Remko Offringa

Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical–basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical–basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical–basal axis formation of the embryo, and thus determine the axiality of the adult plant.

Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux

In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.

Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth

The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.

Cytokinins Act Directly on Lateral Root Founder Cells to Inhibit Root Initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.

Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.

We determined whether T‐DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T‐DNA, one carrying a 5′ deleted NPTII gene and the other a NPTII gene with a 3′ deletion. A restored NPTII gene was found in 1‐4% of the protoplasts that had been cotransformed with both T‐DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T‐DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T‐DNA insertion carrying a defective NPTII gene with a 3′ deletion was transformed via Agrobacterium with a T‐DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T‐DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.

A regulated auxin minimum is required for seed dispersal in Arabidopsis.

Local hormone maxima are essential for the development of multicellular structures and organs. For example, steroid hormones accumulate in specific cell types of the animal fetus to induce sexual differentiation and concentration peaks of the plant hormone auxin direct organ initiation and mediate tissue patterning. Here we provide an example of a regulated local hormone minimum required during organogenesis. Our results demonstrate that formation of a local auxin minimum is necessary for specification of the valve margin separation layer where Arabidopsis fruit opening takes place. Consequently, ectopic production of auxin, specifically in valve margin cells, leads to a complete loss of proper cell fate determination. The valve margin identity factor INDEHISCENT (IND) is responsible for forming the auxin minimum by coordinating auxin efflux in separation-layer cells. We propose that the simplicity of formation and maintenance make local hormone minima particularly well suited to specify a small number of cells such as the stripes at the valve margins.

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

This work identifies the Ser residues located in three evolutionarily conserved TPRXS(N/S) motifs within the PIN1 auxin efflux carrier hydrophilic loop as substrates of the PINOID kinase. It shows that reversible phosphorylation of these Ser residues by PINOID and possibly other kinases is necessary and sufficient for proper PIN1 polar localization, auxin distribution, and regulated plant development. Polar cell-to-cell transport of auxin by plasma membrane–localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of auxin flow is controlled by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identified three evolutionarily conserved TPRXS(N/S) motifs within the PIN1HL and proved that the central Ser residues were phosphorylated by the PINOID (PID) kinase. Loss-of-phosphorylation PIN1:green fluorescent protein (GFP) (Ser to Ala) induced inflorescence defects, correlating with their basal localization in the shoot apex, and induced internalization of PIN1:GFP during embryogenesis, leading to strong embryo defects. Conversely, phosphomimic PIN1:GFP (Ser to Glu) showed apical localization in the shoot apex but did not rescue pin1 inflorescence defects. Both loss-of-phosphorylation and phosphomimic PIN1:GFP proteins were insensitive to PID overexpression. The basal localization of loss-of-phosphorylation PIN1:GFP increased auxin accumulation in the root tips, partially rescuing PID overexpression-induced root collapse. Collectively, our data indicate that reversible phosphorylation of the conserved Ser residues in the PIN1HL by PID (and possibly by other AGC kinases) is required and sufficient for proper PIN1 localization and is thus essential for generating the differential auxin distribution that directs plant development.

Maintenance of Embryonic Auxin Distribution for Apical-Basal Patterning by PIN-FORMED–Dependent Auxin Transport in Arabidopsis

Molecular mechanisms of pattern formation in the plant embryo are not well understood. Recent molecular and cellular studies, in conjunction with earlier microsurgical, physiological, and genetic work, are now starting to define the outlines of a model where gradients of the signaling molecule auxin play a central role in embryo patterning. It is relatively clear how these gradients are established and interpreted, but how they are maintained is still unresolved. Here, we have studied the contributions of auxin biosynthesis, conjugation, and transport pathways to the maintenance of embryonic auxin gradients. Auxin homeostasis in the embryo was manipulated by region-specific conditional expression of indoleacetic acid-tryptophan monooxygenase or indoleacetic acid-lysine synthetase, bacterial enzymes for auxin biosynthesis or conjugation. Neither manipulation of auxin biosynthesis nor of auxin conjugation interfered with auxin gradients and patterning in the embryo. This result suggests a compensatory mechanism for buffering auxin gradients in the embryo. Chemical and genetic inhibition revealed that auxin transport activity, in particular that of the PIN-FORMED1 (PIN1) and PIN4 proteins, is a major factor in the maintenance of these gradients.

PIN Auxin Efflux Carrier Polarity Is Regulated by PINOID Kinase-Mediated Recruitment into GNOM-Independent Trafficking in Arabidopsis

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development.

PINOID-Mediated Signaling Involves Calcium-Binding Proteins

The plant hormone auxin is a central regulator of plant development. In Arabidopsis, the PINOID (PID) protein serine/threonine kinase is a key component in the signaling of this phytohormone. To further investigate the biological function of PID, we performed a screen for PID-interacting proteins using the yeast two-hybrid system. Here, we show that PID interacts with two calcium-binding proteins: TOUCH3 (TCH3), a calmodulin-related protein, and PID-BINDING PROTEIN 1 (PBP1), a previously uncharacterized protein containing putative EF-hand calcium-binding motifs. The interaction between PID and the calcium-binding proteins is significant because it is calcium dependent and requires an intact PID protein. Furthermore, the expression of all three genes (PID, TCH3, and PBP1) is up-regulated by auxin. TCH3 and PBP1 are not targets for phosphorylation by PID, suggesting that these proteins act upstream of PID. PBP1 was found to stimulate the autophosphorylation activity of PID, and calcium influx and calmodulin inhibitors where found to enhance the activity of PID in vivo. Our results indicate that TCH3 and PBP1 interact with the PID protein kinase and regulate the activity of this protein in response to changes in calcium levels. This work provides the first molecular evidence for the involvement of calcium in auxin-regulated plant development.