High levels of auxin signalling define the stem-cell organizer of the vascular cambium.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium1. Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown2. Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium3. Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.

Mobile PEAR transcription factors integrate positional cues to prime cambial growth.

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4-the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

PLETHORA gradient formation mechanism separates auxin responses.

During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip. In addition, auxin is also pivotal for tropic responses. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation.

Auxin influx carriers control vascular patterning and xylem differentiation in Arabidopsis thaliana.

Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants.

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.

Parsimonious Model of Vascular Patterning Links Transverse Hormone Fluxes to Lateral Root Initiation: Auxin Leads the Way, while Cytokinin Levels Out.

An auxin maximum is positioned along the xylem axis of the Arabidopsis root tip. The pattern depends on mutual feedback between auxin and cytokinins mediated by the PIN class of auxin efflux transporters and AHP6, an inhibitor of cytokinin signalling. This interaction has been proposed to regulate the size and the position of the hormones' respective signalling domains and specify distinct boundaries between them. To understand the dynamics of this regulatory network, we implemented a parsimonious computational model of auxin transport that considers hormonal regulation of the auxin transporters within a spatial context, explicitly taking into account cell shape and polarity and the presence of cell walls. Our analysis reveals that an informative spatial pattern in cytokinin levels generated by diffusion is a theoretically unlikely scenario. Furthermore, our model shows that such a pattern is not required for correct and robust auxin patterning. Instead, auxin-dependent modifications of cytokinin response, rather than variations in cytokinin levels, allow for the necessary feedbacks, which can amplify and stabilise the auxin maximum. Our simulations demonstrate the importance of hormonal regulation of auxin efflux for pattern robustness. While involvement of the PIN proteins in vascular patterning is well established, we predict and experimentally verify a role of AUX1 and LAX1/2 auxin influx transporters in this process. Furthermore, we show that polar localisation of PIN1 generates an auxin flux circuit that not only stabilises the accumulation of auxin within the xylem axis, but also provides a mechanism for auxin to accumulate specifically in the xylem-pole pericycle cells, an important early step in lateral root initiation. The model also revealed that pericycle cells on opposite xylem poles compete for auxin accumulation, consistent with the observation that lateral roots are not initiated opposite to each other.

The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress.

Receptor-like kinases are important regulators of many different processes in plants. Despite their large number only a few have been functionally characterized. One of the largest subgroups of receptor-like kinases in Arabidopsis is the cysteine-rich receptor like kinases (CRKs). High sequence similarity among the CRKs has been suggested as major cause for functional redundancy. The genomic localization of CRK genes in back-to-back repeats has made their characterization through mutant analysis unpractical. Expression profiling has linked the CRKs with reactive oxygen species, important signaling molecules in plants. Here we have investigated the role of two CRKs, CRK6 and CRK7, and analyzed their role in extracellular ROS signaling. CRK6 and CRK7 are active protein kinases with differential preference for divalent cations. Our results suggest that CRK7 is involved in mediating the responses to extracellular but not chloroplastic ROS production.

Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium.

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.

Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes.

During primary growth, plant tissues increase their length, and as these tissues mature, they initiate secondary growth to increase thickness.1 It is not known what activates this transition to secondary growth. Cytokinins are key plant hormones regulating vascular development during both primary and secondary growth. During primary growth of Arabidopsis roots, cytokinins promote procambial cell proliferation2,3 and vascular patterning together with the hormone auxin.4-7 In the absence of cytokinins, secondary growth fails to initiate.8 Enhanced cytokinin levels, in turn, promote secondary growth.8,9 Despite the importance of cytokinins, little is known about the downstream signaling events in this process. Here, we show that cytokinins and a few downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors are rate-limiting components in activating and further promoting secondary growth in Arabidopsis roots. Cytokinins directly activate transcription of two homologous LBD genes, LBD3 and LBD4. Two other homologous LBDs, LBD1 and LBD11, are induced only after prolonged cytokinin treatment. Our genetic studies revealed a two-stage mechanism downstream of cytokinin signaling: while LBD3 and LBD4 regulate activation of secondary growth, LBD1, LBD3, LBD4, and LBD11 together promote further radial growth and maintenance of cambial stem cells. LBD overexpression promoted rapid cell growth followed by accelerated cell divisions, thus leading to enhanced secondary growth. Finally, we show that LBDs rapidly inhibit cytokinin signaling. Together, our data suggest that the cambium-promoting LBDs negatively feed back into cytokinin signaling to keep root secondary growth in balance.

Report on the methodology applied by EFSA to provide a quantitative assessment of pest-related criteria required to rank candidate priority pests as defined by Regulation (EU) 2016/2031.

In agreement with Article 6(2) of the Regulation (EU) 2016/2031 on protective measures against pests of plants, the European Commission has been tasked by the Council and European Parliament to establish a list of Union quarantine pests which qualify as priority pests. The prioritisation is based on the severity of the economic, social and environmental impact that these pests can cause in the Union territory. The Commission's Joint Research Centre (JRC) is in charge of developing a methodology based on a multi-criteria decision analysis and composite indicators. In this context, EFSA has provided technical and scientific data related to these pests, in particular: (i) the potential host range and distribution of each of these pests in the Union territory at the level of NUTS2 regions; (ii) parameters quantifying the potential consequences of these pests, e.g. crop losses in terms of yield and quality, rate of spread and time to detection. Expert knowledge elicitation methodology has been applied by EFSA in order to provide those parameters in a consistent and transparent manner.