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

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.

Roles of non-coding RNAs in the hormonal and nutritional regulation in nodulation and nitrogen fixation

“…MiR390, an evolutionarily conserved miRNA, targets the Trans-Acting Short Interference RNA3 ( TAS3 ) and causes the cleavage of TAS3 by ARGONAUTE7 at the 3’ ends most proximal site to produce ta-siRNA ( Hobecker et al., 2017 ). TAS3 -derived ta-siRNAs directly control the cleavage of complementary mRNAs encoding ARF2/3/4 ( Hobecker et al., 2017 ; Kirolinko et al., 2021 ). Knockdown of ARF2/3/4 reduces the initiation and progression of infection events to inhibit nodule formation and downregulate the Nodulation Signaling Pathway 2 ( NSP2 ) ( Kirolinko et al., 2021 ).…”

“…TAS3 -derived ta-siRNAs directly control the cleavage of complementary mRNAs encoding ARF2/3/4 ( Hobecker et al., 2017 ; Kirolinko et al., 2021 ). Knockdown of ARF2/3/4 reduces the initiation and progression of infection events to inhibit nodule formation and downregulate the Nodulation Signaling Pathway 2 ( NSP2 ) ( Kirolinko et al., 2021 ). At the early stage of nodulation, the expression level of miR390 and tasiRNAs are at a low level, whereas TAS3 and ARF2/3/4 are substantially highly expressed to promote the nodule formation ( Hobecker et al., 2017 ).…”

Molecular Cloning, Characterization and Expression Analysis of Auxin Response Factor GhARF2 Gene in Upland Cotton (Gossypium hirsutum L.)

“…In tomato, the SlARF2 gene not only participates in the regulation of fruit ripening, but also participates in the regulation of root architecture (Hao et al, 2015;Ren et al, 2017), and overexpression SlARF2 gene leads to a signi cant increase in the number of lateral roots (Ren et al, 2017). Similarly, the MtARF2 gene in alfalfa has also been con rmed to be related to the development of the roots, and knockout of the MtARF2 gene will result in a decrease in the length of the main roots and an increase in the density of the lateral roots (Kirolinko et al, 2021). In addition, the ARF2 gene is also involved in the regulation of K + uptake of roots under low-potassium conditions in Arabidopsis.…”

FveARF2 negatively regulates fruit ripening and quality in strawberry

“…ARF is a key transcription factor involved in all stages of plant growth and development ( Tian et al., 2004 ). OsARF1 participates in rice coleoptile growth and in Medicago truncatula , MtARF2/3/4 can regulate lateral root formation, while silencing MtARF2/3/4 leads to shorter lateral roots ( Waller et al., 2002 ; Kirolinko et al., 2021 ). In tomato, SlARF5 can affect ovary development through gibberellin and auxin signaling pathways, and silencing SlARF5 results in fruit size and weight reduction ( Liu et al., 2018 ).…”