Mapping and engineering of auxin-induced plasma membrane dissociation in BRX family proteins.

Angiosperms have evolved the phloem for the long-distance transport of metabolites. The complex process of phloem development involves genes that only occur in vascular plant lineages. For example, in Arabidopsis thaliana, the BREVIS RADIX (BRX) gene is required for continuous root protophloem differentiation, together with PROTEIN KINASE ASSOCIATED WITH BRX (PAX). BRX and its BRX-LIKE (BRXL) homologs are composed of four highly conserved domains including the signature tandem BRX domains that are separated by variable spacers. Nevertheless, BRX family proteins have functionally diverged. For instance, BRXL2 can only partially replace BRX in the root protophloem. This divergence is reflected in physiologically relevant differences in protein behavior, such as auxin-induced plasma membrane dissociation of BRX, which is not observed for BRXL2. Here we dissected the differential functions of BRX family proteins using a set of amino acid substitutions and domain swaps. Our data suggest that the plasma membrane-associated tandem BRX domains are both necessary and sufficient to convey the biological outputs of BRX function and therefore constitute an important regulatory entity. Moreover, PAX target phosphosites in the linker between the two BRX domains mediate the auxin-induced plasma membrane dissociation. Engineering these sites into BRXL2 renders this modified protein auxin-responsive and thereby increases its biological activity in the root protophloem context.

Arabidopsis Protein Kinase D6PKL3 Is Involved in the Formation of Distinct Plasma Membrane Aperture Domains on the Pollen Surface.

Certain regions on the surfaces of developing pollen grains exhibit very limited deposition of pollen wall exine. These regions give rise to pollen apertures, which are highly diverse in their patterns and specific for individual species. Arabidopsis thaliana pollen develops three equidistant longitudinal apertures. The precision of aperture formation suggests that, to create them, pollen employs robust mechanisms that generate distinct cellular domains. To identify players involved in this mechanism, we screened natural Arabidopsis accessions and discovered one accession, Martuba, whose apertures form abnormally due to the disruption of the protein kinase D6PKL3. During pollen development, D6PKL3 accumulates at the three plasma membrane domains underlying future aperture sites. Both D6PKL3 localization and aperture formation require kinase activity. Proper D6PKL3 localization is also dependent on a polybasic motif for phosphoinositide interactions, and we identified two phosphoinositides that are specifically enriched at the future aperture sites. The other known aperture factor, INAPERTURATE POLLEN1, fails to aggregate at the aperture sites in d6pkl3 mutants, changes its localization when D6PKL3 is mislocalized, and, in turn, affects D6PKL3 localization. The discovery of aperture factors provides important insights into the mechanisms cells utilize to generate distinct membrane domains, develop cell polarity, and pattern their surfaces.

Dynamic PIN-FORMED auxin efflux carrier phosphorylation at the plasma membrane controls auxin efflux-dependent growth.

The directional distribution of the phytohormone auxin is essential for plant development. Directional auxin transport is mediated by the polarly distributed PIN-FORMED (PIN) auxin efflux carriers. We have previously shown that efficient PIN1-mediated auxin efflux requires activation through phosphorylation at the four serines S1-S4 in Arabidopsis thaliana The Brefeldin A (BFA)-sensitive D6 PROTEIN KINASE (D6PK) and the BFA-insensitive PINOID (PID) phosphorylate and activate PIN1 through phosphorylation at all four phosphosites. PID, but not D6PK, can also induce PIN1 polarity shifts, seemingly through phosphorylation at S1-S3. The differential effects of D6PK and PID on PIN1 polarity had so far been attributed to their differential phosphosite preference for the four PIN1 phosphosites. We have mapped PIN1 phosphorylation at S1-S4 in situ using phosphosite-specific antibodies. We detected phosphorylation at PIN1 phosphosites at the basal (rootward) as well as the apical (shootward) plasma membrane in different root cell types, in embryos, and shoot apical meristems. Thereby, PIN1 phosphorylation at all phosphosites generally followed the predominant PIN1 distribution but was not restricted to specific polar sides of the cells. PIN1 phosphorylation at the basal and apical plasma membrane was differentially sensitive to BFA treatments, suggesting the involvement of different protein kinases or trafficking mechanisms in PIN1 phosphorylation control. We conclude that phosphosite preferences are not sufficient to explain the differential effects of D6PK and PID on PIN1 polarity, and suggest that a more complex model is needed to explain the effects of PID.

SHADE AVOIDANCE 4 Is Required for Proper Auxin Distribution in the Hypocotyl.

The phytohormone auxin is involved in virtually every aspect of plant growth and development. Through polar auxin transport, auxin gradients can be established, which then direct plant differentiation and growth. Shade avoidance responses are well-known processes that require polar auxin transport. In this study, we have identified a mutant, shade avoidance 4 (sav4), defective in shade-induced hypocotyl elongation and basipetal auxin transport. SAV4 encodes an unknown protein with armadillo repeat- and tetratricopeptide repeat-like domains known to provide protein-protein interaction surfaces. C terminally yellow fluorescent protein-tagged SAV4 localizes to both the plasma membrane and the nucleus. Membrane-localized SAV4 displays a polar association with the shootward plasma membrane domain in hypocotyl and root cells, which appears to be necessary for its function in hypocotyl elongation. Cotransfection of SAV4 and ATP-binding cassette B1 (ABCB1) auxin transporter in tobacco (Nicotiana benthamiana) revealed that SAV4 blocks ABCB1-mediated auxin efflux. We thus propose that polarly localized SAV4 acts to inhibit ABCB-mediated auxin efflux toward shoots and facilitates the establishment of proper auxin gradients.

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases.

The Arabidopsis NPF3 protein is a GA transporter.

Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo. NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA-ABA interaction may occur at the level of transport.

Arabidopsis D6PK is a lipid domain-dependent mediator of root epidermal planar polarity.

Development of diverse multicellular organisms relies on coordination of single-cell polarities within the plane of the tissue layer (planar polarity). Cell polarity often involves plasma membrane heterogeneity generated by accumulation of specific lipids and proteins into membrane subdomains. Coordinated hair positioning along Arabidopsis root epidermal cells provides a planar polarity model in plants, but knowledge about the functions of proteo-lipid domains in planar polarity signalling remains limited. Here we show that Rho-of-plant (ROP) 2 and 6, phosphatidylinositol-4-phosphate 5-kinase 3 (PIP5K3), DYNAMIN-RELATED PROTEIN (DRP) 1A and DRP2B accumulate in a sterol-enriched, polar membrane domain during root hair initiation. DRP1A, DRP2B, PIP5K3 and sterols are required for planar polarity and the AGCVIII kinase D6 PROTEIN KINASE (D6PK) is a modulator of this process. D6PK undergoes phosphatidylinositol-4,5-bisphosphate- and sterol-dependent basal-to-planar polarity switching into the polar, lipid-enriched domain just before hair formation, unravelling lipid-dependent D6PK localization during late planar polarity signalling.

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

D6 PROTEIN KINASE activates auxin transport-dependent growth and PIN-FORMED phosphorylation at the plasma membrane.

The directed cell-to-cell transport of the phytohormone auxin by efflux and influx transporters is essential for proper plant growth and development. Like auxin efflux facilitators of the PIN-FORMED (PIN) family, D6 PROTEIN KINASE (D6PK) from Arabidopsis thaliana localizes to the basal plasma membrane of many cells, and evidence exists that D6PK may directly phosphorylate PINs. We find that D6PK is a membrane-bound protein that is associated with either the basal domain of the plasma membrane or endomembranes. Inhibition of the trafficking regulator GNOM leads to a rapid internalization of D6PK to endomembranes. Interestingly, the dissociation of D6PK from the plasma membrane is also promoted by auxin. Surprisingly, we find that auxin transport-dependent tropic responses are critically and reversibly controlled by D6PK and D6PK-dependent PIN phosphorylation at the plasma membrane. We conclude that D6PK abundance at the plasma membrane and likely D6PK-dependent PIN phosphorylation are prerequisites for PIN-mediated auxin transport.

Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport.

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.

Convergence of auxin and gibberellin signaling on the regulation of the GATA transcription factors GNC and GNL in Arabidopsis thaliana.

Plant growth is regulated by a complex network of signaling events. Points of convergence for the signaling cross-talk between the phytohormones auxin and gibberellin (GA), which partly control overlapping processes during plant development, are largely unknown. At the cellular level, auxin responses are controlled by members of the AUXIN RESPONSE FACTOR (ARF) family of transcription factors as well as AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE (AUX/IAA) proteins that repress the activity of at least a subset of ARFs. Here, we show that the two paralogous GATA transcription factors GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED (GNC) and GNC-LIKE (GNL)/CYTOKININ-RESPONSIVE GATA FACTOR1 (CGA1) are direct and critical transcription targets downstream from ARF2 in the control of greening, flowering time, and senescence. Mutants deficient in the synthesis or signaling of the phytohormone GA are also impaired in greening, flowering, and senescence, and interestingly, GNC and GNL were previously identified as important transcription targets of the GA signaling pathway. In line with a critical regulatory role for GNC and GNL downstream from both auxin and GA signaling, we show here that the constitutive activation of GA signaling is sufficient to suppress arf2 mutant phenotypes through repression of GNC and GNL. In addition, we show that GA promotes ARF2 protein abundance through a translation-dependent mechanism that could serve to override the autoinhibitory negative feedback regulation of ARF2 on its own transcription and thereby further promote GA signaling.

D6PK AGCVIII kinases are required for auxin transport and phototropic hypocotyl bending in Arabidopsis.

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.

WAG2 represses apical hook opening downstream from gibberellin and PHYTOCHROME INTERACTING FACTOR 5.

When penetrating the soil during germination, dicotyledonous plants protect their shoot apical meristem through the formation of an apical hook. Apical hook formation is a dynamic process that can be subdivided into hook formation, maintenance and opening. It has previously been established that these processes require the transport and signaling of the phytohormone auxin, as well as the biosynthesis and signaling of the phytohormones ethylene and gibberellin (GA). Here, we identify a molecular mechanism for an auxin-GA crosstalk by demonstrating that the auxin transport-regulatory protein kinase WAG2 is a crucial transcription target during apical hook opening downstream from GA signaling. We further show that WAG2 is directly activated by PHYTOCHROME INTERACTING FACTOR 5 (PIF5), a light-labile interactor of the DELLA repressors of the GA pathway. We find that wag2 mutants are impaired in the repression of apical hook opening in dark-grown seedlings and that this phenotype correlates with GA-regulated WAG2 expression in the concave (inner) side of the apical hook. Furthermore, wag2 mutants are also impaired in the maintenance or formation of a local auxin maximum at the site of WAG2 expression in the hook. WAG2 is a regulator of PIN auxin efflux facilitators and, in line with previous data, we show that this kinase can phosphorylate the central intracellular loop of all PIN-FORMED (PIN) proteins regulating apical hook opening. We therefore propose that apical hook opening is controlled by the differential GA-regulated accumulation of WAG2 and subsequent local changes in PIN-mediated auxin transport.

Gibberellin regulates PIN-FORMED abundance and is required for auxin transport-dependent growth and development in Arabidopsis thaliana.

Plants integrate different regulatory signals to control their growth and development. Although a number of physiological observations suggest that there is crosstalk between the phytohormone gibberellin (GA) and auxin, as well as with auxin transport, the molecular basis for this hormonal crosstalk remains largely unexplained. Here, we show that auxin transport is reduced in the inflorescences of Arabidopsis thaliana mutants deficient in GA biosynthesis and signaling. We further show that this reduced auxin transport correlates with a reduction in the abundance of PIN-FORMED (PIN) auxin efflux facilitators in GA-deficient plants and that PIN protein levels recover to wild-type levels following GA treatment. We also demonstrate that the regulation of PIN protein levels cannot be explained by a transcriptional regulation of the PIN genes but that GA deficiency promotes, at least in the case of PIN2, the targeting of PIN proteins for vacuolar degradation. In genetic studies, we reveal that the reduced auxin transport of GA mutants correlates with an impairment in two PIN-dependent growth processes, namely, cotyledon differentiation and root gravitropic responses. Our study thus presents evidence for a role of GA in these growth responses and for a GA-dependent modulation of PIN turnover that may be causative for these differential growth responses.

The polarly localized D6 PROTEIN KINASE is required for efficient auxin transport in Arabidopsis thaliana.

The phytohormone auxin is a major determinant of plant growth and differentiation. Directional auxin transport and auxin responses are required for proper embryogenesis, organ formation, vascular development, and tropisms. Members of several protein families, including the PIN auxin efflux facilitators, have been implicated in auxin transport; however, the regulation of auxin transport by signaling proteins remains largely unexplored. We have studied a family of four highly homologous AGC protein kinases, which we designated the D6 protein kinases (D6PKs). We found that d6pk mutants have defects in lateral root initiation, root gravitropism, and shoot differentiation in axillary shoots, and that these phenotypes correlate with a reduction in auxin transport. Interestingly, D6PK localizes to the basal (lower) membrane of Arabidopsis root cells, where it colocalizes with PIN1, PIN2 and PIN4. D6PK and PIN1 interact genetically, and D6PK phosphorylates PIN proteins in vitro and in vivo. Taken together, our data show that D6PK is required for efficient auxin transport and suggest that PIN proteins are D6PK phosphorylation targets.

Examining protein stability and its relevance for plant growth and development.

Eukaryotes control many aspects of growth and development such as cell cycle progression and gene expression through the selective degradation of regulatory proteins by way of the 26S proteasome. Generally, proteasomal degradation requires the poly-ubiquitylation of degradation targets by E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. Specificity is brought to the process by E3 ubiquitin ligases, which engage in direct interactions with the degradation substrate to bring it into the proximity of the E2 enzyme. The abundance of genes encoding E3 ligase subunits in plant genomes invites the hypothesis that protein degradation plays an important role in the control of many plant growth processes, and it is therefore not surprising that proteasomal degradation has already been implicated in several important response pathways. However, most of the genes with a predicted role in the ubiquitin-proteasome pathway still remain to be characterized and the identity of their degradation substrates needs to be revealed. In this chapter, we give an overview of the ubiquitin-proteasome system and the pathway proteins that have been examined in Arabidopsis to date. We review the methods required to identify and characterize the proteins that play a role in protein degradation or that are the target for proteasomal degradation.

The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis.

Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.

Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression.

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber-specific and sucrose-inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B-box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA-binding protein designated Storekeeper (STK) that specifically recognises the B-box motif in vitro. Gel shift experiments with an STK-specific antibody suggest that STK is the B-box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B-box motif.