Identification and Comparative Analysis of MicroRNAs Associated with Low-N Tolerance in Rice Genotypes

<div><h3>Background</h3><p>Nitrogen [N] is a critical limiting nutrient for plants and has to be exogenously supplied to many crops, to achieve high yield with significant economic and environmental costs, specifically for rice. Development of low-input nitrogen sustainable crop is necessary for sustainable agriculture. Identification of regulatory elements associated with low-N tolerance is imperative for formulating innovative approaches for developing low-N tolerant crop plants, using gene manipulation. MicroRNAs (miRNAs) are known to play crucial roles in the modulation of gene expression in plants under various environmental conditions.</p> <h3>Methodology/Principal Findings</h3><p>MiRNAs associated with low-N tolerance have not been identified so far. In this study, we investigated microarray-based miRNA expression in low-N tolerant and low-N sensitive rice genotypes under low N condition. Expressions of 32 miRNAs differed significantly in the two genotypes. Of these 32 miRNAs, expressions of nine miRNAs were further validated experimentally in leaves as well as in roots. Of these differentially expressed miRNAs, six miRNAs (miR156, miR164, miR528, miR820, miR821 and miR1318) were reported in leaves and four (miR164, miR167, miR168 and miR528) in roots. Target genes of all the 32 miRNAs were predicted, which encode transcription factors, and proteins associated with metabolic processes or stress responses. Expression levels of some of the corresponding miRNA targets were analysed and found to be significantly higher in low N-tolerant genotype than low-N sensitive genotype. These findings suggested that miRNAs played an important role in low-N tolerance in rice.</p> <h3>Conclusions/Significance</h3><p>Genome-wide differences in expression of miRNA in low N-tolerant and low N-sensitive rice genotypes were reported. This provides a platform for selection as well as manipulation of genotypes for better N utilization efficiency.</p> </div

Functional categorization of target genes on the basis of AmiGO description.

<p>Functional categorization of target genes on the basis of AmiGO description.</p

Differentially expressed miRNA in IC-547557 and Vivek Dhan genotypes of rice under low-N condition.

<p>The values indicate the fold-change decrease/increase in the miRNA expression in IC-547557 over Vivek Dhan. Significance Analysis of Microarrays (SAM) and a criterion of fold change >2 and q value <0.001 was used to report the variation in expression.</p

Heat map and cluster view of known microRNAs from <i>Oryza sativa.</i>

<p>Biological replicates A1 and A2 represent Vivek Dhan and B1 and B2 represent IC-547557. The scale bar represents scale of relative expression levels of microRNAs. Heat map was generated for all 32 variably expressed microRNAs having statistically positive and significant P value ≤0.05. The hierarchal clustering algorithm used is based on the average linkage method which was developed for clustering correlation matrix. These represents fluorescence ratios, cell with log ratio of 0 are coloured black increasing positive log ratios represented as red intensity and increasing negative log ratios with green intensity.</p

Expression profiling analysis of several target genes in the roots of low-N tolerant (IC-547557) and low-N sensitive (Vivek Dhan) rice genotypes under low-N condition.

<p>The expression levels of miRNAs were normalized to the level of actin. Expression levels are given on a logarithmic scale expressed as 40−ΔC_T, where ΔC_T is the difference in qRT-PCR threshold cycle number of the respective miRNA and the reference actin gene; therefore, 40 equals the expression level of actin gene (the number 40 was chosen because the PCR run stops after 40 cycles). The results are averages ± SE of duplicates of three biological replicate. Significance of the changes between IC-547557 and Vivek Dhan under N-limitation was checked with Student’s <i>t</i>-test at the level of p≤0.05. The significant expression difference between the two genotypes is shown as asterisk.</p

Relative expression level of nitrate transporter gene, <i>OsNrt2</i>, in 21-day-old plants of IC-547557 and Vivek Dhan at 0.1 mM

<p> <b>N.</b> Values are mean of duplicates of three biological replicates. SD is shown by bar.</p

Biomass accumulation and physiological status of IC-547557 (low-N tolerant) and Vivek Dhan (low-N sensitive) genotypes of rice, grown at low (0.01 mM) and optimum (0.1 mM) levels of N.

<p>The results are averages ± SE of three biological replicates. Significance (p≤0.05) of the changes found between low-N and optimum-N conditions was checked with Student’s <i>t-</i>test, and is indicated by asterisk.</p