Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5 / LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate deprived conditions are TMO5 and cytokinin dependent. In conclusion, cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

SummaryThe root endodermis surrounds the central vasculature as a protective sheath, analogous to an animal polarised epithelium, and restricts diffusion by its ring-shaped Casparian strips (CS)1. Following a lag phase, individual endodermal cells suberise in an apparently random fashion, leading to a “patchy” suberisation that eventually gives rise to a zone of continuous deposition2. Casparian strips and suberin lamellae affect paracellular and transcellular transport, respectively. Interestingly, most angiosperms maintain some isolated cells in an unsuberised state3. These so-called “passage cells” are speculated to allow uptake across an otherwise impermeable endodermal barrier. Here, we demonstrate that these passage cells are late emanations of a meristematic patterning process that reads-out the underlying non-radial symmetry of the vasculature. This process is mediated by non-cell autonomous cytokinin repression in the root meristem, leading to distinct phloem and xylem pole-associated endodermal cells. The latter can resist ABA-dependent suberisation and give rise to passage cell formation. Our data further demonstrate that during meristematic patterning, xylem pole-associated endodermal cells can dynamically adapt passage cell numbers in response to nutrient status and that passage cells express transporters and locally impact their expression in adjacent cortical cells.

Summary To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [ 1 , 2 ]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [ 3 , 4 , 5 , 6 , 7 ]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 ( LOG3 ) and LOG4 [ 8 , 9 ] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [ 3 , 6 ]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.

In the past decade, tremendous advance has been made in understanding the biosynthesis, perception and signaling pathways of the plant hormone cytokinin. It also became clear that interfering with any of these steps greatly impacts all stages of growth and development. This has spurted renewed effort recently to understand how cytokinin signaling affects developmental processes. As a result, new insights on the role of cytokinin signaling and the downstream targets during e.g. shoot apical meristem, flower, female gametophyte, stomata and vascular development are being unraveled. In this review, we aim to give a comprehensive overview of recent findings on how cytokinin influences growth and development in plants and highlight areas for future research. 2 Cytokinins: from signaling to development Cytokinins, a class of phytohormones with diverse molecular structures [1], were first discovered in the late 1950's while looking for the molecule responsible for the growthpromoting activity of autoclaved herring sperm DNA [2]. Since then, the signaling pathway of cytokinins has been intensely studied. In our current understanding, cytokinins are synthesized by the ISOPENTENYL TRANSFERASE (IPT) and LONELY GUY (LOG) enzymes, whereas cytokinin conjugation act mainly through CYTOKININ OXIDASE (CKX) enzymes.Cytokinins are next perceived by the ARABIDOPSIS HISTIDINE KINASE2-4 (AHK2-4) receptors which initiates a phosphorylation signaling cascade. This phospho-relay starts with auto-phosphorylation of the receptors and will ultimately lead to phosphorylation and activation of B-type ARABIDOPSIS REPSONSE REGULATORS (ARRs) through the ARABIDOPSIS HISTIDINE PROTEINS (AHPs). The active ARRs will then induce cytokinin responsive genes, such as those encoding the cytokinin signaling repressors A-type ARRs or CYTOKININ RESPONSE FACTORS (CRFs). For detailed information on the biosynthesis, transport, perception and signaling cascades of cytokinins, we refer to several excellent recent reviews [1,[3][4][5]. With the now well studied aspect of cytokinin signaling, the next step is to understand how these processes impinge on plant growth and development. Over the past few years, an increasing number of studies highlight the importance of cytokinins during many developmental processes. In this review, we discuss how these insights aid in understanding how cytokinins controls development at a molecular level. Shoot developmentAlready in the early years of cytokinin research, it was clear that these molecules have a big influence on plant growth, especially together with auxin [6,7]. Plants grown on high levels of auxin and cytokinin massively proliferate and dedifferentiate leading to callus (see Glossary).Growing callus on high cytokinin levels induces shoot regeneration [6]. Based on these classical experiments, cytokinins are considered the main class of hormones involved in shoot development. Cytokinin controls shoot cell fateCytokinin signaling was shown to be vital in the context of shoot apical meristem (SAM; see Glossary) develo...

43Signaling from chloroplasts and mitochondria, both dependent on reactive oxygen 44 species (ROS), merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 45 (RCD1). ROS produced in the chloroplasts affect the abundance, thiol redox state and 46 oligomerization of RCD1. RCD1 directly interacts in vivo with ANAC013 and ANAC017 47 transcription factors, which are the mediators of the ROS-related mitochondrial complex 48 III retrograde signa and suppresses activity of ANAC013 and ANAC017. Inactivation of 49 RCD1 leads to increased expression of ANAC013 and ANAC017-regulated genes 50 belonging to the mitochondrial dysfunction stimulon (MDS), including genes for 51 mitochondrial alternative oxidases (AOXs). Accumulating AOXs and other MDS gene 52 products alter electron transfer pathways in the chloroplasts, leading to diminished 53 production of chloroplastic ROS and increased protection of photosynthetic apparatus 54 from ROS damage. RCD1-dependent regulation affects chloroplastic and mitochondrial 55 retrograde signaling including chloroplast signaling by 3'-phosphoadenosine 5'-56 phosphate (PAP). Sensitivity of RCD1 to organellar ROS provides feedback control of 57 nuclear gene expression. 58 157 Nishiyama et al., 2011).To reveal the significance of RCD1 in responses to PSI-produced 158 chloroplastic ROS, rosettes of Arabidopsis were pre-treated overnight in darkness with 159 MV and exposed to light. After the dark period, the plants displayed unchanged PSII 160 photochemical yield (Fv/Fm). Subsequent exposure to three hours of light resulted in 161 decrease of Fv/Fm in wild type (Col-0) (Fig. 1A), but not in the rcd1 mutant. Moreover, 162 tolerance of rcd1 was evident under various concentrations of MV (Fig. 1B). The 163 superoxide anion is an unstable compound that is enzymatically reduced to the more 164 long-lived H2O2. Chloroplastic production of H2O2 in the presence of MV in the light was 165 assessed by staining plants with 3,3′-diaminobenzidine (DAB). After pre-treatment with 166 MV, higher H2O2 accumulation was evident in both Col-0 and rcd1 (Fig. S1A). Subsequent 167 incubation of these plants under light led to a time-dependent increase in the H2O2 168accumulation in Col-0, but not in rcd1. 169 In several MV-tolerant mutants the resistance is based on restricted access of MV to 170 chloroplasts (Hawkes 2014). However, in rcd1 pretreatment with MV led to initial increase 171 in H2O2 production similar to that in the wild type ( Fig. S1A), suggesting that resistance 172 of rcd1 was not due to restricted delivery of MV to PSI. In order to test this directly, 173 oxidation of PSI was assessed by in vivo spectroscopy using DUAL-PAM. Leaves were 174 adapted to far-red light, which is more efficiently used by PSI than PSII. Under these 175 conditions PSI is producing electrons at a faster rate than it is supplied by electrons 176 coming from PSII, and hence the PSI reaction center P700 becomes oxidized. Then a 177 flash of orange light was provided that is efficiently absorbed by PSII. Electrons generated...

Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. Primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and division orientation by modulating cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetic approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity to ensure optimal cell proliferation rates during root vascular development.

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here, we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.