MIR167a transcriptionally regulates ARF6 and ARF8 and mediates drastically plant Pi-starvation response via modulation of various biological processes

Abstract

Plant miRNA members mediate diverse biological processes through regulating target genes at the posttranscriptional or translational level. In this study, tobacco miRNA NtMIR167a and its targets NtARF6 or NtARF8, two auxin response factor (ARF) encoding genes, were subjected to functional evaluation in mediating the plant growth and Pi-starvation response. NtMIR167a showed induced expression in root tissues upon Pi starvation and whose Pi-deprived upregulated expression was restored gradually upon the Pi normal recovery treatment. Conversely, NtARF6 and NtARF8 displayed inverse expression patterns to this miRNA upon low-Pi stress and the Pi recovery treatment, suggesting their regulation under the miRNA at posttranscriptional level. Trangene analysis revealed that the lines with NtMIR167a overexpression and those with NtARF6 and NtARF8 knockdown exhibited modified phenotypes and Pi-associated traits with respect to wild type under both P normal and Pi starvation conditions, together with altered reactive oxygen species (ROS)-associated parameters. Expression analysis revealed that a set of antioxidant enzyme (AE) genes display reduced transcripts in the Pi-deprived lines overexpressing TaMIR167a and knockdown of NtARF6 and NtARF8, suggesting the AE gene expression impacting on the lowered AE activities of transgenic lines. Transcriptome analysis on the NtMIR167 overexpression line revealed a transcriptional variation on quantities of genes that are categorized into diverse functional groups, suggesting that the miRNA/target module-mediated plant growth and Pi starvation response are associated with modulated transcript profiling initiated by auxin signaling controlled by ARF6 and ARF8. Additionally, NtPT1 and NtPT2, two phosphate transporter (PT) genes of tobacco, exhibited downregulated expression in lines overexpressing NtMIR167a or those with knockdown of NtARF6 and NtARF8; antisense expression of these PT genes resulted in reduced P accumulation and inhibited plant growth under Pi starvation treatment. Our results together confirmed that NtMIR167a/NtARF6(8) modules are crucial in mediating plant growth and Pi starvation adaptation via regulating various biological processes including ROS homeostasis and Pi acquisition.