Peptide-Receptor Signaling Controls Lateral Root Development

Lateral root development progresses through different steps with, the peptides and receptors involved in each of these steps triggering downstream mechanisms upon peptide perception.

Plant root systems enable nutrient and water uptake as well as anchorage to the substrate. 18 Nutrient and water availability are known to affect root system architecture, and certain root traits 19 can be linked to nutrient and water use efficiency (Comas et al., 2013;Uga et al., 2013;Li et al., 20 2016; Duque and Villordon, 2019; Sandhu et al., 2019). A better understanding of how root system 21 architecture is determined could therefore be extremely valuable for crop yield enhancement. 22 Regular spacing of lateral roots, as well as initiation and development of lateral root primordia, is 23 tightly regulated in Arabidopsis. However, lateral root development is readily influenced by external 24 cues, ensuring the root system architecture is highly adaptable to different environmental 25 conditions (Tian et al., 2014;. To achieve such strict regulation while 26 maintaining a high degree of flexibility, lateral root development relies on strong intercellular 27 communication networks, mediated by the exchange of molecular messengers over both short and 28 long distances. Several types of molecular messengers are known to regulate plant development, 29 including phytohormones, reactive oxygen species, mobile transcription factors and small non-30 coding RNAs (Van Norman et al., 2011). However, it is becoming increasingly clear that a multitude 31 of secreted signaling peptides and their transmembrane receptors are involved in the control of 32 numerous physiological and developmental processes, including lateral root development. Over the 33 past few years, involvement of multiple peptide signaling pathways has been uncovered in several 34 aspects of lateral root development. Here, we provide an overview of the peptide-receptor 35 modules currently known to play a role in different steps of the lateral root developmental process 36 in Arabidopsis thaliana. 37 38 Signaling peptides controlling lateral root initiation 39 40 TOLS2 signaling controls the lateral inhibition of lateral root founder cell specification 41 The regions of the primary root that experience a peak of DR5:LUC expression in the elongation 42 zone (BOX1) are relatively broad and typically encompass at least ten pericycle cells from which 43 only one pair will become lateral root founder cells (LRFCs) (De Smet et al., 2007;Moreno-Risueno 44 et al., 2010). This has led to the assumption that from early on, inhibitory mechanisms are at play 45 that would avoid overproliferation of the pericycle and prevent initiation of surplus lateral root (LR) 46 primordia. It has recently been shown that TARGET OF LBD SIXTEEN 2 (TOLS2) and a close homolog 47 called PLASMA MEMBRANE INTRINSIC PROTEIN 2 (PIP2), together with their transmembrane 48 2 receptor RECEPTOR-LIKE KINASE 7 (RLK7), are involved in the selection of LRFCs within this region 49 (Toyokura et al., 2019). TOLS2 is expressed in LRFCs and developing LR primordia and its 50 transcription is auxin-inducible via the activity of LATERAL ORGAN BOUNDARIES-DOMAIN 16 51 (LBD16), a transcriptional activator with a demonstrated role in LR formation (Okushima et al., 52 2007;Lee et al., 2009;Goh et al., 2012). TOLS2 overexpression and treatment with synthetic TOLS2 53 or PIP2 peptides reduces the density of pre-branch sites, resulting in a reduction in total LR 54 primordium density. RLK7 is expressed in the pericycle, endodermis and cortex from the 55 differentiation zone onwards. Its expression is strongest in the pericycle but is absent in LRFCs and 56 LR primordia, where TOLS2 is expressed. Loss-of-function rlk7 mutants exhibit an increased pre-57 branch site density and pre-branch sites are often found in close proximity to each other, a 58 phenotype also observed in tols2pip2 double mutants. These findings indicate that TOLS2, PIP2 and 59 RLK7 are negative regulators of LRFC specification, which is in agreement with the presumed role of 60 LBD16 during pre-branch site formation (Goh et al., 2012). Pre-branch site analysis using the 61 DR5:LUC marker indicated that the regions of the primary root that were primed in the elongation 62 zone often contain 2 pairs of LRFCs in close proximity to each other, even in wild-type roots, but 63 that one of them is typically transient and disappears so that LR initiation is inhibited at this site. 64 This inhibition is dependent on the activity of TOLS2 and RLK7. Transcriptome analysis has shown 65 that PUCHI, a transcription factor known to control cell division patterning during LR primordium 66 development (Hirota et al., 2007;Kang et al., 2013;Trinh et al., 2019), is induced by TOLS2 in an 67 RLK7-dependent manner. In agreement with this, puchi mutants display increased pre-branch site 68 densities and increased frequencies of paired pre-branch sites. Furthermore, puchi mutants show a 69 reduced sensitivity to TOLS2 peptide treatments. Consistent with the expression patterns of RLK7, 70 PUCHI is not expressed in LRFCs but is preferentially expressed in the adjacent cells in which RLK7 is 71 thought to be activated by TOLS2 to induce its transcription. Taken together, the TOLS2 signaling 72 peptide thus seems to be involved in a lateral inhibition mechanism that prevents LR primordia 73 from developing in close proximity to each other. This is achieved via the transcriptional activation 74 of PUCHI in the regions flanking a LRFC pair through its receptor RLK7. In case a new pre-branch site 75 is established in close proximity to a pre-existing one, the TOLS2 signaling pathway represses LRFC 76 identity in one of them, thereby ensuring proper LR spacing ( Figure 1). Interestingly, the increased 77 pre-branch site density in rlk7 mutants does not result in an increase in LR density as it does in 78 puchi mutants, suggesting that an additional lateral inhibition mechanism is at play after pre-branch 79 site formation that might also work via PUCHI. 80 Apart from TOLS2, other peptides from the same family were also found to show an inhibitory 81 effect on LR density (Ghorbani et al., 2015), but whether they affect the same pathway or trigger an 82 independent one has yet to be discovered. 83 84 CLE peptides play diverse roles during lateral root initiation 85 The CLAVATA3/ESR-RELATED 1 (CLE) family is a large family of peptides, the most well studied of 86 which is CLAVATA3 (CLV3) which plays an important role in the control of stem cell differentiation 87 in the shoot apical meristem (Clark et al., 1997;Fletcher et al., 1999;Brand et al., 2000;Schoof et 88 al., 2000;Ogawa et al., 2008;Nimchuk et al., 2011). Several CLE genes show distinct but often 89 overlapping expression patterns throughout LR primordium development and some were found to 90 be expressed at the base of the elongation zone, which may indicate a role in the priming of xylem 91 pole pericycle (XPP) cells (Jun et al., 2010;Czyzewicz et al., 2015). Synthetic CLE1, -4, -7, -26 and -27 92 peptide treatments were found to increase emerged LR densities, suggesting a stimulatory role on 93 LR initiation (Czyzewicz et al., 2015). However, these results should be interpreted with caution 94 since this increase coincided with a strong decrease in primary root length, rendering LR density 95 measurements rather unreliable. Furthermore, as will be discussed below, overexpression of CLE1, 96 -4 and -7 has been reported to result in a strong reduction in emerged LR density without affecting 97 primary root length (Araya et al., 2014). Nonetheless, CLE26 peptide treatments can induce the 98 3 formation of a limited number of LRs in arf7arf19 double mutants, in which the canonical auxin 99 signaling required for LR initiation is almost completely inhibited (Okushima et al., 2005), 100 suggesting a stimulatory role of CLE26 on LR initiation downstream of AUXIN RESPONSE FACTOR 7 101 (ARF7) and ARF19.  102  Two other CLE peptides, CLE41 and 44, both encoding the same mature peptide known as  103  TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF), have also been shown to  104 positively regulate LR initiation (Cho et al., 2014). TDIF and its receptor TDIF RECEPTOR (TDR) are 105 known to suppress vascular stem cell differentiation into xylem (Hirakawa et al., 2008) and have 106 been shown to stimulate LR initiation by regulating the transcriptional activity of ARF7 and ARF19 107 (Cho et al., 2014). TDIF peptide treatments increase emerged LR density in a TDR-dependent 108 manner while tdr loss-of-function mutants show reduced emerged LR densities. The kinase domain 109 of TDR was found to interact with BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which has been 110 implicated in the regulation of auxin and brassinosteroid signaling (Pérez-Pérez et al., 2002;Vert et 111 al., 2008). BIN2 gain-of-function mutants show an increased LR density while loss-of-function 112 mutants exhibit a reduction, indicating that BIN2 positively regulates LR development. 113 Furthermore, BIN2 gain-of-function mutants were found to be hypersensitive to auxin (Pérez-Pérez 114 et al., 2002;Cho et al., 2014). BIN2 can directly interact with ARF7 and is able to phosphorylate 115 both ARF7 and ARF19. This BIN2-mediated phosphorylation attenuates the interaction of these 116 ARFs with AUX/IAAs thereby enlarging the pool of free ARF7 and ARF19 transcriptional activators 117 that are able to bind their target gene promoters, including LBD16 and LBD29. BIN2 is expressed in 118 XPP, epidermal and cortex cells, but is restricted to the vasculature in the elongation zone. During 119 LR development, BIN2 expression was observed in LR initiation sites and the basal part of dome-120 shaped LR primordia. Similarly, TDR is expressed in the vasculature, the pericycle and during LR 121 initiation. TDIF peptides on the other hand are mainly expressed in the phloem and their expression 122 can be induced by auxin (Goda et al., 2008), suggesting that auxin-induced TDIF in the phloem 123 travels to the pericycle where it stimulates TDR-induced activation of BIN2, which in turn 124 attenuates the inhibitory activity of Aux/IAAs on ARF7 and ARF19, thereby reinforcing auxin 125 signaling for the initiation of LR development. 126 The receptor-like kinase ARABIDOPSIS CRINKLY 4 (ACR4) was identified as an auxin-inducible 127 regulator of the first asymmetric anticlinal cell division upon LR initiation and is expressed 128 specifically in the small central daughter cells resulting from this initial asymmetric cell division 129 (Malamy and Benfey, 1997;De Smet et al., 2008). A significant increase in LR primordium density 130 can be observed in acr4 mutants as a result of an increased density in LR initiation events. 131 Furthermore, LR primordia are often initiated close to one another, sometimes even opposing each 132 other, and double layered stretches of pericycle cells or fused primordia have also been observed. 133 Despite the increase in total LR primordium density, emerged LR densities are significantly reduced 134 in arc4 mutants. Conversely, ectopic expression of ACR4 specifically in the XPP results in an increase 135 in emerged LR density. Similar to the lateral inhibition mechanism that has been described for the 136 TOLS2-RLK7 signaling module, ACR4 is believed to repress divisions in pericycle cells surrounding 137 LRFCs, while simultaneously promoting the correct organization of the initial LRFC divisions, 138 thereby ensuring proper LR spacing and initiation. A peptide ligand for ACR4 during LR initiation has 139 not yet been identified. However, CLE40 has been proposed as a ligand for ACR4 in the root apical 140 meristem where the CLE40-ACR4/CLV1 signaling module regulates the fate of root stem cells (Stahl 141 et al., 2009;Stahl et al., 2013;Berckmans et al., 2019). 142 143 RALF34 is involved in lateral root initiation and lateral inhibition 144 RAPID ALKALINIZATION FACTOR (RALF) peptides are known to be involved in the regulation of 145 various processes, primarily as inhibitors of cell elongation (Bergonci et al., 2014;Haruta et al., 146 2014). T-DNA knock-down lines of RALF34 display an increase in total LR primordium density, 147 primarily due to an increased density of stage I primordia (Murphy et al., 2016). Additionally, 148 4 primordia were often found in close proximity to one another and aberrant pericycle cell divisions 149 flanking LR primordia were regularly observed. RALF34 starts to be expressed in XPP cells before 150 there is any visible sign of LR initiation and continues to be expressed in LR primordia during the 151 whole developmental process. Additionally, RALF34 is expressed in the pericycle cells flanking LR 152 primordia. This flanking expression, in combination with the increased density of stage I primordia 153 and the aberrant spacing of LR primordia in ralf34 T-DNA knock-down mutants, suggests that 154 RALF34 serves as a negative regulator of LR initiation and probably acts to prevent LR initiation in 155 close proximity to existing primordia. Once again, this points to the involvement of a peptide 156 signaling pathway in a lateral inhibition mechanism that mediates the spatial distribution of LRs  157 along the primary root. The malectin receptor kinase THESEUS 1 (THE1) has been identified as a 158 receptor for RALF34 (Gonneau et al., 2018). THE1 is expressed throughout the stele, including the 159 pericycle, and in developing LR primordia, and the1 loss-of-function mutants display the same 160 defects as ralf34 knock-down mutants. In addition to THE1, RALF34 signaling also seems to require 161 FERONIA (FER), a receptor kinase that is known to perceive other RALF peptides as well (Haruta et 162 al., 2014;Gonneau et al., 2018 interfere with auxin flows, thereby impacting LRFC divisions. It has been shown that aha2 mutants 177 show significantly reduced frequencies of LR initiation (Młodzińska et al., 2015), further suggesting 178 that it could be part of the RALF34-THE1/FER pathway. In agreement with the inhibitory effect of 179 RALF34 on LR initiation, RALF1 and RALF8 have also been found to reduce LR densities (Atkinson et 180 al., 2013;Bergonci et al., 2014), suggesting that other family members share the same function. 181 182 GLV6 disrupts the essential asymmetry of the first LRFC division 183 GOLVEN (GLV) peptides, also known as ROOT GROWTH FACTORS or CLE-LIKE peptides, are mainly 184 known for their role in primary root growth and root apical meristem maintenance, but their 185 expression patterns and overexpression phenotypes indicate that at least some GLV peptides also 186 play an role during LR initiation (Meng et al., 2012;Fernandez et al., 2013;Fernandez et al., 2015). 187 Out of the 11 GLV genes that are encoded in the Arabidopsis genome, 8 are expressed during LR 188 primordium development. These GLV genes are sequentially induced at different stages (Malamy 189 and Benfey, 1997) of primordium development in a fixed order, with GLV6 and GLV10 as the 190 earliest ones, already being expressed upon LR initiation (Fernandez et al., 2015). Overexpression of 191 several GLV genes triggers aberrant anticlinal cell divisions throughout the whole pericycle resulting 192 in a strong reduction in LR densities (Meng et al., 2012;Fernandez et al., 2013). This effect can be 193 mimicked with GLV peptide treatments and is strongest for GLV peptides that are expressed early 194 on during LR development. Due to the early onset of its expression upon LR initiation, the role of 195 GLV6 during LR development has been analyzed more thoroughly (Fernandez et al., 2015). GLV6 196 starts to be expressed in LRFCs prior to the onset of nuclear migration (see BOX1 5 divisions throughout the XPP. Ectopic expression of GLV6 in different root tissue layers indicated 199 that this effect can almost be completely replicated upon expression in the XPP alone but is a lot 200 weaker when expressed in the overlying tissues. This suggests that GLV6 peptides function as 201 autocrine signals that are produced as well as perceived in the XPP. Careful analysis of the effect of 202 GLV6 during LR initiation indicated that the nuclear migration in LRFCs is disrupted upon treatment 203 with GLV6 peptide. As a result, the first anticlinal cell division of LRFCs loses its essential 204 asymmetry, preventing further LR primordium development. GLV6 peptide signaling thus seems to 205 be required for proper cell patterning upon LR initiation. The downstream mechanisms via which 206 GLV6 exerts its effect on LR initiation have yet to be discovered. However, 5 leucine-rich repeat 207 receptor-like kinases (LRR-RLKs), called ROOT GROWTH FACTOR INSENSITIVE (RGI) 1-5 have been 208 identified as receptors for GLV peptides and GLV-RGI signaling was found to stimulate the 209 expression of PLETHORA (PLT) 1 and PLT2 in the root apical meristem, both at the transcriptional 210 and posttranscriptional level (Ou et al., 2016;Song et al., 2016). GLV peptide 211 signaling is thus required for the installation of the typical PLT protein gradient in the RAM, which is 212 essential for the maintenance of the root stem cell niche and transit amplifying cell proliferation 213 (Galinha et al., 2007). Since several PLT transcription factors also play a role during LR development 214 (Hofhuis et al., 2013;Du and Scheres, 2017), it is possible that their expression is modulated via GLV 215 peptide signaling during this process. In addition, GLV peptides were shown to affect root 216 gravitropism by stimulating PIN-FORMED 2 (PIN2) trafficking to the plasma membrane, thereby 217 disrupting the asymmetric auxin distribution in the root tip required for a gravitropic response 218 (Whitford et al., 2012). Since correct distribution of auxin is crucial for LR development, the GLV 219 overexpression phenotypes could potentially result from the disruption of normal auxin fluxes upon 220 LR initiation. As discussed above, several other signaling peptides that inhibit LR initiation upon 221 overexpression or peptide treatment seem to be involved in the lateral inhibition of LR 222 development. Thorough analysis of the LR phenotypes of glv and rgi mutants should determine 223 whether this is also the case for the GLV6-RGI pathway. 224 225 CEP peptides serve as negative regulators of lateral root initiation 226 Several members of the C-TERMINALLY ENCODED PEPTIDE (CEP) family are expressed during LR 227 development (Roberts et al., 2013). As will be discussed below, they have mainly been studied for 228 their role in nitrogen (N) starvation responses and the associated inhibition of LR elongation, but 229 some of them have also been shown to affect LR initiation. Under N-limiting conditions, cep3 loss-230 of-function mutants show increased total LR primordium numbers, while CEP3 peptide treatments 231 result in a decrease, pointing to an inhibitory effect of CEP3 on LR development (Delay et al., 2013). 232 No significant changes in the proportions of the different developmental stages were observed, 233 suggesting that CEP3 inhibits LR initiation but does not affect further development once initiation 234 has taken place. However, the authors report LR primordium numbers instead of densities and 235 since cep3 knock-out and CEP3 peptide treatment increase and decrease primary root length 236 respectively, the reported changes in LR numbers might be an effect of changes in primary root 237 length rather than LR initiation. 238 The role of CEP5 during LR initiation in Arabidopsis has been studied more extensively (Roberts et 239 al., 2016). CEP5 is downregulated by auxin and its expression can be detected in the root tip at the 240 start of the elongation zone and in association with LR primordia. In both cases, CEP5 is 241 predominantly expressed in phloem pole pericycle cells and to a lesser extent in the adjacent 242 phloem. Knocking down CEP5 expression causes an increased density of stage I primordia and a 243 faster progression through the LR developmental stages upon gravistimulation-induced LR 244 initiation. CEP5 overexpression and synthetic peptide treatments on the other hand, result in a 245 decrease in total LR density, indicating a reduction in LR initiation events. Furthermore, aberrant 246 pericycle divisions, as well as fused and closely spaced LR primordia were often observed upon 247 overexpression or peptide treatment. As discussed above for several other peptides, the 248 6 occurrence of these fused and clustered primordia might suggest that CEP5 is also involved in a 249 lateral inhibition mechanism that prevents the development of LR primordia in close proximity to 250 each other. However, in contrast to these other cases (TOLS2, ACR4 and RALF34), the occurrence of 251 closely spaced LR primordia does not coincide with an increase in total LR density. Conversely, the 252 increased occurrence of closely spaced primordia in CEP5 overexpressing seedlings is accompanied 253 by a decrease in total LR density, suggesting another mechanism to be at play that requires further 254 investigation. The LRR-RLKs CEP RECEPTOR1/XYLEM INTERMIXED WITH PHLOEM1 (CEPR1/XIP1) and 255 CEPR2 serve as receptors for CEP1, CEP3 and CEP5 and potentially also other members of the CEP 256 family (Tabata et al., 2014). In the root, CEPR1 starts to be expressed in the phloem pole pericycle 257 and the adjacent phloem from the start of the elongation zone onwards and can only be detected 258 in LRs after emergence. Loss-of-function cepr1 mutants were found to show a reduced sensitivity to 259 CEP5 peptide treatments, supporting the role of CEPR1 as a CEP5 receptor during LR development. 260 However, these mutants display a reduction in total LR density resulting from a reduction in LR 261 initiation events (Roberts et al., 2016), which is similar to the CEP5 overexpression phenotype. It 262 was therefore suggested that CEP5 functions as a negative regulator of CEPR1 activity in the 263 context of LR initiation (Roberts et al., 2016). According to this hypothesis, CEPR1 serves to 264 promote LR initiation while its activity is suppressed upon binding with CEP5 peptide ligands. 265 Conversely, another study reported that cepr1cepr2 double mutants showed an increase in 266 emerged as well as non-emerged LR primordium densities (Dimitrov and Tax, 2018), suggesting that 267 CEPRs might act as negative regulators of LR initiation. while LIKE AUX1 3 (LAX3)-mediated auxin influx in the cortex and epidermis triggers the 299 degradation of IAA14 (Swarup et al., 2008). The breakdown of these Aux/IAAs releases the ARFs in 300 these tissues, leading to the induction of cell wall remodeling genes, resulting in cell separation and 301 spatial accommodation of the underlying LR primordium (Swarup et al., 2008;Vermeer et al., 302 2014). In agreement with this, expression of IDA is auxin-inducible, and the onset of IDA expression 303 is delayed in lax3 mutants. Furthermore, the degradation of pectin in the middle lamella between 304 endodermis and cortex cells is hampered in ida, hae and haehsl2 mutant roots, and expression 305 levels of several cell wall remodeling enzymes were found to be reduced. IDA signaling in floral 306 organ abscission requires the activity of a MPK phosphorylation cascade, comprised of MPKK4/5 307 and MPK6/3 Meng et al., 2016) and it was recently discovered that this MAPK 308 module is also required for IDA-induced cell separation during the spatial accommodation of LR 309 development (Zhu et al., 2019). IDA peptide treatment is able to induce MPK6 and MPK3 310 phosphorylation, but this effect is suppressed in mpkk4mpkk5 and haehsl2 double mutants. 311 Furthermore, pectin degradation in the cell wall between endodermis and cortex cells covering LR 312 primordia does not take place in these mutants and transcript levels of several cell wall remodeling 313 genes are significantly reduced. The IDA-HAE/HSL2 signaling pathway thus seems to function as an 314 auxin-inducible mechanism that triggers cell wall remodeling genes through a downstream MAPK 315 cascade in the cells that surround LR primordia, thereby enabling growing primordia to penetrate 316 the overlying tissues. 317 318 Local inactivation of CIF signaling might be required for primordium growth through the 319 endodermis 320 While the IDA-HAE/HSL2 peptide signaling pathway stimulates LR formation by accommodating its 321 development in the overlying tissues, CASPARIAN STRIP INTEGRITY FACTOR (CIF) peptides seem to 322 have the opposite effect (Ghorbani et al., 2015). Treatment with CIF2, and to a lesser extent also 323 with CIF1 peptides, results in a marked decrease in emerged LR density, while the overall LR 324 primordium density is not affected. Moreover, the density of stage V primordia is unusually high in 325 CIF2-treated roots indicating that LR development is often halted or slowed down at this 326 developmental stage. The transition from a stage IV to a stage V primordium usually entails the 327 transition from a flat-topped to a more dome-shaped primordium. In contrast, the majority of 328 primordia in CIF2-treated roots appear flattened and have difficulty penetrating the overlying 329 tissues, reminiscent of ida and haehls2 mutant phenotypes. CIF2 peptide signaling thus seems to 330 inhibit LR primordium growth by preventing its spatial accommodation in the overlying tissues. As discussed above, several CEP peptides have an inhibitory effect on LR initiation. However, some 349 CEPs were also found to regulate the growth of LR primordia after initiation as well as the 350 elongation of mature LRs. Furthermore, they seem to provide a mechanism to integrate 351 information about the prevailing environmental conditions and adapt the root system accordingly. 352 Several CEP genes are expressed during LR development and it has been shown that CEP1 353 overexpression as well as treatments with synthetic CEP5 peptides result in the inhibition of LR 354 elongation due to a reduction in cell numbers and cell size in the meristematic zone ( LR elongation, but this effect is even stronger in cepr1 single mutants and cepr1cepr2 double 380 mutants which seem hypersensitive to sucrose addition. Moreover, cepr1 mutants show increased 381 LR lengths in high light conditions, suggesting that CEPR1 normally represses LR elongation in 382 response to photosynthesis-derived sugars. Accordingly, expression of several CEP genes is induced 383 upon sucrose addition and CEP5 peptide treatments were shown to reduce LR length in a CEPR1-384 dependent manner. CEP-CEPR signaling thus seems to work as a control mechanism that attenuates 385 LR elongation in response to sucrose in order to balance growth with resource availability. 386 387 CLE peptides inhibit lateral root primordium growth and lateral root elongation upon nitrogen 388 deficiency 389 Apart from CEPs, CLE peptides have also been found to regulate LR primordium development and 390 LR elongation depending on N-availability (Araya et al., 2014). The expression of several CLE 391 peptides (CLE1, -3, -4 and -7) is induced upon N-deficiency and overexpression of these CLEs results 392 in reduced LR lengths without affecting primary root growth, an effect that is strongest in moderate 393 to high-N conditions. Additionally, emerged LR density is strongly decreased in these 394 overexpression lines due to an increase in the frequency of non-emerged LR primordia (stage I-IV) 395 while total LR primordium density was not affected. This indicates that these CLE peptides do not 396 affect LR initiation but inhibit LR primordium growth and LR elongation. Mutants in CLAVATA1 397 (CLV1), the LRR-RLK that is responsible for the perception of CLV3 in the shoot apical meristem 398 9 (Fletcher et al., 1999;Ogawa et al., 2008) One of the most striking common themes that emerged from recent studies is the role of several 420 peptide signaling pathways in lateral inhibition mechanisms that prevent the development of LR 421 primordia in close proximity to each other, indicating that this is an important and tightly regulated 422 process. Interestingly, the involvement of signaling peptides in lateral inhibition seemingly 423 coincides with a role during LR initiation. This suggests that lateral inhibition is one of the first 424 events in the LR developmental process and occurs rather early in the young part of the 425 differentiation zone, probably around the same time at which LR initiation takes place. 426 Some signaling peptides are not merely required for normal LR development but appear to actively 427 modulate the process depending on the environmental conditions. Several peptides from both the 428 CLE and the CEP family prevent the expansion of the root system in N-deficient patches in the soil 429 by inhibiting LR primordium development and/or LR elongation in these conditions. Additionally, 430 some CEPs attenuate the elongation of LRs in response to photosynthesis-derived sugars. 431 Furthermore, expression levels of several CEP peptides were also found to respond to osmotic-and 432 salt stress as well as high CO 2 levels (Delay et al., 2013), suggesting that environmental cues are 433 integrated at the level of these peptides to modulate LR development accordingly. These peptide-434 receptor modules thus seem to serve as molecular mechanisms that underlie the developmental 435 plasticity of the root system, allowing plants to adapt their root system architecture to the 436 environment and to balance growth with their energetic and nutritional status. 437 Signaling peptide families are usually rather big and functional redundancy within peptide families 438 has often been reported. Since single knock-out mutants rarely show striking phenotypes, most of 439 our knowledge about the activity of these peptides is based on gain-of-function studies. until now only been studied in isolation, it is not clear whether peptides and receptors from 449 different families can also act redundantly with one another. Future research should thus 450 determine whether functional overlap and crosstalk between different peptide-receptor pathways 451 exists when they regulate the same process. Therefore, it will be important to unravel the 452 mechanisms that are activated downstream of each peptide-receptor pair during LR development, 453 information that is currently lacking for most peptide signaling pathways. 454 Several peptides and receptors were found not to be expressed in XPP cells or LR primordia, leaving 455 it unclear how they affect LR development. Furthermore, several peptides affect the cells in which 456 they are produced, raising the question as to why a secreted signal is employed in the first place. 457 For a proper understanding of peptide-receptor pathways as mechanisms for intercellular 458 communication, more detailed knowledge of when and where peptides are secreted and perceived 459 is required. 460 Finally, it will be interesting to investigate the involvement of other peptide-receptor pathways in 461 LR development. The Arabidopsis genome is estimated to encode more than 1000 signaling 462 peptides and over 600 RLKs Lease and Walker, 2006), of which only a 463 fraction has been studied so far. Furthermore, it will be paramount to determine whether the 464 peptide-receptor modules that govern LR development in Arabidopsis are conserved in other plant 465 species. It is known that the CLE and CEP peptide families are also represented in Medicago 466 truncatula and that some of these peptides have similar effects on LR development as described in 467 Arabidopsis (Imin et al., 2013;Huault et al., 2014;Patel et al., 2018  Overview of the different steps of lateral root development and the peptide-receptor pairs 479 currently known to be involved in each of these steps. During a DR5:LUCIFERASE (DR5:LUC) 480 maximum in the elongation zone, XPP cells are primed for lateral root formation. In the maturation 481 zone, lateral root initiation takes place, a process that is influenced by the stimulatory and 482 inhibitory effects of multiple peptide signaling pathways. Additionally, several signaling peptides 483 repress lateral root development in the vicinity of pre-existing primordia in a process called lateral 484 inhibition. During later developmental stages, several peptide-receptor pathways affect lateral root 485 primordium growth and its spatial accommodation in the surrounding tissues, allowing for 486 emergence from the primary root. Finally, signaling peptides also control the elongation of lateral 487 roots in response to environmental stimuli. Arabidopsis on a feedback loop regulated by CLV3 activity. • Since the discovery of ACR4 as the first transmembrane receptor with a role in lateral root development, a large number of receptors, together with their peptide ligands, are now known to control several aspects of the lateral root developmental process. • The precise effects of individual signaling peptides on each step of LR development and the molecular mechanisms that are triggered downstream of peptide perception, remain largely unknown for most peptide-receptor pairs but are now slowly being uncovered, sometimes revealing interesting common themes. • It has become clear that some peptidereceptor modules are not merely required for proper lateral root development, but also modulate the process in relation to the environmental conditions as well as the energetic and nutritional status of the plant.

OUTSTANDING QUESTIONS
• Which peptides within a peptide family act redundantly with one another and how is LR development affected in higher-order mutants? • Do peptides and receptors from different families that affect the same LR developmental process act redundantly with one another and/or is there crosstalk between these pathways? • What are the downstream mechanisms activated upon peptide perception and how do they affect LR development? • When and where exactly are peptides and their receptors expressed and what does this mean for the LR developmental process they regulate?
• Can our knowledge on the peptide-receptor modules that regulate LR development in Arabidopsis be translated to other species, including crops?

Development: From Priming to Elongation
In Arabidopsis, lateral root (LR) primordia arise from pairs of xylem pole pericycle (XPP) cells, called LR founder cells (LRFCs), that are primed for future LR development in the elongation zone of the primary root. Here, an auxin-driven oscillatory gene expression mechanism is active, which can be visualized using the DR5:LUCIFERASE (DR5:LUC) auxin signaling reporter. During each DR5:LUC peak, a patch of XPP cells is primed for LR development. These primed patches of cells maintain DR5:LUC expression as they leave the elongation zone and are called "pre-branch sites". This periodic priming of XPP cells gives rise to regularly spaced pairs of LRFCs that are able to produce LR primordia. Subsequently, another peak in auxin signaling activity triggers the migration of LRFC nuclei towards the common cell wall, followed by an asymmetric anticlinal cell division in both LRFCs, yielding two small central cells and two larger daughter cells. Next, a dome-shaped primordium is formed through a series of anticlinal, periclinal and oblique divisions. This process can be subdivided in different stages based on the number of cell layers in the primordium and the position of the tip of the growing primordium relative to the overlying tissues. These developmental stages range from stage I primordia, single-layered primordia resulting from the first asymmetric anticlinal cell divisions of LRFCs, to stage VII primordia, multilayered dome-shaped primordia constituting the final stage before emergence from the primary root.
Smooth progression through these stages is again dependent on auxin distribution and signaling. Furthermore, auxin signaling is also required in cells that cover developing LR primordia. Here, auxin regulates the spatial accommodation of LR primordia, which entails shrinkage of endodermal cells to allow for the swelling of LRFCs, as well as cell wall remodeling in the endodermis, cortex and epidermis, enabling the LR primordia to push through these overlying tissues. Finally, once a LR primordium has emerged from the primary root, LRs start to elongate, during which auxin transport also plays a key role. The development of LRs can thus largely be subdivided into four main steps: priming, primordium initiation, primordium development, and elongation. Furthermore, initiation and primordium development are strongly intertwined with spatial accommodation in the overlying tissues. Signaling peptides are now known to be involved in nearly all of these steps (Figure 1). Germination and cotyledon greening: both processes are discussed together as they are both inhibited by high external sugar supply, but there are also differences in their regulation. P P P