microRNA165 and 166 modulate salt stress response of the Arabidopsis root.

In plants, developmental plasticity allows for the modulation of organ growth in response to environmental cues. Being in contact with soil, roots are the first organ responding to soil abiotic stresses such as high salt concentration. In the root, plasticity relies on changes in the activity of the apical meristem, the region at the tip of the root where a set of self-renewing undifferentiated stem cells sustains growth. We show that salt stress promotes root meristem cells differentiation via reducing the dosage of the microRNAs miR165 and 166. By means of genetic, molecular and computational analysis, we show that the levels of miR165 and 166 respond to high salt concentration, and that miR165 and 166-dependent PHB modulation is fundamental for the response of root growth to this stress. Salt dependent reductions of miR165 and 166 causes rapid increase of the Arabidopsis homeobox protein PHABULOSA (PHB) expression and production of the root meristem pro-differentiation hormone cytokinin. Our data provide direct evidence of how the miRNA-dependent modulation of transcription factors dosage mediates plastic development in plants. In plants, development must be both robust - to ensure appropriate growth - and plastic - to enable the adaptation to external cues. Plastic development largely depends on the modulation of gene expression, controlling the concentration of developmental factors, such as hormones, transcription factors (TFs) and signalling molecules (Garcia-Molina et al, 2013; Hofhuis & Heidstra, 2018; Lopez-Ruiz et al, 2020; Schroder et al, 2021). A classic example of plant developmental plasticity is the adaptation of plant growth to high salt conditions, a stress that inhibits shoot and root development (Flowers et al, 1997). Roots are the first organs sensing salt concentration in soil, where high salt reduces meristem activity and root growth (Dinneny et al, 2008; Geng et al, 2013; Jiang et al, 2016). It has been suggested that the regulation of several plant hormones and miRNAs mediate the plant response to salt stress (Dolata et al, 2016; Geng et al, 2013; Iglesias et al, 2014; Jiang et al, 2016; Nishiyama et al, 2011; Yan et al, 2016). However, the molecular interplays mediating the adaptation of plant roots to salt stress are still vague. Post-embryonic root growth is supported by the activity of the root meristem, a region located at the root tip where self-renewing stem cells divide asymmetrically in the stem cell niche (SCN), originating transit-amplifying daughter cells that divide in the division zone (DZ) (Di Mambro et al, 2018). Once these cells reach a developmental boundary denominated transition zone (TZ), they stop dividing and start to elongate in the so-called elongation/differentiation zone (EDZ) (Di Mambro et al, 2018). A dynamic balance between cell division and cell differentiation ensures continuous root growth, maintaining a fixed number of cells in the DZ. Alterations in this dynamic equilibrium promote or inhibit root growth (Di Mambro et al, 2018; Salvi et al, 2020). microRNA molecules (miRNA) play a key role in the control of root meristem development (Bertolotti et al, 2021a; Skopelitis et al, 2012). Maturation of plant miRNAs depends on the activity of a multiprotein complex comprising the DICER-LIKE1 (DCL1), HYPONASTIC LEAVES1 (HYL1) and SERRATE (SE) proteins that cut pre-miRNA transcripts into 21 nucleotides mature miRNA (Yan et al, 2016). Among miRNAs, miR165 and 166 have been shown to be main regulator of root development (Carlsbecker et al, 2010; Dello Ioio et al, 2012). miR165 and miR166 are pleiotropic regulators of plant developmental processes. miR165 and 166 family consists of nine independent loci (MIR165 A-B and MIR166 A-G) that drive expression of pre-miR165 and 166 in different tissues and at different developmental stages (Miyashima et al, 2011). miR165/166 activity is crucial in the control of robust development, restricting the expression of the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII), including PHABULOSA (PHB) and PHAVOLUTA (PHV), which are involved in root and shoot development, vascular growth, and leaf and embryo polarity (Carlsbecker et al, 2010; Dello Ioio et al, 2012; Di Ruocco et al, 2017; Grigg et al, 2009; McConnell et al, 2001; Skopelitis et al, 2017; Williams et al, 2005). In the root, miR165/166 regulate meristem homeostasis and radial patterning (Carlsbecker et al, 2010; Dello Ioio et al, 2012); pre-miR165a, pre-miR166a and b transcription is promoted by the SCARECROW (SCR) and SHORTROOT (SHR) transcription factors (Carlsbecker et al, 2010) and, thanks to the cell-to-cell mobility, mature miR165 and 166 forms diffuse to patterns both the root vasculature and the ground tissue (Carlsbecker et al, 2010; Miyashima et al, 2011; Skopelitis et al, 2018; Vaten et al, 2011; Bertolotti et al, 2021b). In the root meristem the miR165-166-PHB module promotes the synthesis of the plant hormone cytokinin, an important player in root developmental plasticity regulating cell differentiation rate of meristematic cells via the activation of the ARABIDOPSIS HISTIDINE KINASE3 (AHK3)/ARABIDOPSIS RESPONSE REGULATOR 1/12 (ARR1/12) pathway (Dello Ioio et al, 2007,2008). Here, we show that in response to salt stress miR165 and 166 modulates PHB expression to adjust root meristem activity. Salt exposure results in changes in cytokinin biosynthesis, which further regulates the miR165/166-PHB module. Hence, in addition to the above-described miRNA activity in controlling root robust development, we provide clear evidence that, in response to environmental cues, miRNAs are crucial also in the control of root plastic development, modulating the dosage of transcription factors.