The plant hormone auxin plays a central role in the growth and development of plants. Auxin acts in a concentration dependent manner and polar cell-to-cell transport of this hormone determines its... Show moreThe plant hormone auxin plays a central role in the growth and development of plants. Auxin acts in a concentration dependent manner and polar cell-to-cell transport of this hormone determines its distribution in the tissues of plants. This polar auxin transport is mediated by several families of auxin transporters, including the PIN FORMED (PIN) auxin efflux carriers that determine the direction of transport by their polar localization at the plasma membrane. The plasma membrane abundance and polarity of PINs (and thereby of polar auxin transport) is regulated by their post-translational modification, of which phosphorylation is best studied. PIN proteins in Arabidopsis consist of two transmembrane domains separated by a ‘long’ (PIN1,2,3,4,6,7) or by a ‘short’ (PIN5,8) hydrophilic loop. Phosphorylation of ‘long’ PINs in their central hydrophilic loop by the AGC3 kinases PINOID, WAG1 and WAG2 triggers shootward (apical) or outer-lateral polarity. The AGC1- type D6 kinases also phosphorylate the PIN hydrophilic loop, however this was reported to result in auxin transport activation rather than subcellular polarity establishment. Here we investigate the conservation and phylogeny of AGC3 and D6 kinases and their phosphorylation sites in PINs from the earliest land plants to flowering plants. In early land plants, many of the same proteins and conserved motifs can be found, however it is in monocots and dicots that conservation of PIN phosphorylation by AGC3 and D6 kinases is strongest. The expansion and increased conservation of AGC3 and D6 kinases and PINs in later lineages such as monocot and dicot flowering plants, is in line with their important role in the formation of reproductive organs and in the tropic growth responses that allow plants to adapt to changes in their environment. Show less
What makes plant shoots grow towards the light, and plant roots grow down into the soil? This was a question that Charles Darwin asked himself, and his experiments more than a century ago to find... Show moreWhat makes plant shoots grow towards the light, and plant roots grow down into the soil? This was a question that Charles Darwin asked himself, and his experiments more than a century ago to find the answer laid the basis for the identification of the growth hormone auxin. Auxin, or indole-3-acetic acid (IAA), directs plant growth and development through its polar cell-to-cell transport-driven asymmetric distribution. Cellular IAA concentrations determine cell division, -elongation and -differentiation by facilitating the degradation of the Aux/IAA repressor proteins and thus inducing gene expression. The presumed pathway for the programmed degradation of proteins involves the attachment of a protein called ubiquitin, leading to recognition and destruction by a molecular complex called the proteasome. Here we investigated the role of protein ubiquitination and degradation in auxin action. First, we provided evidence for the longstanding paradigm that Aux/IAA proteins are ubiquitinated prior to their proteasomal degradation. At the same time we showed that ubiquitin labeling is not necessarily required for proteasomal degradation of plant proteins. Moreover, we showed that a regulator of auxin transport polarity is also involved in fine tuning auxin responses through the ubiquitin pathway. Our results place protein ubiquitination at a central position in auxin biology and thus in the movement of plants. Show less
Plant architecture is determined by tightly regulated developmental processes that largely depend on the action of the plant hormone auxin. A major determinant in auxin action, besides its... Show morePlant architecture is determined by tightly regulated developmental processes that largely depend on the action of the plant hormone auxin. A major determinant in auxin action, besides its signaling pathway, is its polar cell-to-cell transport (PAT) throughout the plant. The direction on this transport depends on the localization of the auxin efflux carriers, the PIN proteins. The PINOID (PID) serine/threonine protein kinase is a key regulator of the subcellular localization of the PINs, which are direct phosphorylation targets of the kinase. This thesis describes the functional analysis of three direct interacting partners of PID, two calcium-binding proteins, TOUCH3 (TCH3) and PID BINDING PROTEIN1 (PBP1), and a BTB and TAZ domain (BT) protein. Several studies have already indicated that calcium signaling is induced by auxin application and is necessary for auxin transport. With the isolation of the two calcium-binding proteins TCH3 and PBP1 as interactors of PID, a molecular link between auxin transport and calcium signaling was identified. In this thesis, we show that calcium is involved in the regulation of both the kinase activity and the subcellular localization of PID. In complement to calcium, BTB scaffold proteins are part of the PID protein complex. A detailed analysis of BT protein family in Arabidopsis indicate a functional redundancy among the five members of this family and their requirement for the female gametophyte development. Moreover the BT proteins are required scaffold components in the PID signaling pathway. The functional analyses of the PBPs described in this thesis uncover a new mechanism of protein kinase activity regulation via calcium signaling, and present novel roles for the BT proteins, not only in PID signaling, but also more in general in plant development. Show less
