Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue.

The phloem performs essential systemic functions in tracheophytes, yet little is known about its molecular genetic specification. Here we show that application of the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) specifically inhibits specification of protophloem in Arabidopsis roots by locking the sieve element precursor cell in its preceding developmental state. CLE45 treatment, as well as viable transgenic expression of a weak CLE45 G6T variant, interferes not only with commitment to sieve element fate but also with the formative sieve element precursor cell division that creates protophloem and metaphloem cell files. However, the absence of this division appears to be a secondary effect of discontinuous sieve element files and subsequent systemically reduced auxin signaling in the root meristem. In the absence of the formative sieve element precursor cell division, metaphloem identity is seemingly adopted by the normally procambial cell file instead, pointing to possibly independent positional cues for metaphloem formation. The protophloem formation and differentiation defects in brevis radix (brx) and octopus (ops) mutants are similar to those observed in transgenic seedlings with increased CLE45 activity and can be rescued by loss of function of a putative CLE45 receptor, BARELY ANY MERI-STEM 3 (BAM3). Conversely, a dominant gain-of-function ops allele or mild OPS dosage increase suppresses brx defects and confers CLE45 resistance. Thus, our data suggest that delicate quantitative interplay between the opposing activities of BAM3-mediated CLE45 signals and OPS-dependent signals determines cellular commitment to protophloem sieve element fate, with OPS acting as a positive, quantitative master regulator of phloem fate. stem cell | division plane switching

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.

Brassinosteroids (BRs) trigger an intracellular signaling cascade through its receptors BR INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1) and BRL3. Recent studies suggest that BR-independent inputs related to vascular differentiation, for instance root protophloem development, modulate downstream BR signaling components. Here, we report that protophloem sieve element differentiation is indeed impaired in bri1 brl1 brl3 mutants, although this effect might not be mediated by canonical downstream BR signaling components. We also found that their small meristem size is entirely explained by reduced cell elongation, which is, however, accompanied by supernumerary formative cell divisions in the radial dimension. Thus, reduced cell expansion in conjunction with growth retardation, because of the need to accommodate supernumerary formative divisions, can account for the overall short root phenotype of BR signaling mutants. Tissue-specific re-addition of BRI1 activity partially rescued subsets of these defects through partly cell-autonomous, partly non-cell-autonomous effects. However, protophloem-specific BRI1 expression essentially rescued all major bri1 brl1 brl3 root meristem phenotypes. Our data suggest that BR perception in the protophloem is sufficient to systemically convey BR action in the root meristem context.

Cell polarity is a key feature in the development of multicellular organisms. For instance, asymmetrically localized plasma-membrane-integral PIN-FORMED (PIN) proteins direct transcellular fluxes of the phytohormone auxin that govern plant development. Finetuned auxin flux is important for root protophloem sieve element differentiation and requires the interacting plasma-membrane-associated BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) proteins. We observed ''donut-like'' polar PIN localization in developing sieve elements that depends on complementary, ''muffin-like'' polar localization of BRX and PAX. Plasma membrane association and polarity of PAX, and indirectly BRX, largely depends on phosphatidylinositol-4,5-bisphosphate. Consistently, mutants in phosphatidylinositol-4phosphate 5-kinases (PIP5Ks) display protophloem differentiation defects similar to brx mutants. The same PIP5Ks are in complex with BRX and display ''muffin-like'' polar localization. Our data suggest that the BRX-PAX module recruits PIP5Ks to reinforce PAX polarity and thereby the polarity of all three proteins, which is required to maintain a local PIN minimum.