Peptide signaling pathways in vascular differentiation

CLE peptide and related signaling pathways take up prominent roles in the development of both vascular tissues, xylem and phloem.


Promotion of procambial/cambial cell proliferation by TDIF-TDR 91
TDR is expressed preferentially in procambium and cambium (Hirakawa et al. 2008;92 Fisher and Turner 2007), while CLE41 and CLE44 are expressed specifically in phloem 93 and more widely in its neighboring cell files, respectively (Fig. 1A;Hirakawa et al. 94 2008). Defects in TDR or CLE41 cause the depletion of the procambial cells (Fisher and 95 Turner 2007, Hirakawa et al. 2008, 2010, Etchells and Turner 2010. Ectopic expression 96 of CLE41 under different promoters revealed that expression of CLE41 in or around 97 procambial cells is sufficient to drive vascular cell division, but localized expression of 98 CLE41 in the phloem is required for maintaining properly orientated cell divisions 99 (Etchells and Turner 2010). Thus, phloem-synthesized TDIF regulates the orientation of 100 procambial cell divisions in a non-cell-autonomous fashion. 101 TDIF upregulates WOX4 expression in a TDR-dependent manner (Hirakawa 102 et al. 2010). Genetic analyses showed that WOX4 is required to promote the 103 proliferation of procambial/cambial cells, but not for repressing xylem differentiation in 104 response to the TDIF signal. The WOX14 transcription factor may also function 105 redundantly in Arabidopsis to modulate procambial cell proliferations (Etchells et al., 106 conserved (Hirakawa and Bowman, 2015, Etchells et al., 2015, Kucukoglu et al., 2017 and PttWOX4a and PttWOX4b are specifically expressed in the cambial region during 116 vegetative growth, but not after growth cessation and during dormancy (Kucukoglu et 117 al., 2017). A decrease in PttWOX4a/b levels achieved by RNAi treatment caused severe 118 reduction of the width of the vascular cambium and greatly diminished secondary 119 growth, although primary growth was not affected. 120 Because there is no homologous sequence to Arabidopsis WOX14 in poplar 121 or spruce (Picea) genomes, WOX4 is expected to be a major player of cambial 122 proliferation in woody plants. PtPP2::PttCLE41-PttANT::PttPXY double 123 overexpression lines, in which phloem-specific expression of PttCLE41 and 124 cambium-specific expression of PXY are induced, exhibited highly organized vascular 125 tissue, comparable to that of wild-type controls (Etchells et al., 2015). Because 126 orthologs of CLE41, TDR/PXY, and WOX4 are widely distributed in angiosperm and 127 gymnosperm tree species (Hirakawa andBowman, 2015, Kucukoglu et al., 2017), the 128 TDIF-TDR/PXY-WOX4 pathway is an evolutionarily conserved program for the 129 regulation of the vascular cambium activity in wood formation. Recently two groups 130 identified bifacial, strongly proliferating cambial stem cells that feed both xylem and 131 phloem production during secondary growth in Arabidopsis (Shi et al., 2019, Smetana 132 et al. 2019. In fact, in the cambial stem cells, TDR/PXY and WOX4 genes were actively 133 expressed. Recently, Zhu and others found that PtrCLE20 is expressed in xylem cells 134 and represses cambium activity in poplar (Zhu et al. 2019). The molecular mechanism 135 underlying the xylem-producing CLE peptide-dependent regulation of cambium activity 136 should be analyzed further. 137

Interaction of the TDIF-TDR pathway with phytohormones 138
The role of auxin in the regulation of vascular cambium activity is well established and 139 the auxin concentration gradient peaks over the cambial region and decreases towards 140 the surrounding secondary tissues in hybrid aspen (Populus tremula L. x Populus 141 tremuloides Michx) (Tuominen et al., 1997). WOX4 has been shown to be necessary 142 both for the auxin responsiveness of the cambium cells and for the auxin-dependent 143 increase in the cambium cell division activity (Suer et al., 2011). Local auxin signaling 144 in TDR-positive stem cells stimulates cambium activity (Brackmann et al. 2018). An 145 analysis with a highly sensitive auxin response marker revealed a moderate auxin 146 (Kondo et al., 2014). The loss-of-function mutant of BRASSINAZOLE RESISTANT 1 179 (BZR1), the closest homolog of BES1, also shows a bes1-like phenotype, but a weaker 180 one (Saito et al., 2018). Thus, BES1 and BZR1, which are suppressed by GSK3s, act 181 redundantly in promoting xylem differentiation (Fig. 1A). Further analysis indicated 182 that the GSK3s-BES1/BZR1 signal not only regulates xylem but also phloem 183 differentiation (Kondo et al., 2016, Saito et al., 2018. This finding is consistent with a 184 previous report showing that both bes1-D and bzr1-D partially rescued the phenotype of TDIF-TDR-GSK3s-BES1/BZR1 signaling pathway, because GSK3s and BES1/BZR1 204 are among its key components (Fig. 1A). Indeed, brassinosteroid promotes xylem 205 differentiation (Yamamoto et al, 1997, Caño-Delgado et al., 2004, Kubo et al, 2005, 206 which suggests that brassinosteroid regulates procambial cell fates to counteract TDIF 207 signaling through the reverse regulation of GSK3s. BRASSINOSTEROID 208 INSENSITIVE 1 (BRI1) is the major receptor for brassinosteroid and the BRI1 gene is 209 expressed widely in plants, while its homologs, BRI1-LIKE 1 (BRL1) and 3 (BRL3), are 210 expressed predominantly in vascular tissues. In particular, BRL1 expression is 211 associated with the procambial cells in inflorescence stems (Caño-Delgado et al., 2004). 212 In procambial cells, therefore, the TDIF and brassinosteroid signals may be balanced 213 via GSK3s to determine procambial cell identity. Because brassinosteroid biosynthesis 214 occurs in procambial cells, which is promoted by auxin and cytokinin (Yamamoto et al., 215 2001(Yamamoto et al., 215 , 2007, brassinosteroid may act as an autocrine factor in xylem differentiation 216 from procambial cells. 217

ERECTA and related receptors cooperate with TDR/PXY family receptors 262
The LRR-RLK encoded by the ERECTA (ER) gene is part of a small gene family 263 together with ER-LIKE1 (ERL1) and ERL2. In er erl1 stems, intervening cambial cells 264 are decreased and phloem cells are frequently located adjacent to xylem cells (Uchida 265 and Tasaka, 2013). This also occurs in tdr (Fisher and Turner, 2007;Hirakawa et al., 266 2008), suggesting that ER and ERL1, like TDR, act by suppressing xylem 267 differentiation and promoting cambial cell activity. By contrast, er erl1 hypocotyls 268 display a remarkable expansion of the xylem and an increase in xylem fiber cells (Ragni 269 et al., 2011;Ikematsu et al., 2017). 270 On the one hand, this result supports the idea that ER and ERL1 suppress 271 xylem differentiation but appear to suppress cambial activity. On the other hand, the 272 relative xylem expansion in the Landsberg er (Ler) genotype was found to be due to 273 early cessation of phloem formation and reduced cambial activity (Sankar et al., 2014). 274 The difference between stems and hypocotyls in er erl1 may be explained by the 275 tissue-specific gene regulation of ERL1 and ERL2: In the er pxy pxl1 pxyl2 genetic 276 background, ERL1 and ERL2 gene expression is reduced greatly in stems, while their 277 expression is strikingly enhanced in hypocotyls (Wang, et al., 2019). These findings 278 clearly indicate a tight interaction between PXY family genes and ER family genes (Fig.  279   1A). Indeed, the sextuple mutant, er erl1 erl2 pxy pxl1 pxyl2 shows a complete 280 suppression of secondary growth of hypocotyls, supporting the view that both TDR and 281 ER family genes are crucial factors of secondary growth (Wang, et al., 2019). Although BREVIPEDICELLUS is required for the hypocotyl to gain competency to 291 respond to gibberellin and trigger fiber differentiation in both the wild-type and er erl1, 292 it is still unknown whether the ER/ERL1 pathway is associated with the gibberellin 293 pathway. 294 Ligands for ER and EFR1 that function in xylem development have not been 295 identified. However, it is known that EPIDERMAL PATTERNING FACTOR 1 (EPF1), 296 EPF2, and EPF-LIKE9 (EPFL9) peptides function as ligands for ER and ERL1 in 297 stomata development (Han and Torii, 2016). In stem elongation, EPFL4 and EPFL6, 298 which are produced in epidermal cells, act as ligands for phloem-located ER (Abrash et 299 al., 2011;Uchida et al., 2012). In hypocotyls, EPFL4 and EPFL6 are expressed in 300 xylem parenchyma cells and differentiating xylem cells (Wang, et al., 2019 (Hang and Torii, 2016). Therefore, a specific combination 307 of ER/ERL1, EPF/EPFL peptides, and co-receptors, such as TMM and SERK family 308 proteins, may function in vascular development in stems and hypocotyls. 309

Phloem-related CLE peptide signals 310
An interesting aspect emerging from the analysis of pxy/tdr and related mutants is that 311 they do not lose their capacity to produce proper xylem and phloem tissues, as judged 312 from morphological and molecular-genetic criteria (Fisher and Turner, 2007;Hirakawa 313 et al., 2008). This even holds true for higher order (sextuple) mutants that largely

Root-active CLE peptides are perceived in the phloem 328
The root protophloem is also a system where a key question can be answered: Is CLE 329 peptide signaling required to form functional phloem? Early hints that CLE peptides 330 could play a role in root development came from the observation that several CLE genes 331 are expressed in the root (Jun et al., 2010), and that treatments with many chemically 332 synthesized CLE peptides suppress root growth (Fiers et al., 2005;Ito et al., 2006;333 Kinoshita et al., 2007). Such so-called "root-active" CLE peptides were also employed 334 to identify components that are necessary for CLE peptide perception in the root, and 335 notably identified the receptor-like protein CLV2 and its interacting partner, the 336 pseudokinase SUPPRESSOR OF OVEREXPRESSION OF LLP1-2/CORYNE 337 (SOL2/CRN) (Jeong et al., 1999;Miwa et al., 2008;Muller et al., 2008;Meng and 338 Feldman, 2010). 339 Both clv2 and crn mutants display substantial resistance to root growth 340 inhibition by a range of root-active CLE peptides, and both the CLV2 and CRN genes 341 are essentially expressed at low levels throughout the root (Hazak et al., 2017). 342 However, recently it was shown that restricted expression of CRN in the developing 343 protophloem is sufficient to fully recover CLE perception in a crn null mutant 344 background (Hazak et al., 2017). This matched the observation that root-active CLE 345 peptides a priori suppress protophloem sieve element differentiation 346 (Rodriguez-Villalon et al., 2014;Hazak et al., 2017), which is essential for root growth 347 (Furuta et al., 2014;Rodriguez-Villalon et al., 2014). Given that interactions between 348

CLE peptide signaling as rapid mediators of stress-induced sink shutdown 391
Beyond purely developmental aspects, CLE peptides may also transmit environmental 392 inputs, and these two roles might not be mutually exclusive in the case of individual 393 peptides. Indeed, CLE25 is a prime example in this context. In addition to its expression 394 in the protophloem, CLE25 expression is induced by drought stress and acts as a 395 long-distance signal that is transported through the vasculature to convey this 396 physiological state to the shoot system (Takahashi et al., 2018). Coincidentally, such 397 induction should also suppress protophloem differentiation and thereby root growth 398 (Anne and Hardtke, 2017;Hazak et al., 2017), which could be advantageous in drought 399 conditions. Yet another example is the root-active CLE peptide, CLE14, which is 400 induced upon phosphate starvation and triggers a breakdown of root meristem 401 maintenance and root growth (Gutierrez-Alanis et al., 2017). Since this response 402 requires the CLV2|CRN module, it appears that it is achieved through a shutdown of 403 protophloem formation (Meng and Feldman, 2010;Gutierrez-Alanis et al., 2017;Hazak 404 et al., 2017). 405 In summary, beyond any positive developmental roles in phloem development 406 that largely remain to be discovered, root-active CLE peptides might be mainly 407 involved in the sensing of adverse environmental conditions. From the current data, it 408 seems that their upregulation in response to such adverse conditions suppresses 409 protophloem differentiation and thereby halts root growth. From a physiological 410 perspective, such a mechanism would be a very efficient and rapid way to stop growth 411 and eliminate a metabolic sink that finds itself in sub-optimal conditions. Because 412 phloem transport is essentially auto-regulated, this would "automatically" redirect 413 phloem sap to actively growing roots. Thus, CLE peptide regulation may primarily 414 serve to fine-tune and optimize root system exploration of the wildly heterogenous soil 415 matrix, which could represent a substantial adaptive advantage. 416

Concluding remarks 417
In summary, peptide signaling pathways have been increasingly implicated in vascular 418 development over recent years (Figure 1 summarizes peptide and hormone signaling 419 pathways discussed in this update). From the current data, it appears clear that various 420 peptides and plant hormones not only function as ligands in vascular cell proliferation, 421 but also in both xylem and phloem cell type fate regulation, and both in a 422 cell-autonomous or non-cell autonomous manner. In addition, the emerging picture is 423 becoming considerably complex since the exact impact of a given signaling pathway 424 seems to depend on the developmental stage or the organ. For example, the TDIF-TDR 425 signaling pathway apparently acts in the cambium and actively proliferating 426 procambium of hypocotyls and stems, but not in the root apical meristem. Moreover, 427 the picture is complicated by the observation that at least in the RAM context, 428 CLE45-BAM3 signaling has to be suppressed to permit phloem differentiation, 429 although CLE peptide signaling might be required to initiate proper phloem formation.  • The role of a given signaling pathway can be context-dependent and depend on the developmental stage or the organ. • The complex network composed of multiple intersecting signaling pathways renders the regulation of procambial/cambial cell identity and proliferation robust. • CLE peptide signals might convey environmental inputs as a rapid control of sink organ development.

OUTSTANDING QUESTIONS
• How does crosstalk between different peptide signaling pathways occur mechanistically during vascular development? Do different receptors form a complex on the plasma membrane, and/or are there signaling hubs that connect different signaling pathways? • Are long-distance peptide signaling pathways controlling vascular development? • Are the peptide processing and secretion processes regulated as a function of vascular development? • What are the mechanisms that underlie developmental stage-and organ-specific peptide signaling? • Where do the processed peptides act at high spatio-temporal resolution?