Huseyin Tombuloglu

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.

High-throughput transcriptome analysis of barley (Hordeum vulgare) exposed to excessive boron.

Boron (B) is an essential micronutrient for optimum plant growth. However, above certain threshold B is toxic and causes yield loss in agricultural lands. While a number of studies were conducted to understand B tolerance mechanism, a transcriptome-wide approach for B tolerant barley is performed here for the first time. A high-throughput RNA-Seq (cDNA) sequencing technology (Illumina) was used with barley (Hordeum vulgare), yielding 208 million clean reads. In total, 256,874 unigenes were generated and assigned to known peptide databases: Gene Ontology (GO) (99,043), Swiss-Prot (38,266), Clusters of Orthologous Groups (COG) (26,250), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (36,860), as determined by BLASTx search. According to the digital gene expression (DGE) analyses, 16% and 17% of the transcripts were found to be differentially regulated in root and leaf tissues, respectively. Most of them were involved in cell wall, stress response, membrane, protein kinase and transporter mechanisms. Some of the genes detected as highly expressed in root tissue are phospholipases, predicted divalent heavy-metal cation transporters, formin-like proteins and calmodulin/Ca(2+)-binding proteins. In addition, chitin-binding lectin precursor, ubiquitin carboxyl-terminal hydrolase, and serine/threonine-protein kinase AFC2 genes were indicated to be highly regulated in leaf tissue upon excess B treatment. Some pathways, such as the Ca(2+)-calmodulin system, are activated in response to B toxicity. The differential regulation of 10 transcripts was confirmed by qRT-PCR, revealing the tissue-specific responses against B toxicity and their putative function in B-tolerance mechanisms.

Boron induced expression of some stress-related genes in tomato

Boron (B) is a potential environmental toxicant for plants under excessive conditions. To understand the molecular stress response involved in high B exposure, we focused on the transcript accumulation of three stress-related genes: Hsp90, MT2 and GR1. Transcript accumulations were determined on B-stressed tomato plants by using a quantitative real-time PCR technique. Tomato seedlings were exposed to B ranging from 80 to 5120 μM for 24 h in nutrient solution. Root and shoot transcript accumulations were assessed. Results showed that the genes were over-expressed in B-stressed tomato. The highest relative fold change value was measured on GR1 for both root and shoot (8-10 and 30-34-fold increases, respectively), indicating the activation of the oxidative stress enzyme to tolerate B-stress as an early response. The activation of these genes could be a protection mechanism against to B stress.

Boron and Plants

Boron is found naturally in the earth’s crust in the oxidized form as borax and colemanite, particularly in the oceans, sedimentary rocks, coal, shale, and some soils. It is never found in the elemental form in nature possessing a complex chemistry similar to that of silicon, with properties switching between metals and non-metals. Boron has become an important and strategic element in terms of developing technologies. It is released into the environment mainly through the weathering of rocks, volatilization from oceans, geothermal steam, burning of agricultural refuse and fuel wood, power generators (coal/oil combustion), glass industry, household use of boron-containing products (including soaps and detergents), borax mining and processing, leaching from treated wood and paper, chemical plants, and sewage/sludge disposal, but a major proportion originates from the weathering of rocks. Boron is regarded as an essential element for human beings, animals and plants. Boron occurs in soils at concentrations ranging from 10 to 300 mg kg−1 depending on the type of soil, amount of organic matter, and amount of rainfall. The treatments lead to significant increases in the productivity of some plants but in certain cases a decrease is seen as the boron level increases with the boron content of irrigation water, in particular on the soils with a heavy texture, high CaCO3 and clay content. Lack of boron in plants results in necrosis but excess amounts are said to produce poisonous effects. Turkey produces more than 60% of the world’s borax, with important boron reserves located in Susurluk, Bigadic and Sindirgi regions of Balikesir, Kestelek-Bursa, Emet-Kutahya, the largest reserves occur in Kirka-Eskisehir. Therefore, there is a naturally occurring high level of boron in the ground waters in some of these areas due to the excess amounts of boron given out to the environment during washing and purification processes which result in the pollution of cultivated areas. An attempt will be made here to present an overview of the plant diversity on the boron contaminated soils in Turkey, effects of different concentrations of boron on the germination ability of some plants and possible candidates for phytomining of the soils showing boron toxicity symptoms.

Genome of wild olive and the evolution of oil biosynthesis

Significance We sequenced the genome and transcriptomes of the wild olive (oleaster). More than 50,000 genes were predicted, and evidence was found for two relatively recent whole-genome duplication events, dated at approximately 28 and 59 Mya. Whole-genome sequencing, as well as gene expression studies, provide further insights into the evolution of oil biosynthesis, and will aid future studies aimed at further increasing the production of olive oil, which is a key ingredient of the healthy Mediterranean diet and has been granted a qualified health claim by the US Food and Drug Administration. Here we present the genome sequence and annotation of the wild olive tree (Olea europaea var. sylvestris), called oleaster, which is considered an ancestor of cultivated olive trees. More than 50,000 protein-coding genes were predicted, a majority of which could be anchored to 23 pseudochromosomes obtained through a newly constructed genetic map. The oleaster genome contains signatures of two Oleaceae lineage-specific paleopolyploidy events, dated at ∼28 and ∼59 Mya. These events contributed to the expansion and neofunctionalization of genes and gene families that play important roles in oil biosynthesis. The functional divergence of oil biosynthesis pathway genes, such as FAD2, SACPD, EAR, and ACPTE, following duplication, has been responsible for the differential accumulation of oleic and linoleic acids produced in olive compared with sesame, a closely related oil crop. Duplicated oleaster FAD2 genes are regulated by an siRNA derived from a transposable element-rich region, leading to suppressed levels of FAD2 gene expression. Additionally, neofunctionalization of members of the SACPD gene family has led to increased expression of SACPD2, 3, 5, and 7, consequently resulting in an increased desaturation of steric acid. Taken together, decreased FAD2 expression and increased SACPD expression likely explain the accumulation of exceptionally high levels of oleic acid in olive. The oleaster genome thus provides important insights into the evolution of oil biosynthesis and will be a valuable resource for oil crop genomics.

Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars

MircroRNAs (miRNAs) are small non-coding RNAs about 21 nt in length. These short transcripts regulate developmental and stress responses in plants. Cold stress is one of the most restraining abiotic factors adversely affecting the plant yield. In the present study, some cold stress-related miRNAs (miR167, miR169, miR172, miR393 and miR397) in tomato (Solanum lycopersicum) were assessed at early time points (0, 1, 4 and 16 h) of cold exposure. Relative expression of miRNAs was measured by stem–loop quantitative reverse transcription polymerase chain reaction. The results showed that miR167, miR169, miR172 and miR393 were activated in the early time points of cold treatment. Especially, miR172 was found to have highest expression level. Furthermore, target genes of selected miRNAs were identified and their expression profiles were assessed between cold-sensitive and cold-tolerant cultivars of tomato. It was found that inferred expression patterns of target genes were differentiated between the cultivars. Analysis of cis-acting elements showed that miRNAs had stress-responsive elements. Meanwhile, since no miR393 sequence is available, putative miR393 sequence and its secondary structure were predicted in tomato. These results may provide a framework for further analysis in terms of understanding the response of miRNAs against cold stress in tomato.

Aquaporins in Boron-Tolerant Barley: Identification, Characterization, and Expression Analysis

Barley is an economically important crop widely cultivated in the world. Boron (B) toxicity limits its yield in a variety of regions worldwide. B-tolerant cultivar of barley (Hordeum vulgare, cv Sahara) maintains low B in their roots and leaves in B-toxic conditions and able to resist against it. B import and export to the plant is controlled by several transmembrane proteins such as aquaporins (AQP), which are key players not just for water uptake, but also a wide selection of substrates including B. In this study, we identified and characterized AQPs from B-tolerant barley to understand their possible positive impact in B-toxicity tolerance. In order to filter out the AQP genes, we have mined the RNA-Seq data obtained from barley roots and leaves which are B-stressed and control in this study. A total of 30 AQP were identified within four subfamilies: plasma membrane intrinsic proteins (PIPs) (13), tonoplast intrinsic proteins (TIPs) (11), NOD26-like intrinsic proteins (NIPs) (4), and small basic intrinsic proteins (SIPs) (2). Differential expression was measured in barley AQP transcripts upon excess B treatment. Contribution of each AQP member to B-tolerance was evaluated. Particularly, NIP1:1 was found to be highly up-regulated in roots, in contrast down-regulated in leaf, indicating plasma membrane and vacuole cooperated control of B-regulation. In addition to NIP2:1, NIP2:2 expression was found to be reduced that may contribute to B-toxicity tolerance. Moreover, comparative phylogenetic analysis was conducted using diverse plant AQP members. Comprehensive evaluation of conserved domains, critical residues, and 3D models give insights about the substrate selectivity and passage capability of barley AQPs.

Assessment of Cd-induced genotoxic damage in Urtica pilulifera L. using RAPD-PCR analysis

ABSTRACT Plants can be used as biological indicators in assessing the damage done by bioaccumulation of heavy metals and their negative impact on the environment. In the present research, Roman nettle (Urtica pilulifera L.) was employed as a bioindicator for cadmium (Cd) pollution. The comparisons between unexposed and exposed plant samples revealed inhibition of the root growth (∼25.96% and ∼45.92% after treatment with 100 and 200 µmol/L Cd concentrations, respectively), reduction in the total soluble protein quantities (∼53.92% and ∼66.29% after treatment with 100 and 200 µmol/L Cd concentrations, respectively) and a gradual genomic instability when the Cd concentrations were increased. The results indicated that alterations in randomly amplified polymorphic DNA (RAPD) profiles, following the Cd treatments, included normal band losses and emergence of new bands, when compared to the controls. Also, the obtained data from F1 plants, utilized for analysis of genotoxicity, revealed that DNA alterations, occurring in parent plants due to Cd pollution, were transmitted to the next generation.

Bamboo Biomass: Various Studies and Potential Applications for Value-Added Products

Bamboo being an industrial crop belongs to the family Gramineae, subfamily Bambusiodeae, and grows naturally in many countries including Malaysia. The cultivation of bamboo for commercial purposes in Malaysia is rather limited due to problems in acquiring suitable land for planting, although it has been widely recognized for excellent production of fiber biomass globally. It produces long fibers derived from outer fibrous bark, and has great potential in the biocomposite industries. In other countries like China and Japan, the plantation of bamboo is common and utilized for many purposes including the production of fiber. Usually, the extraction of fiber from bamboo is done in 3–4-year-old bamboo plantations. Fiber is produced through alkaline hydrolysis and multiphase bleaching processes of bamboo stems and leaves followed by chemical treatments of starchy pulp generated during the process. Bamboo fiber has micro-gaps, which makes it softer than cotton and increases its capability to absorb moisture. Other interesting properties of bamboo fiber are highly elastic, bacteriostatic, antifungal, antibacterial, hypoallergenic, hydroscopic, natural deodorizer, resistant to ultraviolet light, and biodegradable which render the bamboo fiber products environmentally friendly. Furthermore, it is highly durable, stable, and tough and has substantial tensile strength. Due to its versatile properties, bamboo fibers are used mainly in textile industry for making garments, bathrobes, and towels. The plants possess a wider range of adaptation to diverse climatic and soil conditions, and due to its rich cellulose content compared to other plants it has been widely utilized for the production of many products. Thus, it is important to gather and document useful information of its properties and a wide range of products it produces.

Proteomic analysis of naturally occurring boron tolerant plant Gypsophila sphaerocephala L. in response to high boron concentration

Gypsophila sphaerocephala is a naturally Boron (B) tolerant species that can grow around the B mining areas in Turkey, where the B concentration in soil reaches a lethal dose for plants (up to ∼8900mgkg-1 (∼140mM). While its interesting survival capacity in extremely B containing soils, any molecular research has been conducted to understand its high tolerance mechanism yet. In the present study, we have performed a proteomic analysis of this plant to understand its high tolerance towards B-stress. Seedlings of G. sphaerocephala were collected from B mining area and were adapted to greenhouse conditions. An excessive level of Boric acid (3mM)was applied to the plantlets for 24h. Total proteins were precipitated by using TCA/Acetone method. 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) analysis of the proteins was carried out. Out of 121 protein spots, 14 were differentially expressed between the control and B-exposed G. sphaerocephala roots. The peptide profile of each protein was determined by MALDI-TOF mass spectrometer after in-gel trypsin digestion. The identified proteins are involved in different mechanisms in the cell such as in antioxidant mechanism, energy metabolism, protein degradation, lipid biosynthesis and signaling pathways, indicating that G. sphaerocephala has multiple cooperating mechanisms to protect itself from high B levels. Overall, this study sheds light on to the possible regulatory switches (gene/s) controlling the B-tolerance proteins and their possible roles in plants defense mechanism.