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Function and evolution of 'green' GSK3/Shaggy-like kinases
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Younousse Saidi, Timothy J. Hearn and Juliet C. Coates
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School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT,
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United Kingdom
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Corresponding author: Coates, J.C. ([email protected]).
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Glycogen synthase kinase 3 (GSK3) proteins, also known as SHAGGY-like kinases,
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have many important cell signalling roles in animals, fungi and amoebae. In
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particular, GSK3s participate in key developmental signalling pathways and also
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regulate the cytoskeleton. GSK3-encoding genes are also present in all land plants
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and in algae and protists, raising questions about possible ancestral functions in
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eukaryotes. Recent studies have revealed that plant GSK3 proteins are actively
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implicated in hormonal signalling networks during development as well as in biotic
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and abiotic stress responses. In this review, we outline the mechanisms of
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Arabidopsis GSK3 action, summarize GSK3 functions in dicot and monocot
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flowering plants, and speculate on the possible functions of GSK3s in the earliest-
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evolving land plants.
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Multifunctional proteins with conserved structure
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Glycogen synthase kinase 3 (GSK3) was originally characterized in the animal insulin
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signalling pathway as a serine/threonine kinase that phosphorylates glycogen synthase,
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the enzyme responsible for the final step in the synthesis of glycogen [1]. GSK3
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functions in many developmental processes, including cell fate specification,
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cytoskeleton movements and programmed cell death (reviewed in [2]), with roles in
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human diseases, including cancer and Alzheimer’s (reviewed in [3]). In particular, GSK3
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is a central player in the animal Wnt signalling pathway, where extracellular Wnt signals
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lead to the inactivation of GSK3. This blocks the activity of GSK3 towards β-catenin (see
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Glossary), which as a result is no longer degraded by the proteasome, but can build up in
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the nucleus and regulate target gene expression [4]. Thus, GSK3 is crucial for
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developmental patterning [5], a role that appears to be conserved in Dictyostelium
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discoideum [6]. Mammalian GSK3 exists as two isoforms, encoded by separate genes,
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GSK3α and GSK3β, which have a conserved kinase domain but divergent N- and C-
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terminal domains, which are important for regulation of function [7].
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GSK3 substrates are diverse and most of them require phosphorylation by another
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kinase before being phosphorylated by GSK3. This is termed as a 'priming'
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phosphorylation, which positions the substrate in a suitable configuration for
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phosphorylation by GSK3. A phosphate-binding pocket in GSK3 interacts with the
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primed phosphorylation [8]. In GSK3β, the phosphate-binding pocket is defined by
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arginine 96, arginine 180 and lysine 205 [8] (Figure 1). GSK3 is an ancient kinase, with
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homologues found in all eukaryotes studied to date. Unlike in animals and Dictyostelium,
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land plant GSK3s are encoded by relatively large multigene families whose members
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share high sequence similarity. In all angiosperm GSK3s so far analysed, the phosphate-
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binding pocket residues are identical to those in GSK3, suggesting that plant GSK3s can
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phosphorylate primed substrates [9] (Figure 1). Accordingly, a proteomic study identified
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ten Arabidopsis thaliana (Arabidopsis) (proteins with putative GSK3 phosphorylation
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sites: five of these were phosphorylated at the corresponding priming site [10].
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In Arabidopsis there are ten GSK3 homologues, also termed Shaggy Kinases
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(AtSK or ASK) in reference to the Drosophila GSK3 homologue [11]. Nomenclature of
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Arabidopsis GSK3s can be confusing. A full complement of names for each Arabidopsis
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protein, along with names for other plant GSK3s that have been studied, is given in Table
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1. In the last decade, substantial progress has been made in understanding how plant
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GSK3s perform their diverse functions. In the following sections we will describe
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currently known plant GSK3 functions, discuss the molecular mechanisms of GSK3
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action in plants, and highlight possible GSK3 functions in early-evolving land plants as
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an exciting area for future research developments.
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GSK3s and brassinosteroid signalling in flowering plants
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Steroid hormone signalling is found in plants, animals and fungi and, therefore, might be
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predicted to have an ancient evolutionary origin [12]. However, the proteins involved in
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plant steroid signalling belong to plant-specific protein families, thus indicating a
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separate origin for steroid signalling in the plant lineage. Brassinosteroid (BR) signalling
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modulates a range of physiological responses in flowering plants, including cell
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expansion, greening, flowering time, fertility and differentiation of vascular tissue
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(reviewed in [13-16]).
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In Arabidopsis, BRs are perceived by a plasma membrane-localized receptor
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kinase, BRI1 [13-15]. The downstream signal transduction involves a gene isolated by
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forward genetic studies, BRASSINOSTEROID-INSENSITIVE 2 (BIN2). Dominant
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mutations in bin2 result in brassinosteroid-insensitive dwarf plants. BIN2 encodes a
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GSK3 with a catalytic domain sharing ~70% identity with animal GSK3β [17-20] (Figure
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1). Gain-of-function bin2 point mutations and over-expression of BIN2 all cause the
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same BR insensitivity phenotype, confirming BIN2 as an inhibitor of BR signalling [17-
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20].
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In the absence of a BR signal, active BIN2 negatively regulates BR-specific
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transcription factors, BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS-
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SUPPRESSOR 1 (BES1/BZR2) (Figure 2). BIN2 phosphorylates BZR1/BES1 on many
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serine and threonine residues, which leads to their proteasomal degradation [21-23],
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reminiscent of the role of animal GSK3 during both Wnt- and Hedgehog signalling [4,
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24]. BIN2 can also promote BZR1/BES1 nuclear export, thus inhibiting BZR1/BES1
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binding to DNA [25-26]. Again, this is a role also demonstrated by activated
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(phosphorylated (Figure 1)) animal and amoebal GSK3, which control the nuclear export
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of the transcription factors NF-ATc and StatA, respectively [27-28]. Interaction between
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BIN2 and BZR1/BES1 is via a specific docking mechanism not seen in other GSK3
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substrates [29], and independent of prime phosphorylation or a scaffold protein [21]. In
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the presence of BR, BIN2 activity towards BES1/BZR1 is inhibited and, consequently,
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unphosphorylated BZR1 and BES1 are no longer degraded and can direct transcription of
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BR-responsive genes (Figure 2) [13-15].
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The exact mechanism by which the BR signal is transduced from the plasma
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membrane to BIN2 has now been elucidated [13-15,30-32] and it demonstrates novel
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modes of inhibition of GSK3 activity. BR is perceived by the BRI1 brassinosteroid
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receptor and BAK1 (BRI1-associated receptor kinase) co-receptor (Figure 2). BR binding
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to BRI1 enables transphosphorylation of BRI1 and BAK1, and activation of BRI1. BRI1
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then phosphorylates BR-signalling kinases (BSKs) at the plasma membrane.
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Phosphorylated BSK binds and activates BSU1 phosphatases, which dephosphorylate
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tyrosine 200 (Y200) in BIN2, inactivating its kinase activity [31] (Figure 2).
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Interestingly, the dominant bin2-1 (E263K) mutation (Figure 1) renders BIN2
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hyperactive by blocking its dephosphorylation by BSU1 [31]. In response to BR, BIN2 is
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also degraded by the proteasome, whereas the bin2-1 mutant protein is considerably
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stabilized [30]. Whether it is the Y200-dephosphorylated form of BIN2 that is
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specifically targeted for degradation has yet to be determined. Y200 in BIN2 is
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homologous to Y216 in human GSK3β (Figure 1), a tyrosine residue absolutely required
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for GSK3 activity. The homologous tyrosine residue in other AtGSK3s is also
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phosphorylated in vivo, and this phosphorylation is required for kinase activity [10]. In
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GSK3β, this residue is autophosphorylated by GSK3 itself just after the protein is
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synthesized, with the help of the Hsp90 chaperone protein [33]. BSU1-mediated
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dephosphorylation of Y200 in BIN2 is the first example of direct dephosphorylation of
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the conserved tyrosine residue of GSK3 as a means of negatively regulating kinase
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activity. Furthermore, most plant GSK3s (all except clade III kinases, Table 1) do not
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contain the N-terminal serine 9 residue found in GSK3β that is key to regulating GSK3β
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inhibition (Figure 1) (reviewed in [2-3]). It is, therefore, possible that a different
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mechanism exists in plants for GSK3 inactivation.
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The mechanism for brassinosteroid perception and transduction involves GSK3
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homologues functioning redundantly for the negative regulation of BR responses [26,34-
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35]. Experiments using loss- and gain-of-function mutants showed that two other GSK3
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homologues, AtSK22/BIL1 and AtSK23/BIL2 (Table 1), function in the same way as
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BIN2 during BR signalling [35]. Together these genes make up sub-clade II of the
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Arabidopsis GSK3s in the flowering plant phylogeny [9,36] (Table 1). Although the
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triple mutant lines of BIN2, BIL1 and BIL2 show a BR-enhanced phenotype, analysis of
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the levels of phosphorylated BZR1 showed that despite knockouts of all clade II GSK3s
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there was still substantial phosphorylation of BZR1 [35]. This indicated a role for
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additional GSK3(s) in the BR signalling pathway, which have been identified as
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AtSK31/ASKθ [37], a member of sub-clade III, and possibly all three of the subclade I
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GSK3s [31]. Therefore, at least seven out of the ten Arabidopsis GSK3 proteins are
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implicated in BR signalling. Importantly, a chemical activator of BR signalling, bikinin,
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specifically inhibits the same subset of seven Arabidopsis GSK3 proteins [38]. BIN2
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orthologues from cotton (Gossypium hirsutum) and rice (Oryza sativa) have been isolated
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and their overexpression in Arabidopsis has been shown to cause severe growth defects
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similar to bin2 gain-of-function (BR-insensitive) mutants [39-40]. This suggests that
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BIN2 orthologues encode isoforms that share a conserved function in BR signalling.
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BIN2 at the crossroads between brassinosteroids, abscisic acid and auxin
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One exciting finding is that GSK3 may mediate the crosstalk between BR signalling and
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other hormone signalling pathways [41-42]. Abscisic acid (ABA) inhibits BR signalling,
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enhances BES1 phosphorylation and induces the expression of BR-suppressed genes
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[41]. This effect was alleviated when BIN2 activity was blocked using lithium chloride
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(LiCl), a GSK3 inhibitor. The effect of ABA on BR signalling was independent of the
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early BR perception events [41]. However, whether ABA has a direct effect on BIN2
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phosphorylation or whether it affects the upstream BSK/BSU components remains to be
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elucidated (Figure 2). Auxin, unlike ABA, has no effect on the phosphorylation state of
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BES1 [43]. However, ARF2, an auxin response factor (ARF) that acts as a transcriptional
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repressor, has numerous putative GSK3 recognition sites and the interaction between
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BIN2 and ARF2 has been confirmed in yeast [42]. In vitro studies also showed that BIN2
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is capable of phosphorylating ARF2 and consequently inhibiting its DNA-binding and
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repressor activity [42]. These findings provide a molecular explanation for the effect of
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BRs on the auxin response by suggesting that BIN2 facilitates binding of activator ARF
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proteins by removing repressor ARFs from regulatory elements in the promoters of
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auxin-responsive genes ([42] and Figure 2). It is possible that hormone signalling may
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impinge upon plant GSK3s in additional ways. For example, AtSK31 gene expression is
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upregulated by auxin in roots [44], although AtSK31 does not have a demonstrated role
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in BR or auxin signalling.
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GSK3 function in floral organs and cell expansion
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Molecular and genetic analysis has confirmed roles for Arabidopsis GSK3s in the
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development of flowers and reproductive organs. AtSK11 and AtSK12 show specific
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strong expression in early floral meristems, which later becomes restricted to sepal
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primordia, petals, carpels and the pollen-containing regions of the anthers [45]. Within
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the carpels, AtSK11 and AtSK12 expression concentrates on adaxial sides (nearest the
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central axis) of the carpels and in ovule primordia, particularly in the integuments and
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megaspore mother cell [45]. A quantitative RT-PCR study of all ten Arabidopsis GSK3s
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revealed that a third GSK3, AtSK31, was also expressed relatively highly in floral organs
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[46]. Moreover, the AtSK31 protein localizes to the nuclei of developing tissues,
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particularly in floral organs, gametophytes and embryos [47].
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Consistent with the expression data, antisense reduction in either AtSK11 or
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AtSK12 transcript levels results in disrupted cell division in the floral meristem, in
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flowers with increased numbers of sepals and petals, and in abnormal carpel development
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[45]. Interestingly, overexpression of different AtSK32 isoforms in wild-type Arabidopsis
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causes varying effects [48]. Overexpression of either wild-type AtSK32 or a kinase-dead
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mutant (K167A) resulted in no detectable phenotype [48]. However, overexpression of a
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mutant specifically unable to phosphorylate primed GSK3 substrates (R178A; Figure 1)
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resulted in plants with short hypocotyls and reduced cell elongation in floral organs.
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Accordingly, AtSK32 (K167A)-overexpressing plants showed a marked downregulation
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of the genes encoding several cell wall-modifying enzymes involved in cell elongation
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[48]. This suggests that GSK3-mediated cell elongation responses may require the action
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of primed GSK3 substrates. It is likely that in addition to floral cell expansion, GSK3s
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may have a more general role in cell elongation in the plant given that BRs are
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instrumental in controlling cell elongation and dominant bin2/ucu1 mutants show cell
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elongation defects in leaves [19]. Given that GSK3 targets BZR1 and BES1 do not
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require a priming phosphorylation [21,30], it seems that new targets of AtSK32 await
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discovery.
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GSK3 roles in stress responses
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Work in several plant species has implicated GSK3s in abiotic stress responses, although
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sometimes in conflicting ways [40,46,49-52] (Table 1). An up-regulation of GSK3
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transcripts by salt stress has been reported in Arabidopsis (AtSK13, AtSK31 and AtSK42)
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[46], wheat (Triticum aestivum) (TaGSK1) [53], rice (OsGSK1) [40] and sugarcane
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(Saccharum officinarum) (SuSK) [52]. The above mentioned Arabidopsis GSKs are also
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induced by osmotic stress whereas rice OsGSK1 transcripts show a strong reduction
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following drought treatment [40]. Confirming the role of GSK3 as an active component
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of the salt stress signalling pathway, Arabidopsis plants overexpressing AtGSK1
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(AtSK22) show a significant upregulation of several salt-stress-responsive genes, even in
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the absence of high salinity [49]. As a result, they exhibit an enhanced resistance to high
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salt conditions. In another study, a rice mutant with a T-DNA insertion in OsGSK1
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exhibited elevated transcript levels of specific stress-responsive genes and a strong
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tolerance to high salt and drought treatments [40]. In Medicago sativa (alfalfa), the kinase
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activity of a plastid localized GSK3 (MsK4) was rapidly increased under hyperosmotic
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conditions [51]. The overexpression of MsK4 in Arabidopsis improved salt tolerance
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through changes in carbohydrate metabolism [51]. MsK4 is most similar to AtSK41,
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which does not respond to stress treatments [46]. However, AtSK31 shows a unique
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upregulation in plants that have been subjected to a dark period [46], making it a
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promising candidate for future studies.
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GSK3s are also important in the plant response to biotic stress. M. sativa MsK1
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shows a change in activity in response to plant defence elicitors. Interestingly, exposure
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to cellulase rapidly inhibited MsK1 activity and triggered its proteasome-dependent
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degradation [54]. The role of GSK3 in modulating the disease response was demonstrated
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when over-expression of MsK1 in Arabidopsis reduced the pathogen-mediated activation
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of AtMPK3 and AtMPK6 MAP kinases and exacerbated the susceptibility of the plant to
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Pseudomonas syringae [54]. This suggests that MsK1 acts as negative regulator of the
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basal defence response and that its elicitor-triggered regulation is essential to activate
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downstream components of plant defence pathways [54]. Another M. sativa GSK3, WIG
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(wound-induced gene), has a kinase activity that is transiently triggered following leaf
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injury [55]. Although none of the Arabidopsis GSKs seem to be transcriptionally induced
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by wounding [46], their kinase activity under mechanical injury has never been tested to
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date. Overall, GSK3 family members seem to have distinct regulatory functions and their
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activity appears to be modulated by various cues. How different stimuli modulate GSK3
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activity during stress responses is largely unknown. One attractive hypothesis is that this
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occurs through phosphorylation/dephosphorylation by upstream components but this has
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yet to be demonstrated. Future research should also focus on isolating the targets of stress
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response-related GSK3s, which should open new possibilities in the quest for crop
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improvement.
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The stress-responsive nature of GSK3s is evolutionarily ancient. GSK3 genes in
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the yeast Saccharomyces cerevisiae, particularly MCK1, are involved in responses to
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environmental stresses, including heat, cold, salt/osmotic stress, nutrient stress, oxidative
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stress and metal ion stress [56-58]. The temperature-sensitive phenotype of a yeast
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quadruple gsk3 mutant is rescued by expression of mammalian GSK3β [59], suggesting
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further cross-kingdom conservation of functionality. Similarly, Arabidopsis AtSK22 can
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rescue the salt-sensitive phenotype of the yeast mck1 mutant [60]. To thrive following
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transition from water to land, plants had to acquire new stress-tolerant adaptations,
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particularly in response to desiccation, light and temperature changes. For this reason, in
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the next section we consider the GSK3s of algae and of plants that evolved early to life
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on land.
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Evolution of plant GSK3 function
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GSK3 genes may have played an instrumental role in the acquisition of stress tolerance
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mechanisms during the adaptation to life outside water because land plants are unusual in
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possessing large GSK3 gene families. GSK3 gene diversification has occurred more than
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once during land plant evolution [36,50]. In seed plants, three GSK3 clades are
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represented in gymnosperms, with divergence into four clades occurring before (or early
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in) angiosperm evolution [36,50]. The moss Physcomitrella patens diverged early from
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the seed plant lineage around 470 million years ago, shortly after plants appeared on land.
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The five Physcomitrella GSK3 genes discovered by cDNA library screening [50] fit into
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a separate subclade that is most similar to flowering plant clades I and IV [36,50].
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Physcomitrella GSK3s share 95% sequence identity across the whole protein [50],
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suggesting a large degree of redundancy and possibly recent gene duplications in moss.
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The complete sequencing of the Physcomitrella [61] and the lycophyte Selaginella
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moellendorffii [62] genomes has made it possible to assess the full complement of GSK3
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gene sequences in the early land plants. We can detect seven putative GSK3 homologues
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in Physcomitrella, and two in Selaginella (J.C. Coates, unpublished). Our tentative
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phylogenies suggest that the Selaginella GSK3 genes duplicated after the divergence of
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Selaginella and Physcomitrella.
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The functions of GSK3s in Physcomitrella and Selaginella are unknown.
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However, as in Arabidopsis and rice, two Physcomitrella genes show increased transcript
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abundance when exposed to sorbitol and polyethylene glycol [50] (Table 1), suggesting a
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conserved role in osmotic stress responses. Interestingly, sequenced genomes of green
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and red algae (Chlamydomonas reinhardtii, Volvox carteri, Porphyra umbilicalis and
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Cyanidioschyzon merolae) each contain only one GSK3 gene, further suggesting that all
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the 'green' GSK3 gene duplications occurred after the evolution of land plants.
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Within the heterokont lineage, we can detect a single GSK3 homologue in the
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sequenced genomes of each of: Ectocarpus siliculosus (multicellular brown alga),
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Aureococcus anophagefferens (unicellular brown alga), Thalassiosira pseudonana
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(diatom), Phaeodactylum tricornutum (diatom), and Phytophthora infestans (oomycete).
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The situation in alveolates is more complex. Single GSK3 homologues exist in
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Plasmodium sp. and Toxoplasma sp. with larger gene families in Paramecium tetraurelia
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and Tetrahymena thermophila. Within excavates a single GSK3 homologue is present in
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Leishmania sp. with two possible homologues in Trypanosoma sp. Together, these data
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suggest a single common ancestor for GSK3. Whether the GSK3s identified in algae and
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other protists function in osmotic stress tolerance is unknown.
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Conclusions and future directions
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GSK3 proteins have extremely diverse functions both between kingdoms and within a
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single species. This suggests that GSK3s have been co-opted into many signalling
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pathways throughout evolution and highlights their pivotal importance in eukaryotic
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signalling. Plant GSK3 proteins are required for growth, development and environmental
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stress tolerance. These are all agronomically important plant traits, making plant GSK3s
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potential targets for future crop manipulation. So far, most functional studies have been
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carried out in Arabidopsis or in other dicots. The exact degree of GSK3 functional
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conservation across land plants is unknown, although rice and cotton BIN2 homologues
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appear to have conserved function when overexpressed in Arabidopsis [39-40].
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Producing dwarf plants was key to the success of the Green Revolution, and cereal semi-
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dwarf mutants with altered BR signalling have been discovered [63-65]. Perhaps cereal
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BIN2 homologues can be manipulated to produce semi-dwarf plants. Indeed, the
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discovery of a GSK3 mutant [48] affecting the phosphorylation of only some substrates
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could be a powerful tool for future development and use in crop manipulation.
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GSK3s play a role in integrating multiple hormonal signals (BR, ABA and auxin).
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In addition to their role in development, these hormones are involved in responding to a
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variety of environmental stresses. Therefore, deciphering the molecular mechanism by
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which GSK3s mediate hormone crosstalk represents an attractive way to modulate plant
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stress tolerance.
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Whether plant GSK3s share conserved molecular functions with GSK3s in other
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systems is still an open question. Key unanswered questions include (i) whether plant
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GSK3s provide a direct link between developmental cell signalling and the cytoskeleton,
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and (ii) whether plant and animal GSK3s share any conserved protein targets and
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upstream regulators. These should be exciting areas for future research in Arabidopsis
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and other species.
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Acknowledgements
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We apologize to colleagues whose work could not be included because of space
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limitations. We thank Jennifer Nemhauser for useful advice, Laura Moody for her helpful
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comments, and three anonymous reviewers for their constructive feedback.
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References
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17
461
Table 1. Land plant GSK3s
Arabidopsis GSK3
clade
Arabidopsis GSK3
Gene identifier
Function/remark
Refs
I
AtSK11/ASKα
At5g26750
[45,31]
AtSK12/ASKγ
At3g05840
AtSK13/ASKε
At5g14640
AtSK21/ASKη/BIN2/UCU1
AtSK22/ASKι/BIL1/AtGSK1
AtSK23/ASKζ/BIL2
AtSK31/ASKθ
At4g18710
At1g06390
At2g30980
At4g00720
AtSK32/ASKβ
AtSK41/ASKκ/AtK-1
AtSK42/ASKδ
At3g61160
At1g09840
At1g57870
Flower development/brassinosteroid
signalling
Flower development/brassinosteroid
signalling
Osmotic stress induced /brassinosteroid
signalling
Brassinosteroid signalling
Brassinosteroid signalling/salt stress
Brassinosteroid signalling
Brassinosteroid signalling/osmotic stress
induced
Flower development
Unknown
Osmotic stress induced
GSK3 gene
MsK1
MsK4
WIG
OsGSK1
TaGSK1
BIN2
SuSK
PpSK2
PpSK4
Highest similarity to
AtSK11
AtSK41
AtSK31
AtSK21/BIN2
AtSK12
AtSK21/BIN2
AtSK42
AtSK13
AtSK13
Function/remark
Pathogen response
Carbohydrate metabolism/salt stress
Wound response
Brassinosteroid signalling/salt stress
Salt stress
Brassinosteroid signalling
Salt/osmotic stress
Osmotic stress induced
Osmotic stress induced
II
III
IV
Plant species
Medicago sativa
Oryza sativa
Triticum aestivum
Gossypium sp.
Saccharum officinarum
Physcomitrella patens
[45,31]
[46,31]
[17,18,19,20]
[35,49]
[35]
[37,46]
[48]
[46]
[54]
[51]
[55]
[40]
[53]
[39]
[52]
[50]
[50]
462
18
463
Glossary
464
Alveolates: a protist lineage including Apicomplexans (unicellular parasites such as
465
Plasmodium (the malaria parasite) and Toxoplasma), marine dinoflagellates and ciliates.
466
Angiosperms: also called flowering plants. A group of land plants with reproductive
467
organs in the form of flowers, which produce seeds enclosed in capsules (or fruits). They
468
are believed to have evolved more recently (in geological terms). Modern agriculture is
469
mostly reliant on species within this group.
470
Bryophytes: Early land plants that do not have vascular tissues. They do not form
471
flowers and thus do not produce seeds. Sexual reproduction leads to the formation of
472
single-celled spores. Bryophytes are characterized by a dominant haploid gametophyte
473
life cycle. This group contain mosses, hornworts and liverworts.
474
β-catenin: is an animal protein (called Armadillo in Drosophila) containing tandem
475
copies of a protein motif called Armadillo repeat domain, which form a conserved three-
476
dimensional structure specialized for protein–protein interaction. β-catenin homologues
477
function in various processes, including intracellular signalling (e.g. Wnt signalling) and
478
cytoskeletal regulation.
479
Cellulase: A class of enzymes that can hydrolyse cellulose and degrade cell walls. They
480
have been shown to induce plant defence responses.
481
Cilia: Either motile or primary (non-motile), structurally related to flagella and are found
482
in animals. Nearly every mammalian cell has a primary cilium. Cilia are emerging as key
483
regulators of development and signalling in animal systems, particularly as homes for
484
Wnt signalling pathway components.
19
485
Cytokinesis: The process of division of the cytoplasm of a single eukaryotic cell during
486
mitosis.
487
Elicitor: Molecules produced by pathogens, or by plants in response to pathogens, that
488
activate plant defence responses.
489
Excavates: A eukaryotic kingdom consisting largely of unicellular species, some of
490
which, such as Trypanosoma and Leishmania, are disease-causing parasites.
491
Ferns: Vascular plants differing from gymnosperms and angiosperms by their mode of
492
reproduction. They reproduce via spores and constitute the earliest plants having true
493
leaves (megaphylls). This group includes horsetails (Equisetum) and lady fern
494
(Athyrium).
495
Flagella: Motile microtubule-containing cellular extensions found on unicells and on
496
motile gametes in animals, plants and protists. They act as environmental sensors and
497
control cell motility and cell–cell interactions.
498
Gymnosperms: A group of land plants that reproduce by means of 'naked seeds' exposed
499
in open structures, such as cones or leaves (in contrast to angiosperms with enclosed
500
seeds). This group includes conifers and Ginkgo.
501
Heterokonts: the eukaryotic phylum containing both unicellular and multicellular brown
502
algae, diatoms, and oomycete pathogens.
503
Lycophytes: Seedless vascular plants with primitive leaves (microphylls) having only a
504
single vascular trace (vein). Lycophytes include the resurrection plant Selaginella
505
lepidophylla and the model plant Selaginella moellendorffii, which has a sequenced
506
genome.
507
20
508
Box 1. Conserved roles for plant GSK3s in cytoskeletal regulation?
509
In animals and yeast, GSK3 functions both in cell signalling and in cytoskeletal
510
regulation. It has yet to be determined whether plant GSK3s have cytoskeletal functions.
511
However, given their instrumental roles in cell expansion and growth, it is tempting to
512
speculate that they regulate cell architecture directly.
513
Crosstalk between developmental signalling and the cytoskeleton?
514
In Caenorhabditis elegans and Drosophila, GSK3 responds to Wnt signals and controls
515
mitotic spindle orientation during cell division [66]. In mammalian cells, GSK3
516
phosphorylates a spindle microtubule-associated protein [67]. This function of GSK3
517
might be ancient given that GSK3 has several key roles in cell cycle progression in yeast
518
[57]. Thus, plant GSK3s may also be required for cell division. However, cytokinesis
519
mechanisms in plants are different from those in animal and yeast cells, suggesting that
520
novel regulatory mechanisms may also have evolved.
521
Animal GSK3 phosphorylates a plethora of microtubule-associated proteins [66],
522
some of which are conserved in plants [68]. When Wnt signals regulate GSK3 activity,
523
this leads to changes in microtubule regulation [66]. Thus, a key avenue for future studies
524
is to determine whether plant microtubule-associated proteins are regulated by GSK3,
525
and whether GSK3 provides a link between the cytoskeleton and cell growth regulation
526
by hormone/BR signalling, similar to that found in animals.
527
Flagellar functions of GSK3
528
Flagella and cilia are evolutionarily ancient organelles that regulate key cellular processes
529
[69]. The unicellular green alga Chlamydomonas is a key model system used to study the
530
molecular basis of flagellar dynamics owing to its ease of genetic manipulation.
21
531
Chlamydomonas has two motile flagella of tightly regulated length. Short-term treatment
532
with lithium, a GSK3 inhibitor, leads to elongation and paralysis of the flagella, whereas
533
longer treatments produce aflagellate cells [70]. Similarly, lithium inhibits flagellar
534
regeneration. This suggests a key role for Chlamydomonas GSK3 in regulating flagellar
535
assembly, maintenance, length and motility. RNAi-mediated knock-down of the single
536
Chlamydomonas GSK3 homologue renders cells aflagellate [70]. The active
537
(phosphorylated) form of Chlamydomonas GSK3 is enriched in the flagella (particularly
538
newly assembling flagella) and is associated with the flagellar microtubules only when
539
phosphorylated [70]. This shows that GSK3 is a key regulator of flagellar dynamics.
540
Whether this function is conserved in animals is not yet known, but given the strong
541
conservation of other flagellar protein functions between algae and animals it is an area
542
worth investigating in the future.
543
Plant cells do not have cilia, and most seed plant cells do not have flagella.
544
However, the motile gametes of the seedless land plants (bryophytes, lycophytes and
545
ferns) and some early seed plants are flagellated. These would be interesting organisms in
546
which to investigate GSK3 function.
547
548
549
Figure 1. Alignment of human GSK3 and a representative Arabidopsis GSK3,
550
AtSK21/BIN2. The kinase domain is highlighted in yellow. The conserved tyrosine
551
whose phosphorylation is required for kinase activity (Y216 in GSK3, Y200 in BIN2) is
552
highlighted in red. The conserved phosphate-binding residues that interact with prime-
553
phosphorylated substrate are highlighted in cyan. The conserved TREE domain is boxed
554
in orange. Highlighted in dark blue within this domain are the three residues that lead to
22
555
BIN2 gain-of-function alleles when point-mutated [bin2-1 and dwf12-2D (E263K); bin2-
556
2 (T261I); ucu1-1, ucu1-2 and dwf12-1D (E264K)] [18-20]. The weak allele ucu1-3
557
(P284S) is highlighted in purple. Other loss-of-function mutations (identified in [35]) are
558
highlighted in magenta (G49D, C183Y, R312K; all are in residues conserved in GSK3).
559
The kinase-dead mutation bin2 (K69R) that suppresses gain-of-function mutations is
560
highlighted in light green. Homologous residues to those identified by mutation in
561
AtSK32 [48] are marked with white arrowheads. The serine 9 residue specific to GSK3
562
and essential to the regulation of its inhibition is marked with a black arrowhead.
563
564
Figure 2. The central role of BIN2 in BR signalling and its interface with other hormone
565
pathways. In the absence of a BR signal, the active (phosphorylated) form of BIN2
566
phosphorylates BES1/BZR1 transcription factors, consequently targeting them for
567
proteasomal degradation. The BR hormone activates the BRI1 receptor and its co-
568
receptor, BAK1. This allows transphosphorylation of the two transmembrane receptors.
569
Active BRI1 phosphorylates BSK kinase, which interacts with and activates BSU1
570
phosphatases. Active BSU1 dephosphorylates BIN2, which promotes BIN2 degradation,
571
and allows unphosphorylated BES1/BZR1 to translocate to the nucleus and modulate BR
572
gene expression. BIN2 also interacts and phosphorylates the transcriptional repressor
573
ARF2. This relieves ARF2 repression on auxin-responsive promoters and leads to a
574
synergistic BR–auxin response. ABA inhibits BR signalling and enhances the
575
phosphorylation state of BES1. This action might be via a direct effect on BIN2 or an
576
upstream component.
577
23