Review: Post-translational cross-talk between brassinosteroid and sucrose signaling

Plant Science 248 (2016) 75–81

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Review article

Review: Post-translational cross-talk between brassinosteroid and sucrose signaling Christina Kühn Humboldt University of Berlin, Institute of Biology, Department of Plant Physiology, Philippstr. 13, Building 12, 10115 Berlin, Germany

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Article history: Received 2 March 2016 Received in revised form 21 April 2016 Accepted 23 April 2016 Available online 26 April 2016 Keywords: Brassinosteroid signaling Sucrose transport Endocytosis

a b s t r a c t A direct link has been elucidated between brassinosteroid function and perception, and sucrose partitioning and transport. Sucrose regulation and brassinosteroid signaling cross-talk at various levels, including the well-described regulation of transcriptional gene expression: BZR-like transcription factors link the signaling pathways. Since brassinosteroid responses depend on light quality and quantity, a light-dependent alternative pathway was postulated. Here, the focus is on post-translational events. Recent identification of sucrose transporter-interacting partners raises the question whether brassinosteroid and sugars jointly affect plant innate immunity and plant symbiotic interactions. Membrane permeability and sensitivity depends on the number of cell surface receptors and transporters. More than one endocytic route has been assigned to specific components, including brassinosteroid-receptors. The number of such proteins at the plasma membrane relies on endocytic recycling, internalization and/or degradation. Therefore, vesicular membrane trafficking is gaining considerable attention with regard to plant immunity. The organization of pattern recognition receptors (PRRs), other receptors or transporters in membrane microdomains participate in endocytosis and the formation of specific intracellular compartments, potentially impacting biotic interactions. This minireview focuses on post-translational events affecting the subcellular compartmentation of membrane proteins involved in signaling, transport, and defense, and on the cross-talk between brassinosteroid signals and sugar availability. © 2016 Published by Elsevier Ireland Ltd.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Phloem loading, unloading and long-distance transport of sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Transcriptional control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3.1. The role of the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3.2. Brassinosteroids, sugars and flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 3.3. Brassinosteroids, sucrose and mycorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Subcellular compartmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 4.1. Endocytosis of brassinosteroid signaling components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 4.2. Endocytosis of sucrose transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Endocytosis and defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 5.1. The role of pattern recognition receptors (PRRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 5.2. The role of SUT2-interacting BAK1 in PRR-mediated immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Abbreviations: BR, brassinosteroids; PRR, pattern recognition receptor; LRR-RLK, leucine rich repeat-receptor like kinase. E-mail address: [email protected] http://dx.doi.org/10.1016/j.plantsci.2016.04.012 0168-9452/© 2016 Published by Elsevier Ireland Ltd.

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1. Introduction A link between brassinosteroid signaling and sugars became obvious very early based on different observations. There is obviously a correlation between sugar levels and the expression level of brassinosteroid-related genes in several plant species. In sugar cane plants, it is known that down-regulation of the receptor kinase BAK1 (BRI1-associated kinase 1) expression resulted in decreased levels of soluble sugars [1]. In Arabidopsis, sugar content is also related to brassinosteroid deficiency [2] and in the tomato dx mutant with defective gene expression of the brassinosteroid biosynthetic sterol reductase DIMINUTO1, and consequently diminished brassinosteroid biosynthesis, the amount of soluble sugars and starch in the fruits are reduced as well [3]. The photomorphogenic bls1 (brassinosteroid, light, sugar 1) mutant in Arabidopsis de-etiolates even in darkness, and is hypersensitive to metabolizable sugars showing mortality, chlorophyll and anthocyanin accumulation on sucrose containing medium; hypersensitivity of the bls1 mutant can be rescued by exogenous epi-brassinolide supply restoring viability on 2% sucrose [4]. It is assumed that sugar hypersensitivity is a secondary effect from brassinosteroid synthesis defects. Brassinosteroid biosynthesis gene CPD expression is light-induced via phytochrome signaling and follows circadian cycles leading to brassinolide accumulation in the middle of the light phase [5]. The cross-talk between brassinosteroids and sugars is assumed to occur via a light-induced pathway [6,7], either by direct interaction between BZRs and the photochrome-interacting factor 4 (PIF4) or DELLA repressors; or alternatively via the photoperiodic pathway in a circadian clock-dependent manner, since brassinosteroid application reduces the period length of circadian cycles [8]. Here, an alternative pathway at the post-translational level is presented, where sucrose transporter proteins have direct protein–protein interactions with components of the brassinosteroid signaling pathway with compartmentation, internalization, or competition with interacting partners. It will also be shown that membrane cycling of components of the sugar and brassinosteroid signaling pathways presents an important parameter in plant immune response.

2. Phloem loading, unloading and long-distance transport of sucrose Long-distance transport of photoassimilates occurs in the phloem. Brassinosteroids are involved in vascular differentiation promoting xylem proliferation and several brassinosteroid receptor proteins are expressed specifically in the vasculature. Whereas the brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is expressed ubiquitously, two additional brassinosteroid receptors, BRI1-like 1 and 3 (BRL1 and BRL3), have restricted expression patterns in the vasculature. Both function in vascular differentiation in Arabidopsis by promoting xylem differentiation and repressing phloem differentiation [9]. Transgenic manipulation of brassinosteroid biosynthetic enzymes suggested that brassinosteroids stimulate the flow of assimilates from source to sink via the vasculature leading to enhanced CO2 assimilation and seed filling in rice [10]. Brassinosteroids also promote grape berry ripening [11]: epi-brassinolide treatment of grape berries increased the amounts of soluble sugars in berries as well as the expression of cell wall invertase (VvcwINV) and that of sucrose transporters VvSUC12 and VvSUC27, suggesting that brassinosteroids promote sucrose phloem unloading and thereby berry ripening [12]. Brassinosteroids specifically induce expression of the extracellular invertase Lin6 in tomato involved in the apoplasmic phloem

unloading of sucrose [6]. Thus, there is an experimental link between brassinosteroids and phloem unloading in several species, however it remains unclear how this cross-talk functions at the molecular level. Brassinosteroids stimulate photosynthetic capacity in tomato plants via activation of enzymes of the Calvin cycle by creating a reducing environment [13]. In transgenic tomato plants overexpressing the gene encoding the brassinosteroid biosynthetic enzyme Dwarf1, the activity of dehydroascorbate reductase and glutathione reductase are increased creating a reducing environment. Inhibiting brassinosteroid synthesis had opposite effects. Since enzymes of the Calvin cycle are redox regulated, the authors concluded that brassinosteroid can increase photosynthetic capacity by inducing a reduced redox status and thus maintaining the activity of the Calvin cycle enzymes such as RubisCO [13]. The direct interaction partners of plant sucrose transporters are also involved in the maintenance of redox homeostasis. Interaction partners of the main sucrose transporter in potato StSUT1, namely a protein disulfide isomerase (StPDI1), and Snakin 1 (SN1), an antimicrobial cell wall protein, seem to be required to maintain the redox homeostasis [14,15]: metabolites of the TCA cycle are down-regulated in StPDI1- or SN1-inhibited transgenic plants, the level of antioxidants as well leading to partially reduced stress tolerance in these plants. Thus, a link between sucrose accumulation, photosynthetic capacity and redox homeostasis might be established via direct protein–protein interactions. 3. Transcriptional control 3.1. The role of the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) Sugars not only positively regulate the transcription of the gene encoding the brassinosteroid-activated transcription factor BRASSINAZOLE RESISTANT1 (BZR1), but also stabilize the BZR1 protein [16]. The function of HEXOKINASE1 (HXK1), the primary glucose sensor, depends on the presence of brassinosteroid, suggesting that brassinosteroid may act downstream of HXK1 [17] potentially via BZR1. BZR1 is assumed to represent a converging node between brassinosteroid and sugar signaling [17] and directly interacts with the dark- and heat-activated transcriptional regulator PIF4 [18]. Thereby hypocotyl elongation is promoted in darkness. In addition, BZR1 also belongs to the interacting transcription factors of the DELLA repressor protein REPRESSOR OF ga1-3 (RGA), a coordinator of multiple signaling pathways [19]. BZR1-BAM proteins are transcription factors containing a BZR1 DNA-binding domain combined with ß-amylase domains involved in starch breakdown [20], and BZR1-BAMs counteract brassinosteroid signaling, potentially via competition for BZR1 promoter binding sites [20]. The ß-amylase-like domain of BZR1-BAMs may require an intact substrate binding site to function as a transcriptional activator, and might be a metabolic sensor [21]. 3.2. Brassinosteroids, sugars and flowering Sucrose is required for flower induction, and brassinosteroids also promote flowering in Arabidopsis. Many brassinosteroiddeficient mutants are phenotypically late-flowering (brs1, det2, cpd, bls1). Conversely, mutants that are impaired in brassinosteroid conversion in their inactive form flower early (bas1, sob7) [22]. As already mentioned, the sugar hypersensitivity of the late-flowering bls1 mutant can be rescued by external brassinosteroid supply [4]. It is assumed that sugars and brassinosteroids interact with each other in the flower inducing signal transduction pathway, potentially involving the BZR1 and BZR2 transcription factors [22].

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3.3. Brassinosteroids, sucrose and mycorrhiza Colonization of tomato roots by arbuscular mycorrhizal (AM) fungi increases transcription of all three sucrose transporter genes from tomato [23] and down-regulation of SlSUT2 expression resulted in increased mycorrhizal colonization [24]. The sucrose transporter is localized at the periarbuscular membrane of colonized root cells: therefore a retrieval function of sucrose from the periarbuscular space was postulated. Interestingly, the SlSUT2 protein directly interacts with several components of the brassinosteroid signaling pathway as well as with the Dwarf1/DIM1 protein involved in brassinosteroid biosynthesis. Therefore, it cannot be excluded that brassinosteroids per se affect efficiency of root colonization by beneficial fungi. This hypothesis is supported by the fact that dwarf1/dim1 tomato mutants defective in brassinosteroid biosynthesis [24] as well as dwarf1/dim1 rice mutants defective in the brd2-1 gene [25] have reduced mycorrhization. A direct link between brassinosteroid signaling and mycorrhizal colonization has not yet been elucidated. It might be either that a synergistic effect between sucrose partitioning and brassinosteroid signaling is responsible for these effects or that brassinosteroid indirectly impacts mycorrhizal colonization via regulation of the carbon flux. It is important to investigate whether brassinosteroids are involved in mycorrhiza-induced resistance against pathogens as it has been discussed for jasmonate [26,27]. The membrane steroid binding protein 1 (MSBP1) and a BAK1like receptor kinase were identified among the SlSUT2-interacting proteins as the two strongest interaction partners. Whereas BAK1 is a BRI1-associated receptor kinase, the MSBP1 protein is assumed to have an inhibitory function in brassinosteroid signaling in Arabidopsis [28]. Interestingly, MSBP1 is critical for the development of intact arbuscules and hyphopodia in mycorrhizal roots of Medicago truncatula plants [29]. 4. Subcellular compartmentation Cross-talk between different signaling pathways does not necessarily have to occur at the transcriptional level, but can also occur at the post-translational level. Phosphorylation or endocytosis of membrane proteins is a rapid way to modulate sensitivity and permeability of a given membrane. In the case of brassinosteroid/auxin cross-talk, it is known that common subcellular compartments are one possibility to connect brassinosteroid receptors with auxin carriers. This cross-talk involves several auxin transporting systems such as influx and efflux carriers. The impact of brassinosteroid on auxin signaling occurs on the one hand at the transcriptional level by repressing the expression strength of PIN3, PIN4, PIN7 and LAX transporters [30,31]. Brassinosteroids also affect protein levels of auxin efflux carrier PIN2 and PIN4, but this time in the opposite direction: brassinosteroids seem to have a stabilizing effect on those protein levels [32]. Brassinosteroids control root meristem size via maintenance of cell cycle activity and promotion of cell expansion. Whereas the stele-localized PIN efflux carriers PIN1, PIN3 and PIN7 are not affected at the transcriptional level by brassinosteroids, a positive effect on PIN2 and PIN4 protein levels is observed [32]. To what extent the brassinosteroid effect on growth is mediated via PINmediated auxin redistribution is still unknown 4.1. Endocytosis of brassinosteroid signaling components The brassinosteroid signaling pathway was among the first systems with evidence for the involvement of endocytic trafficking in specific signaling outputs in plants [33]. BRI1 belongs to the leucine-

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rich-repeats receptor-like kinases (LRR-RLKs) and constitutively cycles at the plasma membrane independently of brassinosteroid binding. BRI1 endocytosis is induced by heterodimer formation with the brassinosteroid co-receptor BAK1 followed by auto- and trans-phosphorylation of both kinases. Endocytosis of plant membrane proteins can occur via different mechanisms: the clathrin-mediated endocytosis and the membrane microdomain-associated endocytosis (reviewed in [34]). Extracellular signals and environmental cues determine the endocytic routes of membrane proteins. Membrane microdomains are assumed to be involved in the regulation of signal transduction via endocytosis [34]. BRI1 endocytosis is clathrin-dependent but can also be attenuated by disruption of membrane microdomains [35]. It was speculated that supply of exogenous brassinosteroids increases the recruitment of BRI1 receptor molecules in membrane microdomains and that brassinosteroids stimulate BRI1 endocytosis via the membrane microdomain-associated mechanism. The balance between specific endocytic mechanisms would thereby allow the regulation of receptor internalization depending on ligand activation. The glucose-induced endocytosis of the brassinosteroid receptor BRI1 is another possibility where intrinsic and extrinsic signals are integrated: glucose enhances brassinosteroid signaling to induce root deviation response by increasing receptor BRI1 endocytosis from the plasma membrane to early endosomes. Glucose and brassinosteroids act thereby synergistically to regulate glucoseinduced root directional growth [36]. Endosidin 1 is a vesicle trafficking inhibitor thought to stabilize the actin cytoskeleton, thereby affecting vesicle trafficking. The BRI1 receptor shares the same endocytic compartment with a specific set of auxin carriers. Endosidin 1 specifically induces the agglomeration of the auxin transporters PIN1, AUX1 and of the brassinosteroid receptor BRI1 in so called “endosidin bodies”, whereas other marker proteins are not found in this cellular compartment. Brassinosteroid signaling was thereby inhibited by endosidin 1 [37]. This is an example how components of the brassinosteroid and the auxin signaling pathway may get in contact via enclosure in the same subcellular compartment. Interestingly, endosidin 1 shortens the period length of the circadian clock perhaps via actin-associated processes in a light-dependent manner [38]. Other components of the brassinosteroid signaling pathway undergo endocytosis, and the assumed function of the MSBP1 protein in this pathway is the inhibition of brassinosteroid signaling by removing BAK1 from the plasma membrane, thereby preventing interaction and co-internalization of BAK1 with BRI1 [28]. A potential role in vesicle trafficking and stimulation of PIN2 cycling and redistribution consequently affecting auxin redistribution is also proposed for MSBP1 [39]. 4.2. Endocytosis of sucrose transporters Sucrose transporters also undergo endocytosis in a brefeldin A-sensitive manner [40]. SUT1 recruitment in membrane microdomains can be observed in yeast endocytic mutants. The disruption of membrane microdomains by sterol depletion abolished the ability of StSUT1 to be internalized [41], suggesting that SUT1 recruitment in membrane microdomains is a pre-requisite for its internalization. The internalization of SUT1 seems to be actin-dependent and is inducible by high substrate-concentrations [14]. Several of the StSUT1-interacting proteins were also detected in membrane microdomains such as StPDI1 [42]. As mentioned before, SlSUT2 interacts with BAK1 and MSBP1, with DIM1 and AUX1 [24]. Interestingly, AUX1 was also detected in the

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Fig. 1. Hypothetical model explaining interconnection of brassinosteroid signaling with auxin and sucrose transporters at the post-translational level by subcellular compartmentation or direct interaction. Integral membrane proteins at both, the plasma membrane as well as in endocytotic vesicles depend on various stimuli and depend on the actin cytoskeleton. AUX1: auxin transporter1; BAK1: BRI1-associated kinase 1; BRI1: brassinosteroid insensitive 1; DIM1: diminuto; HVA22: Hordeum vulgare aleurone protein; MSBP1: membrane steroid binding protein 1; PDI1: protein disulfide isomerase 1; PIN2: auxin efflux carrier; REM: remorin; SUT: sucrose transporter; TIL: temperature-induced lipocalin.

detergent-resistant membrane (DRM) fraction of potato source leaves showing reduced detergent solubility as expected for microdomain-associated proteins [42]. DIM1 is an integral membrane protein and was found in the DRM fraction of tobacco plants [43] and – together with remorins – also in the interactom of two

plasmodesmal reticulon proteins [44]. Future work will answer the question whether these sucrose transporter-interacting proteins are also able to undergo membrane microdomain-associated endocytosis.

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Microbe/pathogen-associated molecular paerns Brassinosteroid or other steroid signal? MSBP1

brassinosteroids

MSBP1

BAK1

BRI1

EF-Tu

FLS2

EFR

BAK1 SERK1

BAK1BIK1

other PAMPs ?

BAK1

Unknown ligand ?

BAK1

BKK1

SlSUT2

BAK1

flagellin

BKK1

inhibion of sucrose transport?

inhibion of BR signalling

BR signalling

?

Paern -triggered immunity (PTI)

BAK1

Programmed cell death (PCD)

Fig. 2. Multiple functions of the SlSUT2-interacting BAK1 protein. Scheme was modified from Zipfel, 2008 [63]. The co-receptor BAK1 positively regulates brassinosteroid signalling. It is suggested that BAK1 together with BRI1, or alternatively with MSBP1 or SlSUT2 is internalized in a microdomain-dependent manner. FLS2 and EFR signalling also require BAK1 in pattern-triggered immunity (PTI). Together with BKK1, BAK1 is also involved in cell death control in a brassinosteroid-independent manner. BIK1: Botrytis-induced kinase 1; BKK1: BAK1-like kinase; SERK1: somatic embryogenesis receptor kinase 1.

All known sucrose transporters from Solanum tuberosum [45] and Arabidopsis [46] interact with each other. A complete network of protein-protein-interactions is drawn in Fig. 1, based on known commonly shared subcellular compartments or direct interactions. Most of the SUT-interacting proteins have been found in membrane microdomains (Fig. 1). The properties and composition of the plant plasma membrane lining plasmodesmal connections share common features with membrane microdomains. Both are enriched in sphingolipids and phytosterols, and plasmodesmata typically accumulate microdomain proteins such as remorin and GPI-anchored proteins including the callose-binding protein [47]. Remorin proteins are enriched in 70 nm-membrane microdomains and clusters at plasmodesmata [48]. Remorins can physically interact with the movement protein of Potato Virus X (PVX) and seem to inhibit virus movement through plasmodesmata. A protective role against viral spread within the plant can be assumed. Remorins may also have a role in ecto- and endo-mycorrhizal interactions. RNA-seq transcriptomics revealed that transcription of remorin genes is up-regulated in ectomycorrhizal symbiosis [49] perhaps via their influence on plasma membrane targeting or activity of host plant sugar transporters involved in carbon supply towards the fungus [50]. Moreover, remorin genes are among the late-induced genes during AM fungal infection, and are proposed to have a scaffolding role in signaling complexes within the membrane [51]. Thereby remorins might affect endocytosis of signaling components that needs to be internalized in a microdomain-dependent manner.

5. Endocytosis and defense 5.1. The role of pattern recognition receptors (PRRs) Endocytic events contribute to various aspects of plant-microbe interactions, microbe recognition, root colonization and immune responses. Membrane microdomains seem to have an important role for endocytosis herein [52]. Vesicular membrane trafficking gains more and more attention when describing and analyzing plant interactions with mutualistic or pathogenic microbes [50]. The main focus thereby is on the secretion, the recycling and endo-

cytosis of sugar transporters, reactive oxygen species producers, and pattern recognition receptors (PRRs). Surface-localized receptors recognize potential microbial interactors via perception of microbe-derived molecules. These PRRs can be removed from the plasma membrane in a ligand-binding dependent manner and regulation of the number of receptors at the cell surface in one way to regulate plants immunity. Activationdependent endocytosis of PRRs seems to be a conserved mechanism across PRR families [53], and a switch between different endocytic pathways is possible depending on different exogenous stimuli.

5.2. The role of SUT2-interacting BAK1 in PRR-mediated immunity An alternative function of BAK1 besides being the brassinosteroid co-receptor is brassinosteroid-independent and concerns induction of cell death in a brassinosteroid-independent manner. Hence, BAK1 together with BKK1 have dual physiological roles: they positively regulate brassinosteroid signaling and negatively regulate a brassinosteroid-independent cell death pathway (Fig. 2) [54]. In addition to these two pathways, BAK1 is also involved in pattern-triggered immunity (PTI) by direct interaction with LRR-RLKs such as FLS2 or EFR. These receptors perceive Microbe/Pathogen-Associated Molecular Patterns (MAMPs/PAMPs) in the apoplastic space and assign specificity of the defense response in a ligand-dependent manner [55,56] (Fig. 2). BAK1 from Arabidopsis itself is a LRR-RLK required for MAMP/PAMP recognition and response, and potentially serves as a common adaptor of these signalling complexes [57,58]. The internalization of LRR-receptor kinases together with interacting co-receptors after ligand binding or in response to transphosphorylation seems to be crucial for the signal transmission and the intracellular response via induction of downstream located signalling cascades [59]. It is assumed that BAK1 affects the equilibrium between plasma membrane-located BRI1 homodimers and internalized BRI1-BAK1 heterodimers thereby favouring endocytosis of the heteromeric complex [60]. It is possible that the brassinosteroid signaling pathway intersects with that of similar receptors, and that signal integration occurs via direct interactions of the components of different signaling pathways [61]. BAK1 seems to play an integrative role in

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this by its promiscuous interaction with several other LRR-receptor kinases in a ligand-depending manner. It was shown that brassinosteroids modulate plant immunity at multiple levels, and that direct interaction between the PRRreceptor FLS2 and BAK1 potentially compete with BAK1 binding to the BRI1 receptor [62]. A similar competitive scenario as assumed for the BAK1 protein–protein interactions can be imagined regarding the interaction and internalization of plant sucrose transporters. Sucrose transporters have different affinities towards their substrates, and interact with each other resulting in heterodimeric complexes. Sucrose transporter SlSUT2 is mainly retained in intracellular compartments, and interacts also with brassinosteroid signalingrelated membrane proteins like MSBP1 and BAK1 in intracellular vesicles. It is possible that components of the brassinosteroid and the sugar signaling pathway share the same endocytic compartment and reach the plasma membrane only in response to specific stimuli.

6. Outlook Regarding the direct protein–protein interaction between sucrose transporters and components of the brassinosteroid signaling pathway there are still two different scenarios to be tested: (i) on the one hand the efficiency of sucrose transport across a given membrane might depend on the efficiency of plasma membrane targeting of the transporter, potentially depending on direct interaction with an “escort” protein, or (ii) alternatively the ligandinduced internalization of a surface localized receptor complex is required to initiate a downstream signaling cascade. In both scenarios, competition between interacting proteins might affect the stoichiometry of the protein complexes. Future investigations should reveal the role of SUT phosphorylation, homo- and heterodimerization for endocytosis, exocytosis and activity of those complexes. The composition and stoichiometry of those complexes could finally determine the mode of endocytosis and their final subcellular target compartment and destiny.

Acknowledgements I would like to acknowledge Arnulf Knittel for proof-reading and Philipp Franken for helpful discussion. We apologize to all authors that could not be cited due to the limitation in space.

References [1] R. Vicentini, M. Felix Jde, M.C. Dornelas, M. Menossi, Characterization of a sugarcane (Saccharum spp.) gene homolog to the brassinosteroid insensitive1-associated receptor kinase 1 that is associated to sugar content, Plant Cell Rep. 28 (2009) 481–491. [2] F. Schroder, et al., Consequences of induced brassinosteroid deficiency in Arabidopsis leaves, BMC Plant Biol. 14 (2014) 309. [3] J. Lisso, T. Altmann, C. Mussig, Metabolic changes in fruits of the tomato dx mutant, Phytochemistry 67 (2006) 2232–2238. [4] A. Laxmi, L.K. Paul, J.L. Peters, J.P. Khurana, Arabidopsis constitutive photomorphogenic mutant bls1, displays altered brassinosteroid response and sugar sensitivity, Plant Mol. Biol. 56 (2004) 185–201. [5] S. Bancos, et al., Diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis, Plant Physiol. 141 (2006) 299–309. [6] M. Goetz, D.E. Godt, T. Roitsch, Tissue-specific induction of the mRNA for an extracellular invertase isoenzyme of tomato by brassinosteroids suggests a role for steroid hormones in assimilate partitioning, Plant J. 22 (2000) 515–522. [7] U. Schlüter, D. Köpke, T. Altmann, C. Müssig, Analysis of carbohydrate metabolism of CPD antisense plants and the brassinosteroid-deficient cbb1 mutant, Plant Cell Environ. 25 (2002) 783–791. [8] S. Hanano, M.A. Domagalska, F. Nagy, S.J. Davis, Multiple phytohormones influence distinct parameters of the plant circadian clock, Genes Cells 11 (2006) 1381–1392.

[9] A. Cano-Delgado, et al., BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis, Development 131 (2004) 5341–5351. [10] C.Y. Wu, et al., Brassinosteroids regulate grain filling in rice, Plant Cell 20 (2008) 2130–2145. [11] G.M. Symons, et al., Grapes on steroids. Brassinosteroids are involved in grape berry ripening, Plant Physiol. 140 (2006) 150–158. [12] F. Xu, Z.M. Xi, H. Zhang, C.J. Zhang, Z.W. Zhang, Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during veraison, Plant Physiol. Biochem.: PPB/Soc. Fr. Physiol. Vegetale 94 (2015) 197–208. [13] X.J. Li, et al., Overexpression of a brassinosteroid biosynthetic gene Dwarf enhances photosynthetic capacity through activation of Calvin cycle enzymes in tomato, BMC Plant Biol. 16 (2016) 33. [14] E. Eggert, et al., A sucrose transporter-interacting protein disulfide isomerase affects redox homeostasis and links sucrose partitioning with abiotic stress tolerance, Plant Cell Environ. (2015), http://dx.doi.org/10.1111/pce.12694. [15] V. Nahirnak, et al., Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition, Plant Physiol. 158 (2012) 252–263. [16] Y.Q. Zhang, et al., Brassinosteroid is required for sugar promotion of hypocotyl elongation in Arabidopsis in darkness, Planta 242 (2015) 881–893. [17] Y. Zhang, J. He, Sugar-induced plant growth is dependent on brassinosteroids, Plant Signal. Behav. 10 (2015) e1082700. [18] E. Oh, J.Y. Zhu, Z.Y. Wang, Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses, Nat. Cell Biol. 14 (2012) 802–809. [19] R. Zentella, et al., O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis, Genes Dev. 30 (2016) 164–176. [20] H. Reinhold, et al., beta-amylase-like proteins function as transcription factors in Arabidopsis, controlling shoot growth and development, Plant Cell 23 (2011) 1391–1403. [21] S. Soyk, et al., The enzyme-like domain of Arabidopsis nuclear beta-amylases is critical for DNA Sequence recognition and transcriptional activation, Plant Cell 26 (2014) 1746–1763. [22] I.G. Matsoukas, Interplay between sugar and hormone signaling pathways modulate floral signal transduction, Front. Genet. 5 (2014) 218. [23] K. Boldt, et al., Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza, J. Plant Physiol. 168 (2011) 1256–1263. [24] M. Bitterlich, U. Krügel, K. Boldt-Burisch, P. Franken, C. Kühn, The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation, Plant J. 78 (2014) 877–889. [25] M. Bitterlich, U. Krügel, K. Boldt-Burisch, P. Franken, C. Kühn, Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza, Plant Signal. Behav. 9 (2014). [26] A. Nair, S.P. Kolet, H.V. Thulasiram, S. Bhargava, Systemic jasmonic acid modulation in mycorrhizal tomato plants and its role in induced resistance against Alternaria alternata, Plant Biol. (Stuttg) 17 (2015) 625–631. [27] A. Hilou, H. Zhang, P. Franken, B. Hause, Do jasmonates play a role in arbuscular mycorrhiza-induced local bioprotection of Medicago truncatula against root rot disease caused by Aphanomyces euteiches? Mycorrhiza 24 (2014) 45–54. [28] L. Song, Q.M. Shi, X.H. Yang, Z.H. Xu, H.W. Xue, Membrane steroid-binding protein 1 (MSBP1) negatively regulates brassinosteroid signaling by enhancing the endocytosis of BAK1, Cell Res. 19 (2009) 864–876. [29] H. Kuhn, H. Kuster, N. Requena, Membrane steroid-binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula, New Phytol. 185 (2010) 716–733. [30] J.L. Nemhauser, T.C. Mockler, J. Chory, Interdependency of brassinosteroid and auxin signaling in Arabidopsis, PLoS Biol. 2 (2004) E258. [31] J.L. Nemhauser, J. Chory, BRing it on: new insights into the mechanism of brassinosteroid action, J. Exp. Bot. 55 (2004) 265–270. [32] Y. Hacham, A. Sela, L. Friedlander, S. Savaldi-Goldstein, BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers, Plant Signal. Behav. 7 (2012) 68–70. [33] N.G. Irani, S. Di Rubbo, E. Russinova, In vivo imaging of brassinosteroid endocytosis in Arabidopsis, Methods Mol. Biol. 1209 (2014) 107–117. [34] L. Fan, R. Li, J. Pan, Z. Ding, J. Lin, Endocytosis and its regulation in plants, Trends Plant Sci. 20 (2015) 388–397. [35] L. Wang, et al., Spatiotemporal dynamics of the BRI1 receptor and its regulation by membrane microdomains in living Arabidopsis cells, Mol. Plant 8 (2015) 1334–1349. [36] M. Singh, A. Gupta, A. Laxmi, Glucose and phytohormone interplay in controlling root directional growth in Arabidopsis, Plant Signal. Behav. 9 (2014) e29219. [37] S. Robert, et al., Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 8464–8469. [38] R. Toth, et al., Prieurianin/endosidin 1 is an actin-stabilizing small molecule identified from a chemical genetic screen for circadian clock effectors in Arabidopsis thaliana, Plant J. 71 (2012) 338–352. [39] X. Yang, L. Song, H.W. Xue, Membrane steroid binding protein 1 (MSBP1) stimulates tropism by regulating vesicle trafficking and auxin redistribution, Mol. Plant 1 (2008) 1077–1087.

C. Kühn / Plant Science 248 (2016) 75–81 [40] U. Krügel, et al., Transport and sorting of the Solanum tuberosum sucrose transporter SUT1 is affected by posttranslational modification, Plant Cell 20 (2008) 2497–2513. [41] J. Liesche, H.X. He, B. Grimm, A. Schulz, C. Kühn, Recycling of Solanum sucrose transporters expressed in yeast, tobacco, and in mature phloem sieve elements, Mol. Plant 3 (2010) 1064–1074. [42] U. Krügel, et al., The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase, Mol. Plant 5 (2012) 43–62. [43] S. Mongrand, et al., Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane, J. Biol. Chem. 279 (2004) 36277–36286. [44] V. Kriechbaumer, et al., Reticulomics: protein–protein interaction studies with two plasmodesmata-localized reticulon family proteins identify binding partners enriched at plasmodesmata, endoplasmic reticulum, and the plasma membrane, Plant Physiol. 169 (2015) 1933–1945. [45] A. Reinders, et al., Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element, Plant Cell 14 (2002) 1567–1577. [46] W.X. Schulze, A. Reinders, J. Ward, S. Lalonde, W.B. Frommer, Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system, BMC Biochem. 4 (2003) 3. [47] S. Mongrand, T. Stanislas, E.M. Bayer, J. Lherminier, F. Simon-Plas, Membrane rafts in plant cells, Trends Plant Sci. 15 (2010) 656–663. [48] S. Raffaele, et al., Remorin a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement, Plant Cell 21 (2009) 1541–1555. [49] M.T. Tarkka, et al., OakContigDF159.1, a reference library for studying differential gene expression in Quercus robur during controlled biotic interactions: use for quantitative transcriptomic profiling of oak roots in ectomycorrhizal symbiosis, New Phytol. 199 (2013) 529–540. [50] N. Leborgne-Castel, K. Bouhidel, Plasma membrane protein trafficking in plant-microbe interactions: a plant cell point of view, Front. Plant Sci. 5 (2014). [51] C. Kistner, et al., Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis, Plant Cell 17 (2005) 2217–2229.

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[52] N. Leborgne-Castel, T. Adam, K. Bouhidel, Endocytosis in plant-microbe interactions, Protoplasma 247 (2010) 177–193. [53] M. Frescatada-Rosa, S. Robatzek, H. Kuhn, Should I stay or should I go? Traffic control for plant pattern recognition receptors, Curr. Opin. Plant Biol. 28 (2015) 23–29. [54] K. He, et al., BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways, Curr. Biol.: CB 17 (2007) 1109–1115. [55] G. Vert, Plant signaling: brassinosteroids, immunity and effectors are BAK!, Curr. Biol. 18 (2008) R963–965. [56] L. Wirthmueller, A. Maqbool, M.J. Banfield, On the front line: structural insights into plant-pathogen interactions, Nat. Rev. Microbiol. 11 (2013) 761–776. [57] Y. Belkhadir, L. Yang, J. Hetzel, J.L. Dangl, J. Chory, The growth-defense pivot: crisis management in plants mediated by LRR-RK surface receptors, Trends Biochem. Sci. (2014). [58] Y. Belkhadir, et al., Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 297–302. [59] J. Li, et al., BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling, Cell 110 (2002) 213–222. [60] E. Russinova, et al., Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1), Plant Cell 16 (2004) 3216–3229. [61] W. Wang, M.-Y. Bai, Z.-Y. Wang, The brassinosteroid signaling network—a paradigm of signal integration, Curr. Opin. Plant Biol. 21 (2014) 147–153. [62] Z.Y. Wang, Brassinosteroids modulate plant immunity at multiple levels, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 7–8. [63] C. Zipfel, Pattern-recognition receptors in plant innate immunity, Curr. Opin. Immunol. 20 (2008) 10–16.