Elucidation of Novel cis-Regulatory Elements and Promoter Structures Involved in Iron Excess Response Mechanisms in Rice Using a Bioinformatics Approach

Iron (Fe) excess is a major constraint on crop production in flooded acidic soils, particularly in rice cultivation. Under Fe excess, plants activate a complex mechanism and network regulating Fe exclusion by roots and isolation in various tissues. In rice, the transcription factors and cis-regulatory elements (CREs) that regulate Fe excess response mechanisms remain largely elusive. We previously reported comprehensive microarray analyses of several rice tissues in response to various levels of Fe excess stress. In this study, we further explored novel CREs and promoter structures in rice using bioinformatics approaches with this microarray data. We first performed network analyses to predict Fe excess-related CREs through the categorization of the gene expression patterns of Fe excess-responsive transcriptional regulons, and found four major expression clusters: Fe storage type, Fe chelator type, Fe uptake type, and WRKY and other co-expression type. Next, we explored CREs within these four clusters of gene expression types using a machine-learning method called microarray-associated motif analyzer (MAMA), which we previously established. Through a comprehensive bioinformatics approach, we identified a total of 560 CRE candidates extracted by MAMA analyses and 42 important conserved sequences of CREs directly related to the Fe excess response in various rice tissues. We explored several novel cis-elements as candidate Fe excess CREs including GCWGCWGC, CGACACGC, and Myb binding-like motifs. Based on the presence or absence of candidate CREs using MAMA and known PLACE CREs, we found that the Boruta-XGBoost model explained expression patterns with high accuracy of about 83%. Enriched sequences of both novel MAMA CREs and known PLACE CREs led to high accuracy expression patterns. We also found new roles of known CREs in the Fe excess response, including the DCEp2 motif, IDEF1-, Zinc Finger-, WRKY-, Myb-, AP2/ERF-, MADS- box-, bZIP and bHLH- binding sequence-containing motifs among Fe excess-responsive genes. In addition, we built a molecular model and promoter structures regulating Fe excess-responsive genes based on new finding CREs. Together, our findings about Fe excess-related CREs and conserved sequences will provide a comprehensive resource for discovery of genes and transcription factors involved in Fe excess-responsive pathways, clarification of the Fe excess response mechanism in rice, and future application of the promoter sequences to produce genotypes tolerant of Fe excess.

Comparative Transcriptomics of Lowland Rice Varieties Uncovers Novel Candidate Genes for Adaptive Iron Excess Tolerance

Iron (Fe) toxicity is a major challenge for plant cultivation in acidic waterlogged soil environments, where lowland rice is a major staple food crop. Only few studies have addressed the molecular characterization of excess Fe tolerance in rice, and these highlight different mechanisms for Fe tolerance. Out of 16 lowland rice varieties, we identified a pair of contrasting lines, Fe-tolerant Lachit and -susceptible Hacha. The two lines differed in their physiological and morphological responses to excess Fe, including leaf growth, leaf rolling, reactive oxygen species generation and Fe and metal contents. These responses were likely due to genetic origin as they were mirrored by differential gene expression patterns, obtained through RNA sequencing, and corresponding gene ontology term enrichment in tolerant vs. susceptible lines. Thirty-five genes of the metal homeostasis category, mainly root expressed, showed differential transcriptomic profiles suggestive of an induced tolerance mechanism. Twenty-two out of these 35 metal homeostasis genes were present in selection sweep genomic regions, in breeding signatures, and/or differentiated during rice domestication. These findings suggest that Fe excess tolerance is an important trait in the domestication of lowland rice, and the identified genes may further serve to design the targeted Fe tolerance breeding of rice crops.

Differential responses to iron stress in blackgram (Vigna mungo L.) varieties

The intention behind conducting this study was to evaluate the toxicological response of black gram (Vigna mungo) varieties exposed to iron stress at an early seedling stage. Iron is a micronutrient required to plant for various metabolic activity. However, its toxicity or deficiency can cause harmful effects to the plants. The experiment was conducted in the Department of Life Science and Bioinformatics of Assam University, Silchar during kharif season of 2019. Two blac gram varieties and three iron concentrations (control, 10µM and 2mM) were evaluated in laboratory conditions.The results clearly showed that significant difference was observed in the growth of roots and shoots and total biomass of the plants grown in three different iron conditions. An enhancement was observed in MDA, H2O2, and O2- and in IPU-073 than in Shekhar-1. At iron-deficiency, H2O2, MDA, O2- radical content of IPU-073 increased by 88.3, 79 and 50.1% whereas, in Shekhar-1, it increased by 70.5, 26.8 and 36.6% in roots respectively which was found to be higher than Fe-excess condition in both roots and shoots. The reduction rate of Chlorophyll content was lower in Shekhar-1 (9.1%) than IPU 073 (31.8%). The comparison between these two varieties showed that Shekhar-1 performed better than IPU-073 under different iron stress condition

How Does Rice Defend Against Excess Iron?: Physiological and Molecular Mechanisms

Iron (Fe) is an essential nutrient for all living organisms but can lead to cytotoxicity when present in excess. Fe toxicity often occurs in rice grown in submerged paddy fields with low pH, leading dramatical increases in ferrous ion concentration, disrupting cell homeostasis and impairing growth and yield. However, the underlying molecular mechanisms of Fe toxicity response and tolerance in plants are not well characterized yet. Microarray and genome-wide association analyses have shown that rice employs four defense systems to regulate Fe homeostasis under Fe excess. In defense 1, Fe excess tolerance is implemented by Fe exclusion as a result of suppression of genes involved in Fe uptake and translocation such as OsIRT1, OsYSL2, OsTOM1, OsYSL15, OsNRAMP1, OsNAS1, OsNAS2, OsNAAT1, OsDMAS1, and OsIRO2. The Fe-binding ubiquitin ligase, HRZ, is a key regulator that represses Fe uptake genes in response to Fe excess in rice. In defense 2, rice retains Fe in the root system rather than transporting it to shoots. In defense 3, rice compartmentalizes Fe in the shoot. In defense 2 and 3, the vacuolar Fe transporter OsVIT2, Fe storage protein ferritin, and the nicotinamine synthase OsNAS3 mediate the isolation or detoxification of excess Fe. In defense 4, rice detoxifies the ROS produced within the plant body in response to excess Fe. Some OsWRKY transcription factors, S-nitrosoglutathione-reductase variants, p450-family proteins, and OsNAC4, 5, and 6 are implicated in defense 4. These knowledge will facilitate the breeding of tolerant crops with increased productivity in low-pH, Fe-excess soils.

Physiology and Biochemistry of Fe Excess in Acidic Asian Soils on Crop Plants

Proper transport of iron is very crucial for plant growth and development as it participates in various complex processes in plants like absorption, translocation etc. It also acts as an important component for processes like photosynthesis and respiratory electron transport chain in mitochondria, chloroplast development, and chlorophyll biosynthesis. Asian soils suffer from iron toxic condition and that adversely affects the growth and yield of the plant. This review describes the importance of iron in plant growth and different strategies adopted by plants for iron uptake. It also focuses on different methods and approaches on how plant can cope against acidic soils.