS) generated significant Gene Ontology (GO) terms related to DNA binding and catalytic activites. Genome-wide miRNA expression analysis of peach by deep sequencing approach helped to expand our understanding of miRNA function in response to drought stress in peach and Rosaceae. A set of differentially expressed miRNAs could pave the way for developing new strategies to alleviate the adverse effects of drought stress on plant growth and development.

Genome-wide analysis of the bZIP transcription factors in cucumber.

bZIP proteins are one of the largest transcriptional regulators playing crucial roles in plant development, physiological processes, and biotic/abiotic stress responses. Despite the availability of recently published draft genome sequence of Cucumis sativus, no comprehensive investigation of these family members has been presented for cucumber. We have identified 64 bZIP transcription factor-encoding genes in the cucumber genome. Based on structural features of their encoded proteins, CsbZIP genes could be classified into 6 groups. Cucumber bZIP genes were expanded mainly by segmental duplication rather than tandem duplication. Although segmental duplication rate of the CsbZIP genes was lower than that of Arabidopsis, rice and sorghum, it was observed as a common expansion mechanism. Some orthologous relationships and chromosomal rearrangements were observed according to comparative mapping analysis with other species. Genome-wide expression analysis of bZIP genes indicated that 64 CsbZIP genes were differentially expressed in at least one of the ten sampled tissues. A total of 4 CsbZIP genes displayed higher expression values in leaf, flowers and root tissues. The in silico micro-RNA (miRNA) and target transcript analyses identified that a total of 21 CsbZIP genes were targeted by 38 plant miRNAs. CsbZIP20 and CsbZIP22 are the most targeted by miR165 and miR166 family members, respectively. We also analyzed the expression of ten CsbZIP genes in the root and leaf tissues of drought-stressed cucumber using quantitative RT-PCR. All of the selected CsbZIP genes were measured as increased in root tissue at 24th h upon PEG treatment. Contrarily, the down-regulation was observed in leaf tissues of all analyzed CsbZIP genes. CsbZIP12 and CsbZIP44 genes showed gradual induction of expression in root tissues during time points. This genome-wide identification and expression profiling provides new opportunities for cloning and functional analyses, which may be used in further studies for improving stress tolerance in plants.

Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress

The WRKY superfamily of transcription factors was shown to be involved in biotic and abiotic stress responses in plants such as wheat (Triticum aestivum L.), one of the major crops largely cultivated and consumed all over the world. Drought is an important abiotic stress resulting in a considerable amount of loss in agronomical yield. Therefore, identification of drought responsive WRKY members in wheat has a profound significance. Here, a total of 160 TaWRKY proteins were characterized according to sequence similarity, motif varieties, and their phylogenetic relationships. The conserved sequences of the TaWRKYs were aligned and classified into three main groups and five subgroups. A novel motif in wheat, WRKYGQR, was identified. To putatively determine the drought responsive TaWRKY members, publicly available RNA-Seq data were analyzed for the first time in this study. Through in silico searches, 35 transcripts were detected having an identity to ten known TaWRKY genes. Furthermore, relative expression levels of TaWRKY16/TaWRKY16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY59, TaWRKY61, and TaWRKY82 were measured in root and leaf tissues of drought-tolerant Sivas 111/33 and susceptible Atay 85 cultivars. All of the quantified TaWRKY transcripts were found to be up-regulated in root tissue of Sivas 111/33. Differential expression of TaWRKY16, TaWRKY24, TaWRKY59, TaWRKY61 and TaWRKY82 genes was discovered for the first time upon drought stress in wheat. These comprehensive analyses bestow a better understanding about the WRKY TFs in bread wheat under water deficit, and increased number of drought responsive WRKYs would contribute to the molecular breeding of tolerant wheat cultivars.

Boron Stress Responsive MicroRNAs and Their Targets in Barley

Boron stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. In this study, miRNAs associated with boron stress were identified and characterized in barley. miRNA profiles were also comparatively analyzed between root and leave samples. A total of 31 known and 3 new miRNAs were identified in barley; 25 of them were found to respond to boron treatment. Several miRNAs were expressed in a tissue specific manner; for example, miR156d, miR171a, miR397, and miR444a were only detected in leaves. Additionally, a total of 934 barley transcripts were found to be specifically targeted and degraded by miRNAs. In silico analysis of miRNA target genes demonstrated that many miRNA targets are conserved transcription factors such as Squamosa promoter-binding protein, Auxin response factor (ARF), and the MYB transcription factor family. A majority of these targets were responsible for plant growth and response to environmental changes. We also propose that some of the miRNAs in barley such as miRNA408 might play critical roles against boron exposure. In conclusion, barley may use several pathways and cellular processes targeted by miRNAs to cope with boron stress.

miR408 overexpression causes increased drought tolerance in chickpea

Drought stress limits yield severely in most of the crops. Plants utilize complex gene regulation mechanisms to tolerate water deficiency as well as other abiotic stresses. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are progressively recognized as important regulators of gene expression acting at post-transcriptional level. miR408, conserved in terrestrial plants, targets copper related genes. Although, expression level of miR408 is influenced by various environmental factors including drought stress, the biological action of miR408 is still unclear. To examine the miR408 function upon drought stress in chickpea, transgenic lines overexpressing the miR408 were generated. Induced tolerance was observed in the plants with enhanced miR408 expression upon 17-day water deficiency. Expression levels of miR408 target gene together with seven drought responsive genes were measured using qRT-PCR. Here, the involvement of miR408 in drought stress response has been reported. The overexpression leading plantacyanin transcript repression caused regulation of DREB and other drought responsive genes.

miRNA-based drought regulation in wheat

MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. Drought is a common environmental stress influencing crop growth and development. To date, it has been reported that a number of plant miRNA are involved in drought stress response. In this study, we comparatively investigated drought stress-responsive miRNAs in the root and leaf of bread wheat (Triticum aestivum cv. Sivas 111/33) by miRNA microarray screening. miRNA microarray analysis showed that 285 miRNAs (207 upregulated and 78 downregulated) and 244 miRNAs (115 upregulated and 129 downregulated) were differentially expressed in leaf and root tissues, respectively. Among the differentially expressed miRNAs, 23 miRNAs were only expressed in the leaf and 26 miRNAs were only expressed in the root of wheat growth under drought stress. Upon drought treatment, expression of miR159, miR160, miR166, miR169, miR172, miR395, miR396, miR408, miR472, miR477, miR482, miR1858, miR2118, and miR5049 were found to be significantly differentiated in bread wheat. The regulatory network analysis showed that miR395 has connections with a number of target transcripts, and miR159 and miR319 share a number of target genes. Drought-tolerant and drought-sensitive wheat cultivars showed altered expression pattern upon drought stress in terms of investigated miRNA and their target transcript expression level.

Regulation of the alkaloid biosynthesis by miRNA in opium poppy

Opium poppy (Papaver somniferum) is an important medicinal plant producing benzylisoquinoline alkaloids (BIA). MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) of approximately 21 nucleotides. They are noncoding, but regulate gene expression in eukaryotes. Although many studies have been conducted on the identification and functions of plant miRNA, scarce researches on miRNA regulation of alkaloid biosynthesis have been reported. In this study, a total of 316 conserved and 11 novel miRNAs were identified in opium poppy using second-generation sequencing and direct cloning. Tissue-specific regulation of miRNA expression was comparatively analysed by miRNA microarray assays. A total of 232 miRNAs were found to be differentially expressed among four tissues. Likewise, 1469 target transcripts were detected using in silico and experimental approaches. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates carbohydrate metabolism and genetic-information processing. Additionally, miRNA target transcripts were mostly involved in response to stress against various factors and secondary-metabolite biosynthesis processes. Target transcript identification analyses revealed that some of the miRNAs might be involved in BIA biosynthesis, such as pso-miR13, pso-miR2161 and pso-miR408. Additionally, three putatively mature miRNA sequences were predicted to be targeting BIA-biosynthesis genes.

Transcriptome-wide identification of R2R3-MYB transcription factors in barley with their boron responsive expression analysis

MYB family of transcription factors (TF) comprises one of the largest transcription factors in plants and is represented in all eukaryotes. They include highly conserved MYB repeats (1R, R2R3, 3R, and 4R) in the N-terminus. In addition to this, they have diverse C-terminal sequences which help the protein gain wide distinct functions, such as controlling development, secondary metabolism, hormonal regulation and response to biotic and abiotic stress. Stress-responsive roles of the MYB TFs were reported for drought, salt, wounding, cold, freezing, dehydration and osmotic stresses. This study describes the identification of barley R2R3-MYB TFs including their expression analysis in tissues under control and Boron (B) toxic conditions. Conserved motifs for MYB proteins were searched into barley full-transcriptome RNA-seq data and a total of 320 protein sequences were filtered as MYB TFs in which 51 of them corresponded to R2R3 MYB TFs. Using various bioinformatics tools, their conserved domain structures, chromosomal distributions, gene duplications, comparative functional analysis, as well as phylogenetic relations with Arabidopsis thaliana, were conducted. Beside the RNA-seq data-based expression pattern analysis of 51 R2R3 MYB TFs, quantitative analysis of selected R2R3 MYB TF genes was assessed in control and B-stressed root and leaf tissues. Critical B-induced R2R3 MYB TFs were identified. It was concluded that the results would be useful for functional characterizations of R2R3-type MYB transcription factors that are possibly involved in both B stress and divergent regulation mechanisms in plants.

A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.)

The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2–21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-target-caused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.

Manipulating the Biosynthesis of Bioactive Compound Alkaloids for Next-Generation Metabolic Engineering in Opium Poppy Using CRISPR-Cas 9 Genome Editing Technology

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is a powerful RNA-guided genome editing tool. CRISPR/Cas9 has been well studied in model plant species for targeted genome editing. However, few studies have been reported on plant species without whole genome sequence information. Currently, no study has been performed to manipulate metabolic pathways using CRISPR/Cas9. In this study, the type II CRISPR/SpCas9 system was used to knock out, via nonhomologous end-joining genome repair, the 4′OMT2 in opium poppy (Papaver somniferum L.), a gene which regulates the biosythesis of benzylisoquinoline alkaloids (BIAs). For sgRNA transcription, viral-based TRV and synthetic binary plasmids were designed and delivered into plant cells with a Cas9 encoding-synthetic vector by Agrobacterium-mediated transformation. InDels formed by CRISPR/Cas9 were detected by sequence analysis. Our results showed that the biosynthesis of BIAs (e.g. morphine, thebaine) was significantly reduced in the transgenic plants suggesting that 4′OMT2 was efficiently knocked-out by our CRISPR-Cas9 genome editing approach. In addition, a novel uncharacterized alkaloid was observed only in CRISPR/Cas9 edited plants. Thus, the applicabilitiy of the CRISPR/Cas9 system was demonstrated for the first time for medicinal aromatic plants by sgRNAs transcribed from both synthetic and viral vectors to regulate BIA metabolism and biosynthesis.