The plant hormone auxin regulates plant growth and development through polar cell-to-cell transport-generated maxima and minima. PIN FORMED (PIN) auxin efflux carriers determine the direction of... Show moreThe plant hormone auxin regulates plant growth and development through polar cell-to-cell transport-generated maxima and minima. PIN FORMED (PIN) auxin efflux carriers determine the direction of this auxin flow through their asymmetric placement on the plasma membrane (PM). In Arabidopsis thaliana (Arabidopsis), the PM-associated protein kinase PINOID (PID) regulates PIN localisation and thereby auxin transport polarity by phosphorylating these carriers in their central cytoplasmic loop. PID in turn interacts with the calmodulin-like protein TOUCH3 (TCH3) in response to elevated cytosolic calcium, which dissociates PID from the PM and inhibits its kinase activity. In this thesis, we show that PID also interacts with 10 other CALMODULIN/CALMODULIN-LIKE proteins, which together with TCH3 form a confined clade in the CaM/CML family. The CaM/CML binding domain in PID was found to map to an amphipathic alpha-helix inserted in the catalytic kinase core. Disruption of this alpha-helix did not affect PID kinase activity, but impaired both its PM association and the interaction with the CaM/CMLs, making the kinase “untouchable”. Expression of “untouchable” PID versions in the pid mutant background revealed that proper calcium-CaM/CML-PID signalling is essential to maintain the robust spiral phyllotaxis that is typical for the Arabidopsis inflorescence. Show less
Polar cell-to-cell transport of plant hormone auxin mediated by plasma membrane (PM)-localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information... Show morePolar cell-to-cell transport of plant hormone auxin mediated by plasma membrane (PM)-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 directed by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identified three evolutionarily conserved TPRXS(N/S) motifs within the PINHL, and proved that the central serine residues located in three motifs were phosphorylated by the PINOID (PID) serine/threonine kinase protein and its closely related AGC3 kinases WAG1 and WAG2. Loss-of-phosphorylation PIN1 or PIN2 protein (serine to alanine mutation) induced auxin-related plant defects in inflorescence development or root gravity-response, respectively, correlating with their apical-to-basal polarity changing in their expressing plant tissues. Furthermore, phosphorylation at the conserved serine residues is important for PIN protein PM localization, as loss-of-phosphorylation PIN1 protein exhibited internalized signals in plant embryos and enhanced vacuolar accumulation in dark-incubated Arabidopsis protoplast cells. Our data indicate that phosphorylation of our identified conserved serine residues in the PIN1HL by AGC3 kinases is required for proper PIN polar localization, and is thus essential for generating the differential auxin distribution that directs plant development. Show less
In view of their predominant sessile lifestyle, plants need to be able to adapt to changes in their environment. Environmental signals such as light and gravity modulate plant growth and... Show moreIn view of their predominant sessile lifestyle, plants need to be able to adapt to changes in their environment. Environmental signals such as light and gravity modulate plant growth and architecture by redirecting polar cell-to-cell transport of auxin, thus causing changes in the distribution of this plant hormone. The PIN auxin efflux carriers are key drivers of auxin transport that determine the direction of auxin flow through their asymmetric subcellular distribution. An important component in PIN polarity establishment is the plant protein kinase PINOID (PID). PID instructs apical (shoot meristem facing) PIN polarity by phosphorylating the central hydrophylic loop of PIN proteins (PINHL). In this thesis we investigated modulation of PID activity by the calcium binding proteins TCH3 and PBP1, and by the protein kinase PDK1. All three proteins were found to regulate both the enzymatic activity and the sub-cellular localisation of PID in response to calcium and phospholipids, respectively, and as such they are likely to be involved in translating environmental signals into PIN polarity changes. In addition, we show that PID and its close homologs act both redundantly and differentially in orienting plant development by instructing the subcellular distribution of PINs. 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
