Comprehensive Analysis of Calcium Sensor Families, CBL and CIPK, in Aeluropus littoralis and Their Expression Profile in Response to Salinity.

Plants have acquired sets of highly regulated and complex signaling pathways to respond to unfavorable environmental conditions during evolution. Calcium signaling, as a vital mechanism, enables plants to respond to external stimuli, including abiotic and biotic stresses, and coordinate the basic processes of growth and development. In the present study, two calcium sensor families, CBL and CIPK, were investigated in a halophyte plant, Aeluropus littoralis, with a comprehensive analysis. Here, six AlCBL genes, and twenty AlCIPK genes were studied. The analysis of the gene structure and conserved motifs, as well as physicochemical properties, showed that these genes are highly conserved during evolution. The expression levels of AlCBL genes and AlCIPK genes were evaluated under salt stress in leaf and root tissue. Based on the real-time RT-PCR results, the AlCIPK gene family had a higher variation in mRNA abundance than the AlCBL gene family. AlCIPK genes were found to have a higher abundance in leaves than in roots. The results suggest that the correlation between AlCBL genes and AlCIPK is tissue-specific, and different correlations can be expected in leaves and roots. Based on these correlations, AlCIPK3.1-AlCBL4.1 and AlCIPK1.2-AlCBL4.4 can be co-expressed in the root tissue, while AlCBL10 has the potential to be co-expressed with AlCIPK5, AlCIPK26, and AlCIPK12.3 in the leaf tissue. Our findings reveal valuable information on the structure and function of calcium sensor families in A. littoralis, a halophyte plant, that can be used in future research on the biological function of CBLs and CIPKs on salt stress resistance.

Karyology and Genome Size Analyses of Iranian Endemic Pimpinella (Apiaceae) Species.

Pimpinella species are annual, biennial, and perennial semibushy aromatic plants cultivated for folk medicine, pharmaceuticals, food, and spices. The karyology and genome size of 17 populations of 16 different Pimpinella species collected from different locations in Iran were analyzed for inter-specific karyotypic and genome size variations. For karyological studies, root tips were squashed and painted with a DAPI solution (1 mg/ml). For flow cytometric measurements, fresh leaves of the standard reference (Solanum lycopersicum cv. Stupick, 2C DNA = 1.96 pg) and the Pimpinella samples were stained with propidium iodide. We identified two ploidy levels: diploid (2x) and tetraploid (4x), as well as five metaphase chromosomal counts of 18, 20, 22, 24, and 40. 2n = 24 is reported for the first time in the Pimpinella genus, and the presence of a B-chromosome is reported for one species. The nuclear DNA content ranged from 2C = 2.48 to 2C = 5.50 pg, along with a wide range of genome sizes between 1212.72 and 2689.50 Mbp. The average monoploid genome size and the average value of 2C DNA/chromosome were not proportional to ploidy. There were considerable positive correlations between 2C DNA and total chromatin length and total chromosomal volume. The present study results enable us to classify the genus Pimpinella with a high degree of morphological variation in Iran. In addition, cytological studies demonstrate karyotypic differences between P. anthriscoides and other species of Pimpinella, which may be utilized as a novel identification key to affiliate into a distinct, new genus - Pseudopimpinella.

Genome-Wide Analysis of Potassium Channel Genes in Rice: Expression of the OsAKT and OsKAT Genes under Salt Stress.

Potassium (K+), as a vital element, is involved in regulating important cellular processes such as enzyme activity, cell turgor, and nutrient movement in plant cells, which affects plant growth and production. Potassium channels are involved in the transport and release of potassium in plant cells. In the current study, three OsKAT genes and two OsAKT genes, along with 11 nonredundant putative potassium channel genes in the rice genome, were characterized based on their physiochemical properties, protein structure, evolution, duplication, in silico gene expression, and protein-protein interactions. In addition, the expression patterns of OsAKTs and OsKATs were studied in root and shoot tissues under salt stress using real-time PCR in three rice cultivars. K+ channel genes were found to have diverse functions and structures, and OsKATs showed high genetic divergence from other K+ channel genes. Furthermore, the Ka/Ks ratios of duplicated gene pairs from the K+ channel gene family in rice suggested that these genes underwent purifying selection. Among the studied K+ channel proteins, OsKAT1 and OsAKT1 were identified as proteins with high potential N-glycosylation and phosphorylation sites, and LEU, VAL, SER, PRO, HIS, GLY, LYS, TYR, CYC, and ARG amino acids were predicted as the binding residues in the ligand-binding sites of K+ channel proteins. Regarding the coexpression network and KEGG ontology results, several metabolic pathways, including sugar metabolism, purine metabolism, carbon metabolism, glycerophospholipid metabolism, monoterpenoid biosynthesis, and folate biosynthesis, were recognized in the coexpression network of K+ channel proteins. Based on the available RNA-seq data, the K+ channel genes showed differential expression levels in rice tissues in response to biotic and abiotic stresses. In addition, the real-time PCR results revealed that OsAKTs and OsKATs are induced by salt stress in root and shoot tissues of rice cultivars, and OsKAT1 was identified as a key gene involved in the rice response to salt stress. In the present study, we found that the repression of OsAKTs, OsKAT2, and OsKAT2 in roots was related to salinity tolerance in rice. Our findings provide valuable insights for further structural and functional assays of K+ channel genes in rice.

NaOH low-salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis.

PREMISE OF THE STUDY: High-yield pure chloroplast DNA (cpDNA) is necessary for whole genome sequencing. Chloroplast extraction with traditional high-salt methods causes damage to nuclei and destroys the integrity of organelles, which leads to high genomic contamination from the nucleus and mitochondria. To overcome this issue, we modified a traditional high-salt method to obtain a new approach called the NaOH low-salt method (NLS). METHODS AND RESULTS: The NLS method is based on the mild alkaline lysis of plant cells, followed by homogenization with ultrasonic waves and fractionation under reduced osmotic pressure. Results showed that this modified protocol worked efficiently to extract the intact chloroplast from Aeluropus littoralis and other grasses to obtain high-quality pure cpDNA, which was confirmed by fluorescent microscopy, qPCR, and Illumina paired-end sequencing analysis. CONCLUSIONS: Compared with high-salt methods, the NLS method has proven robust for extraction of intact chloroplasts and preparation of high-yield pure cpDNA from grasses.

Purified sulforaphane from broccoli (Brassica oleracea var. italica) leads to alterations of CDX1 and CDX2 expression and changes in miR-9 and miR-326 levels in human gastric cancer cells.

BACKGROUND: Genetic alterations and epigenetic modifications are two main factors involved in gastric carcinogenesis, progression, and metastasis. Several miRNAs such as miRNA-9 and miRNA-326 may play important role in gastric cancer by targeting the 3'UTR of the caudal type homeobox (CDX) 1 and 2 mRNA respectively. The use of herbal medicines has been widely considered in the treatment of cancers such as gastric cancer. Sulforaphane extracted from broccoli may indirectly prevent cancer through affecting different signaling pathways. The aim of this study was to evaluate the effect of different concentrations of sulforaphane extracted from broccoli sprout (SEBS) on viability, death pattern, and expression alterations of CDX1/2 as well as miRNA-9 and miRNA-326 in normal (HF2FF) and gastric cancer cell lines. METHODS: Two gastric cancer cell lines (AGS and MKN45) and HF2FF normal cell line were cultured and treated with different concentrations (31.25, 62.5, 125, and 250 µg/ml) of the purified sulforaphane. Expression levels of CDX1 and CDX2 as well as miRNA-9 and miRNA-326, and mechanisms leading to cell death were assessed by Taqman real time PCR assay and flow cytometry, respectively. RESULTS: Significant dose-dependent and anti-proliferative effects of the SEBS were observed on AGS and MKN45 cells after 48 h with an IC50 value of about 112 and 125 µg/ml, respectively (P < 0.001). Apoptotic cells were observed in AGS and MKN45 cells but not HF2FF after 48 h of treatment with SEBS. Furthermore, significant changes in expression of CDX1, CDX2, miR-9 and miR-326 in the gastric cancer lines (AGS and MKN45), were observed under different concentrations of SEBS. CONCLUSION: Our present study suggests that the SEBS may influence gastric cancer cell lines at specific doses and change their proliferation rate by altering the expression of CDX1, CDX2, miR-9, and miR-326.