Cytokinins regulate spatially specific ethylene production to control root growth in Arabidopsis

Two principal growth regulators,cytokinins and ethylene,are known to interact in the regulation of plant growth.However,information about the underlying molecular mechanism and positional specificity of cytokinin/ethylene crosstalk in the control of root growth is scarce.We have identified the spatial specificity of cytokinin-regulated root elongation and root apical meristem(RAM)size,both of which we demonstrate to be dependent on ethylene biosynthesis.Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE(IPT)in proximal and peripheral tissues leads to both root and RAM shortening.By contrast,IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to that of mock-treated controls.We show that cytokinins regulate two steps specific to ethylene biosynthesis:production of the ethylene precursor 1-aminocyclopropane-1-carboxylate(ACC)by ACC SYNTHASEs(ACSs)and its conversion to ethylene by ACC OXIDASEs(ACOs).We describe cytokinin-and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes.Using direct ethylene measurements,we identify ACO2,ACO3,and ACO4 as being responsible for ethylene biosynthesis and ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis.Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2(ARR2),a member of the multistep phosphorelay cascade,and the C-terminal portion of ETHYLENE INSENSITIVE 2(EIN2-C),a key regulator of canonical ethylene signaling,is involved in the cytokinin-induced,ethylene-mediated control of ACO4.We propose tight cooperation between cytokinin and ethylene signaling in the spatially specific regulation of ethylene biosynthesis as a key aspect of the hormonal control of root growth.

High temperature increases centromeremediated genome elimination frequency and enhances haploid induction in Arabidopsis

Double haploid production is the most effective way to create true-breeding lines in a single generation.In Arabidopsis,haploid induction via mutation of the centromere-specific histone H3(cenH3)has been shown when the mutant is outcrossed to the wild-type,and the wild-type genome remains in the haploid progeny.However,factors that affect haploid induction are still poorly understood.Here,we report that a mutant of the cenH3 assembly factor Kinetochore Null2(KNL2)can be used as a haploid inducer when pollinated by the wild-type.We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold.We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines,suggesting that haploidinducing lines in crops can be identified in a naturally occurring or chemically induced mutant population,avoiding the generic modification(GM)approach at any stage.Furthermore,a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress,even though it did not induce haploids under standard conditions.Thus,we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

ETR1 Integrates Response to Ethylene and Cytokinins into a Single Multistep Phosphorelay Pathway to Control Root Growth

Cytokinins and ethylene control plant development via sensors from the histidine kinase(HK)family.However,downstream signaling pathways for the key phytohormones are distinct.Here we report that not only cytokinin but also ethylene is able to control root apical meristem(RAM)size through activation of the multistep phosphorelay(MSP)pathway.We found that both cytokinin and ethylene-dependent RAM shortening requires ethylene binding to ETR1 and the HK activity of ETR1.The receiver domain of ETR1 interacts with MSP signaling intermediates acting downstream of cytokinin receptors,further substantiating the role of ETR1 in MSP signaling.We revealed that both cytokinin and ethylene induce the MSP in similar and distinct cell types with ETR1-mediated ethylene signaling controlling MSP output specifically in the root transition zone.We identified members of the MSP pathway specific and common to both hormones and showed that ETR1-regulated ARR3 controls RAM size.ETR1-mediated MSP spatially differs from canonical CTR1/EIN2/EIN3 ethylene signaling and is independent of EIN2,indicating that both pathways can be spatially and functionally separated.Furthermore,we demonstrated that canonical ethylene signaling controls MSP responsiveness to cytokinin specifically in the root transition zone,presumably via regulation of ARR10,one of the positive regulators of MSP signaling in Arabidopsis.

Enquiry into the Topology of Plasma Membrane- Localized PIN Auxin Transport Components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regard- less of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immuno- localization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane （TM） bundles of five m-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.