Project description:The orphan crop, Eragrostis tef, was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants to tolerate water loss to 50% RWC before adverse effects in leaf tissues were observed. Proteomic analysis using iTRAQ mass spectrometry and appropriate database searching enabled the detection of 5727 proteins, of which 211 proteins, including a number of spliced variants, were found to be differentially regulated with imposed stress conditions. Validation of the stress-related proteins, fructose-bisphosphate aldolase (FBA), glutamine synthetase (GLN) and the protective antioxidant proteins, monodehydroascorbate reductase (MDHAR) and peroxidase (POX), using immunodetection and enzymatic assay confirmed protein presence according to iTRAQ findings and showed increased protein abundance levels and enzymatic activity in response to water-deficit. GO-term enrichment and analysis revealed terms involved in biotic and abiotic stress response, signaling, transport, cellular homeostasis and pentose metabolic processes, to be enriched in tef up-regulated proteins, while terms linked to ROS-producing processes under water-deficit, such as photosynthesis and associated light harvesting reactions, manganese transport and homeostasis, the synthesis of sugars and cell wall catabolism and modification, to be enriched in tef down-regulated proteins.
Project description:miRNAs play an important role in growth, development, stress resilience and epigenetic modifications of plants. However, the effect of calcium nutrition on miRNA expression in orphan crops such as tef ((Eragrostis tef) remained unknown. In this study, we analyzed genome-wise miRNAs of roots and shoots in response to long-term calcium deficiency in tef. MiRNA-seq followed by bioinformatic analysis identified a large number of small RNAs with size ranging from 17bp to 35bp. A total 1380 miRNAs were identified in plants experiencing long-term Ca deficiency, while 1495 miRNAs were identified in the control plants. Among the miRNAs identified in this study, 161 miRNAs were known, and 348 miRNAs were novel while the remaining miRNAs were uncharacterized. Putative target genes and their functions were predicted for all the known and novel miRNAs. Based on gene ontology (GO) analysis, the target genes were found to have various biological and molecular functions including calcium uptake and transport. Pairwise comparison of differentially expressed miRNAs revealed that some of the miRNAs were specifically enriched in roots or shoots of low calcium treated plants as compared to control plants. Further characterization of the miRNAs and their targets detected in this study will help in identifying Ca deficiency responses not only in tef but also in related orphan crops
Project description:Calcium (Ca2+) is one of the essential mineral nutrients for plant growth and development. However, the effects of long-term Ca2+ deficiency in orphan crops such as tef (Zucc.) Trotter] which accumulate high levels of Fe in the grains remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly Ethiopia and Eritrea. It is rich in mineral nutrients (Ca+2, Fe, Zn and Mn), vitamins and essential amino acids. In this study, tef plants were grown in hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+ and plant growth parameters and whole genome transcriptome were analyzed. Ca+2-deficient plants exhibited symptoms including leaf necrosis, leaf curling, and growth stunting. Ca2+ deficiency significantly decreased root and shoot Ca+2, potassium (K) and copper content in both root and shoots while it significantly increased root iron (Fe2+) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels including the cyclic nucleotide-gated channels (CNGC) and glutamate receptor-like channels (GLRs), Ca2+-transporters, Ca2+-binding proteins (CBP) and Ca2+-dependent protein kinases (CDPK) were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters including members of vacuolar Fe transporters (VIT), yellow stripe-like (YSL), natural resistance-associated macrophage protein (NRAMP) and oligo-peptide transporters (OPT) were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.
Project description:Here we adapt native elongating transcript sequencing (NET-seq) to develop transcription elongation factor associated nascent elongating transcript sequencing (TEF-seq). In this the RNA polymerase II (Pol2) transcription elongation complex (TEC) is immunoprecipitated via associated transcription elongation factors (TEFs). Sequencing from the 3' end of the Pol2-associated nascent transcript shows the position of the final incorporated nucleotide giving strand-specific, single nucleotide resolution maps of the level of TEF association with Pol2 during transcription.