A universal protocol for high-quality DNA and RNA isolation from diverse plant species.

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and ß-mercaptoethanol (ßME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15-100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.

Expression profiling of yield related genes in rice cultivars under terminal drought stress.

BACKGROUND: Rice crop may experience a significant reduction in yield-up to 50%-due to two occurrences during drought stress: unsuccessful peduncle elongation in panicle exertion and ineffective grain filling. The comprehension of mechanisms that promote drought tolerance during these growth phases is crucial for the production of rice that can withstand drought conditions, thus averting a decrease in crop yield. METHODS AND RESULTS: The expression of two xyloglucan endo transhydrolase/glucosylase genes (OsXTH 5 and 19) in peduncle tissue and a sucrose transporter gene (OsSUT1) in flag leaf sheath were assessed. An experiment was carried out in a factorial arrangement based on completely randomized design in which, factor A was two rice cultivars (Vandana as tolerant and Tarom mahalli as local susceptible to drought) and factor B was five drought stress treatments (full irrigation, drought stress duration in 72 and 96 h, re-watering after 120 and 192 h). Results showed that expression of OsXTH19 and OsXTH5 genes were upregulated in both Vandana and Tarom mahalli cultivars due to stress treatments. OsXTH19 expression was found to decrease while OsXTH5 expression increased during re-watering treatments. It is likely that the persistence of peduncle growth in the drought-tolerant Vandana cultivar can be attributed to the presence of OsXTH19 under drought conditions and OsXTH5 after re-watering. The expression of OsSUT1 in flag leaf sheath of Vandana in re-watering treatments was reached 8-60-fold re-watering. CONCLUSIONS: Peduncle elongation was attributed to two XTH genes under drought stress condition. Panicle exertion may be promoted by sustaining peduncle growth despite drought stress. Consequently, this may led to reduce in non fertile florets and decrease in grain yield by 50%. As grain filling depend to expression of OsSUT1 in flag leaf sheath under drought stress, to improve rice cultivars under aerobic production system and drought stress, it is advised to apply these findings in rice breeding programs.

Identification of drought-tolerant hub genes in Iranian KC-2226 genotype of Aegilops tauschii using transcriptomic analysis.

Aegilops tauschii, as a donor of D genome to the bread wheat with a valuable source of resistance to different biotic and abiotic stresses, is used to improve the quality of wheat cultivars. Every genotype has a specific genetic content, the investigation of which can lead to the identification of useful genes such as stress tolerance genes, including drought. Therefore, 23 genotypes of Ae. tauschii were selected to evaluate their morphological and physiological traits under greenhouse conditions. Among them, a superior tolerant genotype (KC-2226) was chosen for transcriptomic analysis. Our result showed that 5007 and 3489 genes were deferentially up- and downregulated, respectively. Upregulated genes were involved in photosynthesis, glycolysis/gluconeogenesis, and amino acid biosynthesis whereas downregulated genes were often engaged in DNA synthesis, replication, repair and topological changes. The result of protein-protein interaction network analysis showed that AT1G76550 (1.46), AT1G20950 (1.42), IAR4 (1.19), and PYD2 (1.16) among upregulated genes and THY-1 (44), PCNA1 (41) and TOPII (22) among down-regulated genes had the highest interactions with other genes. In conclusion, Ae. tauschii employs elevated transcription of specific genes involved in photosynthesis, glycolysis and gluconeogenesis and amino acid biosynthesis pathways rather than genes active in DNA synthesis and repair to provide the energy needed for the plant to survive under stress conditions.

Genome-wide survey of catalase genes in Brassica rapa, Brassica oleracea, and Brassica napus: identification, characterization, molecular evolution, and expression profiling of BnCATs in response to salt and cadmium stress.

Catalase (CAT, EC 1.11.1.6), one of the most important antioxidant enzymes, can control excess levels of H2O2 produced under oxidative stress in plants. In this study, 16, 8, and 7 CAT genes in the genome of Brassica napus, B. rapa, and B. oleracea were identified, respectively. Phylogenetic studies showed that CATs could be divided into two main groups, each containing specific monocotyledon and dicotyledon subgroups. Motifs, gene structure, and intron phase of CATs in B. napus, Brassica rapa, and Brassica oleracea are highly conserved. Analysis of codon usage bias showed the mutation pressure and natural selection of the codon usage of CATs. Segmental duplication and polyploid were major factors in the expansion of this gene family in B. napus, and genes have experienced  negative selection during evolution. Existence of hormones and stress-responsive cis-elements and identifying miRNA molecules affecting CATs showed that these genes are complexly regulated at the transcriptional and posttranscriptional levels. Based on RNA-seq data, CATs are divided into two groups; the first group has moderate and specific expression in flowers, leaves, stems, and roots, while the second group shows expression in most tissues. qRT-PCR analysis showed that the expression of these genes is dynamic and has a specific expression consistent with other CAT genes in response to salinity and cadmium (Cd) stresses. These results provide information for further investigation of the function of CAT genes in response to stresses and the development of tolerant  plants.

Triterpenoid gene expression and phytochemical content in Iranian licorice under salinity stress.

Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), ß-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), ß-amyrin 11-oxidase (CYP88D6), and ß-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h post-treatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm.

Gene Delivery to Tobacco Root Cells with Single-Walled Carbon Nanotubes and Cell-Penetrating Fusogenic Peptides.

Development of efficient, easy, and safe gene delivery methods is of great interest in the field of plant biotechnology. Considering the limitations of the usual transfection methods (such as transgene size and plant type), several new techniques have been tested for replacement. The success of some biological and synthetic nanostructures such as cell-penetrating peptides and carbon nanotubes in transferring macromolecules (proteins and nucleic acids) into mammalian cells provoked us to assess the ability of an engineered chimeric peptide and also arginine functionalized single-walled carbon nanotube in gene delivery to intact tobacco (Nicotiana tabacum var. Virginia) root cells. It was suggested that the engineered peptide with its special cationic and hydrophobic domains and the arginine functionalized single-walled carbon nanotube due to its nano-cylindrical shape can pass plant cell barriers while plasmid DNA (which codes green fluorescent protein) has been condensed on them. The success of gene delivery to tobacco root cells was confirmed by fluorescence microscopy and western blotting analysis.

Metabolic Engineering of Glycyrrhizin Pathway by Over-Expression of Beta-amyrin 11-Oxidase in Transgenic Roots of Glycyrrhiza glabra.

Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121GUS-9:CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.

Responses of pomegranate cultivars to severe water stress and recovery: changes on antioxidant enzyme activities, gene expression patterns and water stress responsive metabolites.

The biochemical and molecular responses of five commercially well-known pomegranate cultivars to severe water stress were studied. The cultivars were subjected to 14-day water stress by withholding irrigation, followed by re-watering for 7 days. Results showed clear differences in metabolites contents and activities of antioxidant enzymes among various pomegranate cultivars during severe water stress and recovery. According to our results, increased accumulation of proline in pomegranate was not related to osmotic adjustment during severe water stress. Except for 'Ghojagh', leaves grown under severe water stress conditions showed symptoms of oxidative stress such as reduced chlorophyll concentration. The improved performance of 'Ghojagh' under drought stress may be associated with an efficient osmotic adjustment. The up- or down regulated expression of cytosolic glutathione reductase (cytosolic GR) and glutathione peroxidase were observed under drought conditions. Moreover, the suppressed expression of cytosolic GR was also noted. Comparatively, 'Rabab' exhibited higher antioxidant capacity and an efficient ROS-scavenging mechanism under drought stress. Lower levels of membrane lipid peroxidation in 'Ghojagh' and 'Rabab' under drought stress and the marked reduction of malondialdehyde concentration after re-watering represents that these cultivars have a good tolerance to drought stress. As a first step towards the study of the biochemical and molecular responses of pomegranate plants to water stress, this research provides new information into the mechanisms of drought tolerance in the plants.

Identification of CBF14 and NAC2 Genes in Aegilops tauschii Associated with Resistance to Freezing Stress.

Low temperature as one of the most important environmental factors limits the productivity of plants across the world. Aegilops, as a wild species of Poaceae, contains low temperature-responsive genes. In this study, we analyzed morphological (wilting, chlorosis, and recovery) and physiological (ion leakage) characteristics to identification of a cold-tolerant genotype. In this experiment, we introduced two transcription factors (TFs) in Aegilops species for the first time. Bioinformatics analysis demonstrated that our nucleotide sequences have high similarity with CBF14 (C-repeat-binding factor) and NAC2 (NAM, ATAF, and CUC) in Triticum aestivum. Based on the physiological and morphological data, one genotype (Aladizgeh) was identified as the most resistant genotype which was selected for further gene expression analysis. The real-time PCR results indicated that the CBF14 gene was not expressed 3 h following cold treatment, but the highest expression was observed after 6, 12, and 24 h of cold treatment; however, a sudden decrease was observed in its expression after 30 h. The NAC2 gene also was not expressed 3 h after cold stress, but the highest expression was at 24 h and similar to the CBF14 gene; its expression suddenly decreased after 30 h. Our results indicated that this genotype can tolerate -4 °C for 3 h, but the CBF14 and NAC2 genes were activated when treated for longer durations. Expression of TFs studied in this experiment had decreased after 30 h, in which cell death seems to be the important reason.

Effects of an extracted lectin from Citrullus colocynthis L. (Cucurbitaceae) on survival, digestion and energy reserves of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae).

Lectins are the heterogeneous proteins in plants that serve as storage proteins via defensive mechanisms against herbivores. In the current study, a lectin was extracted and purified from seeds of Citrullus colocynthis by Sepharose 4B-Galactose and DEAE-cellulose fast flow chromatographies. Different concentrations of the lectin were added to artificial diet of Ectomyelois ceratoniae larvae finding out its effect on some biological parameters, digestive physiology and amount of storage macromolecules. It was found that CCA (C. colocynthis Agglutinin) increased life span from 23.44 days in control to 28.59 days in the treated individuals. Survival of larvae on control and CCA diets were 93.3 and 66.6%, respectively. Different concentrations of CCA significantly affected α-amylase and general proteolytic activities except for TAG-lipase activity. Activities of all specific proteases decreased when larvae were fed on different concentrations of CCA except for aminopeptidase. Meanwhile, amount of storage macromolecules in the larvae fed on different concentrations of CCA statistically decreased vs. control. These results demonstrated that CCA could intervene in physiology of E. ceratoniae and survival of larvae. Therefore, it can be taken into consideration in IPM of the pest through plant breeding programs.