Submergence-responsive MicroRNAs are potentially involved in the regulation of morphological and metabolic adaptations in maize root cells

Abstract

Background and aims: Anaerobic or low oxygen conditions occur when maize plants are submerged or subjected to flooding of the soil. Maize survival under low oxygen conditions is largely dependent on metabolic, physiological and morphological adaptation strategies; the regulation mechanisms of which remain unknown. MicroRNAs (miRNAs) play critical roles in the response to adverse biotic or abiotic stresses at the post-transcriptional level. The aim of this study was to understand submergence-responsive miRNAs and their potential roles in submerged maize roots.

Methods: A custom muParaflo microfluidic array containing plant miRNA (miRBase: http://microrna.sanger.ac.uk) probes was used to explore differentially expressed miRNAs. Small RNAs from treated roots were hybridized with the microarray. The targets and their cis-acting elements of small RNA were predicted and analysed by RT-PCR.

Key results: Microarray data revealed that the expression levels of 39 miRNAs from nine maize and some other plant miRNA families were significantly altered (P < 0.01). Four expression profiles were identified across different submergence time-points. The zma-miRNA166, zma-miRNA167, zma-miRNA171 and osa-miRNA396-like were induced in the early phase, and their target genes were predicted to encode important transcription factors, including; HD-ZIP, auxin response factor, SCL and the WRKY domain protein. zma-miR159, ath-miR395-like, ptc-miR474-like and osa-miR528-like were reduced at the early submergence phase and induced after 24 h of submergence. The predicted targets for these miRNAs were involved in carbohydrate and energy metabolism, including starch synthase, invertase, malic enzyme and ATPase. In addition, many of the predicted targets were involved in the elimination of reactive oxygen species and acetaldehyde. Overall, most of the targets of induced miRNAs contained the cis-acting element, which is essential for the anaerobic response or hormone induction.

Conclusions: Submergence-responsive miRNAs are involved in the regulation of metabolic, physiological and morphological adaptations of maize roots at the post-transcriptional level.

Fig. 1.

The similarity of sequences among miRNA members from different species. The miRNA in parentheses is the homologue of the miRNA before the parentheses. Shaded nucleotides show differences with zma-miRNA or its homologue. (–) indicates the absence of a nucleotide.

Fig. 2.

The clustering of differentially expressed miRNAs for roots submerged for 12, 24 or 36 h relative to controls. miRNA from maize roots under normal growth condition was used as the reference. The colour saturation reflects the magnitude of the log₂ expression ratio (Cy5/Cy3) for each transcript. Red colour means higher transcript levels than the reference, whereas green means lower transcript levels than the reference.

Fig. 3.

RT-PCR of representative target genes of miRNAs at different time-points in maize roots. (A) The expression of miRNAs associated with targets, expressed relative to the control value. (B) RT-PCR of representative targets of miRNAs. RT-PCR was performed with primers specific for each of the target genes. RT-PCR of γ-tubulin cDNA was used as a positive control.

Fig. 4.

The potential regulating network of submergence-responsive miRNAs in roots cell. The stress-responsive miRNAs could be involved in three pathways to reprogramme complex procedures of metabolism and physiology. (1) Up-regulated miR399 directly targeted cleavage of mRNAs of starch synthase and amino-transferase, prevented metabolic intermediates and glucose from synthesizing starch and amino acid. Accumulated low molecular weight carbohydrate could act as substrate of glycolysis pathway. In addition, the down-regulated miR159, ath-miR395-like and ptc-miR474-like could enhance accumulation of many anaerobic-responsive enzymes involved in carbohydrate and energy metabolism; these accumulated enzymes enhance glycolysis and thus ATP supply. The aerobic metabolism in roots cells would be shifted into anaerobic metabolism during the early submergence phase. (2) Accumulation of SOD and ALDH due to decreasing of osa-miR528-like would aid elimination of reactive oxygen species and acetaldehyde, and thus survival of root cells. (3) The accumulated miR166 and miRNA167 together with down-regulated miR159 could modulate hormone homeostasis via regulating transcripts of HD-ZIP, ARF and GAMYB and then trigger adventitious root formation and lateral root development. The plant has acquired morphological characteristics to adapt to the low-oxygen environment.