Auxin Response Factor 2 (ARF2), ARF3, and ARF4 Mediate Both Lateral Root and Nitrogen Fixing Nodule Development in Medicago truncatula.

Cristina Kirolinko,María Eugenia Zanetti,Andreas Niebel,Karen Hobecker,Jiangqi Wen,Kirankumar S. Mysore,Flavio Antonio Blanco

doi:10.3389/fpls.2021.659061

Abstract

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.

Highlights

Auxins play essential roles in diverse aspects of plant growth and development, including cell elongation, cell polarity, vascular tissue differentiation, embryo patterning, apical dominance, and leaf shape (Salehin et al, 2015)
The tree includes M. truncatula Auxin Response Factors (ARFs) members that have been involved in root development and nodulation in this legume, i.e., MtARF10, MtARF16, and MtARF17 (Bustos-Sanmamed et al, 2013; Breakspear et al, 2014), as well as best M. truncatula homologs of ARF members implicated in these processes in other plant species, such as AtARF5, AtARF7, and AtARF19 in lateral root development (Okushima et al, 2005; Wilmoth et al, 2005; De Smet et al, 2010) and GmARF6 and GmARF8 in nodulation (Wang et al, 2015)
The amino acid sequences of M. truncatula ARF members were retrieved from the recently released version of the M. truncatula genome MtrunA17r5.0ANR (Pecrix et al, 2018). This phylogenetic analysis verified that MtARF2, MtARF3, MtARF4a, and MtARF4b clustered in the same clades as their Arabidopsis counterparts (Supplementary Figure 2)

Summary

Introduction

Auxins play essential roles in diverse aspects of plant growth and development, including cell elongation, cell polarity, vascular tissue differentiation, embryo patterning, apical dominance, and leaf shape (Salehin et al, 2015). It was recently shown that Nuclear Factor YA (NF-YA) genes are important regulators of auxin signaling during nodule development via the direct control of SHORT INTERNODES/STYLISH (STY) transcription factor genes and their downstream targets, YUCCA1 and YUCCA11, involved in auxin biosynthesis (Shrestha et al, 2020) This implies that auxin biosynthesis, signaling, and responses must be integrated with the nodulation program, which is initiated by the LysM domain receptor kinases MtNFP (Nod Factor Perception, Amor et al, 2003) and MtLYK3 (Smit et al, 2007) in M. truncatula and followed by the activation of a number of transcription factors such as the ERF transcription factor MtERN1 (Ethylene response factor Required for Nodulation 1) (Middleton et al, 2007) and the three subunits of the NF-Y heterotrimeric complex (Combier et al, 2006; Laporte et al, 2014; Baudin et al, 2015). Auxins interact with the ethylene signaling pathway during root nodule symbiosis, since the ethylene-insensitive sikle (skl) mutant, which forms numerous infection threads (ITs) and nodules under symbiotic conditions (Penmetsa and Cook, 1997), exhibited altered auxin transport during nodulation (Prayitno et al, 2006)

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