Target-Mimicry-Based miR167 Diminution Confers Salt-Stress Tolerance During In Vitro Organogenesis of Tobacco (Nicotiana tabacum L.)

Abstract

In response to environmental and cellular cues, the spatiotemporal expression of differentially expressed miRNAs (DEmiRs) during in vitro regeneration of plants is prominent. Nevertheless, the influence of stress conditions on the functional imprints of DEmiRs during plant organogenesis is inadequately understood. Target-mimicry based constitutive diminution of miR167 genes in transgenic tobacco revealed their functional contributions in salt-stress tolerance during organogenesis. Up to 61.6% relative increment in the shoot emergence on leaf explants of miR167 mimic lines (cultured on 12.5 and 25 mM NaCl concentrations) exhibited greater magnitude of organogenesis under stress, most probably through upregulated auxin signalling. Relative expression analysis of auxin transporter genes AUX1, PIN1 and PIN2 revealed synergistic effect of percent miR167 diminution and salt-stress (up to 12.5 mM) resulting into enhanced callogenesis and reduced organogenesis. Biochemical assessment of in vitro grown miR167 mimic shoot initials revealed an increase in the relative water content, chlorophyll and antioxidant activity, which suggested a shift in the molecular equilibrium in response to miR167 diminution-associated signalling pathways. Remarkably, independent miR167 diminution lines exhibited expression variations among associated miR156, miR159, and miR394 and validated by the expression analysis of selected target genes SPL9, ARF6 and ARF8. The expression ratios of these miRNAs revealed their coordinated regulatory network in response to salt-stress during organogenesis. These results demonstrate a link between miR167 diminution and salt-stress tolerance in mimic lines, and suggest that a regulatory shift in the cellular metabolism may contribute to salt-stress tolerance in plants. These observations may create new avenues for strategic utilization of such genic resources for future crop research.