Genome-wide identification of microRNAs in response to low nitrate availability in maize leaves and roots

Abstract

Background: Nitrate is the major source of nitrogen available for many crop plants and is often the limiting factor for plant growth and agricultural productivity especially for maize. Many studies have been done identifying the transcriptome changes under low nitrate conditions. However, the microRNAs (miRNAs) varied under nitrate limiting conditions in maize has not been reported. MiRNAs play important roles in abiotic stress responses and nutrient deprivation.

Methodology/principal findings: In this study, we used the SmartArray™ and GeneChip® microarray systems to perform a genome-wide search to detect miRNAs responding to the chronic and transient nitrate limiting conditions in maize. Nine miRNA families (miR164, miR169, miR172, miR397, miR398, miR399, miR408, miR528, and miR827) were identified in leaves, and nine miRNA families (miR160, miR167, miR168, miR169, miR319, miR395, miR399, miR408, and miR528) identified in roots. They were verified by real time stem loop RT-PCR, and some with additional time points of nitrate limitation. The miRNAs identified showed overlapping or unique responses to chronic and transient nitrate limitation, as well as tissue specificity. The potential target genes of these miRNAs in maize were identified. The expression of some of these was examined by qRT-PCR. The potential function of these miRNAs in responding to nitrate limitation is described.

Conclusions/significance: Genome-wide miRNAs responding to nitrate limiting conditions in maize leaves and roots were identified. This provides an insight into the timing and tissue specificity of the transcriptional regulation to low nitrate availability in maize. The knowledge gained will help understand the important roles miRNAs play in maize responding to a nitrogen limiting environment and eventually develop strategies for the improvement of maize genetics.

Figure 1. The Chronic (∼15 days) and transient (2 hr) low nitrate regulated mature miRNA families and species identified in leaves and roots.

Those regulated miRNAs with fold change>2 or <0.5 and q value<0.001 are shown. Left circle: the responsive miRNAs in leaves; Right circle: the responsive miRNAs in root tips; Light blue shadow: the chronic responsive miRNAs; Yellow shadow: the transient responsive miRNAs; White background: both chronic and transient responsive miRNAs. Words in red: up-regulated miRNAs; Words in blue: down-regulated miRNAs. miRNA families are represented by numbers and their species are represented by the letters.

Figure 2. Verification of the low nitrate responsive pattern of the mature miRNAs identified in the roots of maize seedlings and their predicted target genes by qRT-PCR.

The X axis is the time of hours (h) after transfer to low N, and the 15D sample is from plants grown under chronic low N. The expression level is expressed as the mean of relative fold changes of triplicate biological replicates and the vertical bars represent standard derivation of the mean (n = 3).

Figure 3. Verification of the low nitrate responsive pattern of the mature miRNAs identified in leaves and their predicted target genes by qRT-PCR.

The X axis is the time in hours (h) after transfer to low N, and the 15D sample is from plants grown under chronic low N. The expression level is expressed as the mean of relative fold changes of triplicate biological replicates and the vertical bars represent standard derivation of the mean (n = 3).

Figure 4. The potential regulatory network for low nitrate responsive miRNAs in maize leaves and roots.