Molecular Properties of Disordered Plant Dehydrins- Membrane Interaction and Function in Stress

Molecular properties of disordered plant dehydrins- Membrane interaction and function in stress

Sylvia Eriksson

Molecular properties of disordered plant dehydrins

Membrane interaction and function in stress

Sylvia Eriksson

Abstract

Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experi- ments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K- segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with exten- sions (Lti30) or modified (Rab18).

Keywords: abiotic stress; dehydrin; intrinsically disordered proteins; Lea- proteins; phospholipids

Publications

This thesis is based on following papers, which will be referred to by their roman numerals.

Paper I Mouillon J. M., Eriksson S. K. and Harryson P. Mimicking the plant cell interior under water stress by macromolecular crowding: disordered dehydrin proteins are highly resistant to structural collapse. Plant Physiol. 2008, 148(4): 1925-1937.

Paper II Eriksson S. K. *, Kutzer M. *, Procek J., Gröbner G. and Harryson P. Tuna- ble membrane binding of the intrinsically disordered dehydrin Lti30, a cold- induced plant stress protein. Plant Cell 2011, 23(6): 2391-2404.

Paper III Eriksson S.K., Eremina N., Barth A., Danielsson J. and Harryson P. Mem- brane-induced folding of the plant stress dehydrin Lti30. Plant Physiol. 2016, 171(2): 932-943.

Paper IV Eriksson S.K., Danielsson J and Harryson P. Membrane binding of disordered plant dehydrins is tuned by phosphorylation and coordination of Ca2+ and Zn2+ ions. Manuscript

Paper V Eriksson S.K. and Harryson P. Rab18 dehydrin –a stressed induced conditional peripheral membrane protein. Specific interaction with PI(4,5)P2 Manuscript

* These authors contributed equally

Paper I, II and III are Copyright by the American Society of Plant Biologists.

Table of Contents

4'3"" ''''''''''''''''''''''''''''''''''''''''''36 4'4"" '''''''''''''''''''''''''''''''''''''''''''''''''''''37 5'3 ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''39 5'4 ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''39 5'5 # '''''''''''''''''''''''''''''''''''''''''''''''''3@ 6'3 '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''43 SDPDQ&413!"3"!2315"&04 5&0$4 %2"310"4DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDQP SDPDR&413!"32315"&04E&05"3 5&108&5%)")30"4DDDDDDDDDDDDDDDDDDDDDDDDDQQ 6'4 ! &"# #%'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''45 7'3 ! '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''46 7'4 #'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''47 TDQDP")30"&05"3 5&109!"%9!3&04DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDQU TDQDQ0@9)" 3912315" 5&10 5&7&599!"%9!3&04DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDQU TDQDR145F5304(5&10(2%142%139(5&101#!"%9!3&04DDDDDDDDDDDDDDDDDDDDDDDDQU TDQDS%"!"%9!3&044)"5(4 7"0$"34DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDQU 7'5 # ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''49 8'3 # &&%) ! "'''''''''''''''''''''''''''''''''''''''''''''''''''''''52 UDPDQ&)& '&0$ "((6(3!"%9!35&10DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDRQ 8'4 #''''''''''''''''''''''''''''''''''''''''55

8'552! # ''''''''''''''''58 UDRDP,5&RI&4215"05(&2&!&0!"38&5%%&$%7"4& ("$$3"$5&10 2312"04&59DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDRV UDRDQ,5&RI %0$"45%"(&2&!2%4"5304&5&105")2"3563"8%"0 &0!&0$512%142%1(&2&!7"4& ("4DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDRV UDRDR,5&RI&0!6 "4536 563"6210)")30"&0!&0$DDDDDDDDDDDDDDDDDDDDDDDDDRW 8'6 # ## 4A $4A'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''5@ UDSDP"%9!3&02%142%139(5&10560"45%")")30"&05"3 5&10DDDDDDRX UDSDQ1QY0!0QY&104 113!&05"!"%9!3&04&0!&0$512%142%1(&2&!4 DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDRX UDSDR%142%139(5"!!"%9!3&04&0!&0$(&2&!4&05%"23"4"0 "1#1QY 0!0QYF 1)&0"!"##" 54ADDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDSP 8'7 3@ #%( " ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''63

UDTDPPW&0!&0$51&GSBTHQ&4 113!&05"!95%"9F4"$)"05 51$"5%"38&5%#(0'&0$3$&0&0"4DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDSR

UDTDQ:18 0PW!&4 3&)&05""58""0!&##"3"05&Q0!&ADDDSR UDTDRPWC 10!&5&10(2"3&2%"3()")30"2315"&0A<)1!"(1# 2144&("#60 5&101#PW&05%"451)5$63! "((4DDDDDDDDDDDDDDDDDDDDDDDDSS !"# #"" ! $ #!"

Abbreviations

Lea Late Embryogenesis Abundant PC Phosphatidylcholine PG Phosphatidylglycerol PS Phosphatidylserine PA Phosphatidic acid PI Phosphatidylinositol PIP Phosphatidylinositol phosphate

PIP2 Phosphatidylinositol bisphosphate LUV Large Unilamellar Vesicles ABA Abscisic acid CD Circular Dichroism NMR Nuclear Magnetic Resonance FTIR Fourier Transform Infrared Spectroscopy

Amino acids A Ala Alanine C Cys Cysteine D Asp Aspartate E Glu Glutamate F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine

1. Introduction

During recent years the world has been exposed to abnormal weather fluctu- ations causing drought fields and a severe damage of food production. Ex- tended periods of drought have occurred mainly in Africa, North America, China, Australia and Brazil. As the same time as arable crops are reduced, the world’s population and demand for food are increasing.

Plants are on a regularly basis naturally exposed to cycles in temperature, soil salinity and drought. When these conditions become stressful to the plant, it is called abiotic stress, which may affect both plant growth and crop production. This thesis elucidates the function of a group of plant stress proteins, the dehydrins, studied at a molecular level in an attempt to reveal their role in plant stress survival.

The outer border of a cell consists of a cell membrane that distinguishes it from its surroundings. Inside are a wide variety of proteins that perform different cellular reactions such as enzymes and proteins, which control gene expression. Alterations in temperature, water and salinity lead to a reduction in cellular water content to which the cell is sensitive. Even small changes in water levels affect the performance of both cell membranes and proteins. When conditions are changing fast and growth conditions are poor, the plant is considered to be under stress. If the stress persists the plant may finally die. However, desiccation can also be a natural condition such as in pollen or seeds (Ingram and Bartels 1996).

Most plants are rooted to the ground and cannot move when they are exposed to stress; they have to stay and adopt. They have therefor developed several protection mechanisms to maintain cellular functions. This response system includes: inductions of different stress proteins like the dehydrins and chaperones and molecules that maintain cell turgor (sugars, betaine, proline) (Ingram and Bartels 1996).

Expression of the plant specific dehydrins is induced during different water stresses. Dehydrins are intrinsically disordered proteins and lack a fixed 3D structure. The precise in vivo function of dehydrins is still unclear, but under periods of stress, most plant produce high amounts of dehydrins, which must reflect an important role in plant survival. This proposal is experimentally

11 confirmed, since plants overexpressing dehydrins have been shown to have a higher survival rate when stressed compared to wild plant types (Houde et al., 2004; Puhakainen et al., 2004; Brini et al., 2007).

The focus of this thesis lies within the discussion of the molecular functions of the dehydrins, in particular membrane binding. The following chapters include a short introduction to plant stress and stress defense. Finally, the results are discussed with regard to the following matters:

1. Are the dehydrins stable in their disordered state? What structural effects can be detected in the presence of different crowding agents that mimic stressed cells?

2. Regarding membrane binding of dehydrins, which dehydrins bind and how? A model is proposed where the K-segment together with the positive net charge of the dehydrins is the central driving force for membrane interaction.

3. The additive effects of phosphorylation and calcium and zinc coordination in lipid binding of the dehydrins.

4. The specific interaction of the dehydrin Rab18 to the phosphatidylinositol lipids PI(4,5)P2 and PI(5)P. Finally, a mechanistic role for Rab18 in guard cells opening and closing during drought is proposed.

12 2. Water-related stress in plants

Water encompasses 90 % of the mass in most plants and even a minor decrease in this level will lead to the cellular adjustment necessary for sus- tained growth.

The way in which different plant species respond to stress varies, and each response is often multifaced. Therefore, only a short overview of plant stress will be given below before discussing the dehydrins role within. In brief, each plant species has its own kind of stress-induced proteins, and various forms of stress will express specific response proteins. Many plants also have different stress thresholds and what is damaging for one species is a perfect condition for another. To make the picture more complex, most plants have developed several protective systems that can be activated in parallel (Ingram and Bartels 1996).

Low temperature, salinity rich soil and drought are all embraced in the class of water related stress. Signals for cellular stress are probably perceived in the plasma membrane through the activation of different receptors, which leads to metabolic adjustments and genetic adaption (Mahajan and Tuteja 2005).

Cold stress comprises two types of emphases: freezing and low temperature. Plants like maize, cotton, banana and tomato are plants that are very sensitive to cold and negative effects can progress at temperatures below 10-15 °C (Mahajan and Tuteja 2005). At low temperatures, adaptation responses are activated and cells become more tolerant of freezing (Mahajan and Tuteja 2005).

Salt stress is normally equal to high concentrations of Na+, and it gives rise to hyperionic and hyperosmotic stress. The salinity-effect leads to dehydration within the cell. High salt concentration also changes soil porosity, which makes it difficult for roots to acquire water and nutrients (Mahajan and Tuteja 2005).

Drought stress may arise from an inadequate amount of rainfall, soil mois- ture depletion or when evaporation from leaves due to high temperature or winds exceeds the influx of water.

2000). In unsaturated fatty acid, double bonds create a kink in the fatty acid tail that promotes higher flexibility and Tm (Rawicz et al., 2000).

Approximately 20 % of the mass in a hydrated membrane bilayer consists of bound water molecules (Crowe et al., 1992). Drought physically affects membrane lipids, hence two primary effects emerge: a change in lipid phase transition temperature and membrane fusion (Crowe et al., 1992). How can drought change the fluid-to-gel phase? When water is removed the lipid head groups are packed tighter together, which leads to an increase in the van der Waal´s interaction between the fatty acid chains. This will in turn lead to a massive increase in the lipid phase transition temperature (Crowe et al., 1992). A bilayer that is rehydrated in the gel phase is well known to be leaky during rehydration (Blok et al., 1975), and a key response for plants during dehydration and rehydration is to protect and diminish changes in the phase transition to prevent leakage.

Freezing affects cells the same way as drought since this causes water to form solid ice crystals. Freezing may cause lesions in the bilayer that could be lethal to the cell (Uemura et al., 1995). To prevent this, plants can adjust the lipid composition of their membrane in response to various stresses. For example, the plasma membrane in the leaves of Arabidopsis thaliana consists of a total of 46.8 mol% phospholipids under normal growth conditions, but the content of phospholipids increases when cold (2 °C for 1 week) to 57.1 mol%, and the proportion of di-unsaturated lipids is also increasing (Uemura et al., 1995).

High Na+ concentrations will create high osmotic pressure, leading to disor- ganization and finally leakage of the membrane (Mahajan and Tuteja 2005).

2.2 How water stress affects proteins

Proteins are large polypeptide chains. Folded proteins mainly rely on the hydrophobic effect to retain a stable structure and be in the functional state. Hence, at low water levels many proteins malfunction, and some are degraded or refolded. Chaperones are activated to aid refolding, and proteases are responsible for a catalytic breakdown of proteins if needed. Both chaperones and proteases are up-regulated during water stress (Schaffer and Fischer 1988; Guerrero et al., 1990; Wang et al., 2004). Under desiccation stress the translation rate of proteins is relatively low, and the plant is more reliant on restoring its proteins than under normal conditions (Cooke et al., 1979).

15 At low temperature the hydrophobic forces that keep multimeric enzymes together are reduced. The result is that many enzymes dissociate into their subunits and may not again reassemble into a functional quaternary structure. The loss of enzyme activity will not only affect reactions directly but also inhibit normal protein transcription and translation rates (Guy et al., 1998).

High salt strength mainly affects proteins in two ways. First, the protein surface changes, making the protein less soluble, and this affects the interaction driving forces (Kurnik et al., 2012). Second, the water shell that sur- rounds the protein is dissolved, which will lead to precipitation of the protein (Arakawa and Timasheff 1984).

16 3. Plants response to dehydration

3.1 Osmotic adjustments Plants adjust their osmosis during stress through the production of various soluble sugars and compatible solutes (Bohnert et al., 1995).

Sugars The production of sugars have several outcomes in plant cells, for instance, they are known to inhibit leakage to stabilize membranes, maintain cell turgor and protect proteins from dehydration stress (Madden et al., 1985; Kaplan and Guy 2004; Valluru and Van den Ende 2008). Enzymes responsible for sugar metabolism increase during drought stress (Ingram and Bartels 1996). It is not uncommon for plant species to produce unique sugars for each stress type (Dinakar and Bartels 2013).

Amino acids During drought and salinity stress, an increase of certain amino acids is seen, mainly proline and γ-amino butyric acid (GABA) (Renault et al., 2010). Proline not only act as an osmolyte, it also binds dangerous oxygen radicals (Krasensky and Jonak 2012). GABA is a non-protein amino acid that regu- lates plant growth during biotic (a stress caused by another living organism, for example fungi) and abiotic stress (Ramesh et al., 2015).

3.2 Abscisic acid Abscisic acid (ABA) is a multifaceted plant phytohormone produced during drought stress. It acts as a signal for gene expression of kinases, phosphatas- es and ABA receptors, all with possible roles in maintaining the cell structure under stress (Zhu 2002). In addition, ABA also induces stress related proteins, including certain dehydrins (Nylander et al., 2001). One major effect of ABA is to endorse stomatal closure, see Figure 2. Stomata guard cells are specialized cells that can open and close to regulate gas exchange (Kim et al., 2010). During active photosynthesis these cells are open and the plant can take up CO2 while O2 and water vapor outflow. During drought, the loss of water is devastating and plants have a complex signal system to control the closing of the guard cells (Sirichandra et al., 2009).

17 protects citric synthase (CS) and lactate dehydrogenase (LDH) from aggregation and inactivation during freezing and drought stress (Goyal et al., 2005).

Lea Group 3 Group 3 Lea-proteins are characterized by a several-repeated 11-amino-acid segment (TAQAAKEKAGE) and are found in plants, nematodes and pro- karyotes (Dure 1993). They are disordered, but change the structure to an α- helix in the presence of sucrose, glycerol, methanol or when dried (Battaglia et al., 2008). For example, cold induced chloroplastic COR15a from Ara- bidopsis thaliana, adopt an α-helix structure when dried. In its α-helical state COR15a can bind to membranes (Thalhammer et al., 2010). COR15a was early suggested to have a protective role of the bilayer (Steponkus et al., 1998).

19 4. Intrinsically Disordered Proteins

Archetypically, the main hypothesis is that a polypeptide folds into an or- dered 3D structure and then becomes functional. To reach its native state, a folded protein is reliant on the hydrophobic core in combination with stabilizing hydrogen interactions together with other electrostatic forces (Uversky 2009). When a folded protein unfolds, the function is often lost with the loss of structure.

Intrinsically disordered proteins (IDPs) lack a well-defined 3D-structure and are in this respect the opposite of folded proteins. Nevertheless, the proteins have been proven functional and regulate many cellular reactions. Disor- dered proteins usually contain a low quantity of hydrophobic amino acids such as the following: Trp, Phe, Ile, Leu, Val and Cys. They also typically possess a high number of hydrophilic and charged amino acids: Ala, Arg, Gly, Ser, Pro, Gln and Lys (Dunker et al., 2001; Uversky 2009). The usually high net-charge of these proteins is suggested to contribute to an electrostatic repulsion between residues, and this leads to a more extended polypeptide. This, in combination with a natural lack of hydrophobic amino acids means that the driving force for folding is low (Uversky et al., 2000).

The disordered conformation can embrace the entire protein or only cover partial regions. A minimum stretch of 30 amino acids is required to qualify as a disordered region (Tompa 2012). According to prediction, they com- prise 10-35 % of proteins in prokaryotic cells and 15-45 % of proteins in eukaryotic cells (Tompa 2012). Single-cell eukaryotes have the highest number of disordered proteins among organisms (Pancsa and Tompa 2012). In Arabidopsis thaliana, 8 % of the proteins are predicted to be fully disordered and 29 % partly disordered (Dunker et al., 2000).

Some IDPs undergo a disorder-to-order transition upon binding, i.e. they fold when they bind to their target molecule (Kriwacki et al., 1996; Dunker and Obradovic 2001). However, many stay disordered or only adopt a local secondary structure when they bind to their target (Borg et al., 2007; Sigalov and Hendricks 2009).

20 Due to the flexible structure, the structural behavior of IDPs is difficult to monitor and the usual methods used include: Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD), Fourier Transform Infrared Spectroscopy (FTIR), Small Angle X-ray Scattering (SAXS) and Small Angle Neutron Scattering (SANS).

4.1 Functions of disorder proteins How come the plant cell can induce such a high quantity of IDPs, such as the Lea-proteins, and for what purpose? Specific functions of many disordered proteins are still to be found, but several examples of their role in cell signaling, cell cycle control, and transcriptional and translational regulation have been verified (Wright and Dyson 1999; Dyson and Wright 2002). Binding targets such as metals, proteins, membranes and nucleic acids have been reported (Davidson et al., 1998; Bourhis et al., 2004; Love et al., 2004; Tompa and Csermely 2004; Uversky 2011). It seems as if disordered proteins can accomplish most protein functions, but with one exception, enzyme activity. This occurs naturally since most enzymes require a fixed and folded active site.

4.1.2 Disordered proteins acting as chaperones Chaperones are proteins or RNA molecules that assist folding or prevent the aggregation of other macromolecules (Csermely 1997). For plants exposed to dehydration stress, functioning chaperones are mandatory for survival. Many chaperones are partially or totally disordered (Uversky et al., 2005). Some disordered proteins including the Lea-proteins, are suggested to act as chaperones by minimizing the collision rate of unfolded proteins. This would slow down aggregation and formation of protein clusters, since they act as molecular shields (Goyal et al., 2005).

For example, the disordered anhydrin from the nematode Aphelenchus avenae protects the activity of citrate synthase and keep it from aggregating during drought. Through NMR studies it was determined that the anhydrin did not interact directly with the enzyme, and the authors suggested that it functions as a molecular shield (Chakrabortee et al., 2010). Another example is the Lea-like protein CDeT11-24 from the resurrection plant Craterostig- ma plantagineum that protects the activity of both citrate synthase and lactate dehydrogenase under desiccation stress. Whether CDeT11-24 works as a molecular shield or a molecular chaperon that interacts directly with the enzyme was not tested experimentally (Petersen et al., 2012).

21 structural change is also connected to translocation from soluble to membrane bound (Lee et al., 2002; Zhu and Fink 2003).

4.2 Advantages for being disordered: Why do disordered proteins not fold directly? Since many IDPs seem to at least partly fold as they bind to their cellular targets, how come they do not fold directly? What are the biological advantages of being disordered? Gunasekaran et al. 2003 suggested that IDPs take up 15-30 % less space compared to folded proteins; this is due to the fact that a stable monomeric protein with the same extensive interface needs to increase their size 2-3 times. This is not due to actual protein size, but the putative promiscuity is suggested to be more of an advantage. For example, some IDPs bind multiple targets and adopt several different conformations depending on their interaction partner, so-called moonlighting. Each fold may then be connected to a different function (Kriwacki et al., 1996; Cheng et al., 1999; Tompa et al., 2005). In this way IDPs conserve energy and minimize crowding effects for the cell.

Due to a relatively open structure, IDPs become easier targets for proteases, and they are degraded more than 100 times faster than folded proteins. Hence, this means that they can decrease in concentration fast, which can be advantageous for signaling molecules that want to get back to resting levels (Dunker et al., 2008; Kovacs et al., 2008).

23 5. Dehydrins

Dehydrins (Lea group 2) are plant specific IDPs and share molecular charac- teristics with Lea-proteins and IDPs in general. Diverse dehydrins are specifically up-regulated, either by low temperature, freezing, drought, salinity or by the plant phytohormone ABA.

5.1 Conserved segments Dehydrins vary in size from 9.6 kDa to 70 kDa (Labhilili et al., 1995; Graether and Boddington 2014). The sequence contains highly conserved segments are repetitively scattered: the K-segment (EKKGIM- DKIKEKLPG), the S-segment (SSSSSSSSDS), the Y-segment ((V/T)D(E/Q)YGNP) and the charged peptide (EEDEDGEKKKKEKKKK- KI), see Figure 4 (Close 1996; Mouillon et al., 2006). Although highly conserved, no function has been directly linked to any of the segments. Howev- er, they form the base for the classification of the dehydrin subfamilies. This is determined by how the segments are organized in the sequence: SKn, Kn, YnSKn and YnKn (Close 1996). There is no clear correlation between the dehydrin subclass and the kind of stress they are expressed upon. Some broad generalizations can be made: the Kn dehydrins seem to often be induced by low temperature, while the YnSKn dehydrins appear to be induced by drought (Close 1996). The correlation of function and segment is not clear and needs to be investigated further. The main proposal for function is that the K-segment binds to membranes (Close 1996). Indeed, experimental data suggest that K- segments are involved in membrane interaction (Koag et al., 2009; Clarke et al., 2015) and Kn dehydrins seem to bind to membranes more often than others subclasses (Graether and Boddington 2014).

By definition, all dehydrins contain at least one copy of the K-segment, but this segment is often repeated several times (Neven et al., 1993; Close 1996). The exact amino-acid composition of the K-segment in different dehydrins varies somewhat (Close 1996; Graether and Boddington 2014). The K-segment was proposed early to form an amphipathic α-helix that interacts and stabilizes membranes or proteins (Dure 1993; Danyluk et al., 1998; Koag et al., 2003). The short peptide of the K-segment in solution, in the

24 absence of binding partners, was found to be fully disordered (Mouillon et al., 2006; Atkinson et al., 2016).

The Ser-rich stretch that exists in certain dehydrins is called the S-segment. This segment is suggested to contain phosphorylation sites for kinases. Sev- eral dehydrins have been shown to be phosphorylated both in vivo and in vitro (Plana et al., 1991; Jiang and Wang 2004).

The Y-segment is usually found at the N-terminal in many dehydrins. It contains similarities to the sequence of the nucleotide-binding site of chap- erons in bacteria and plants. However, evidence for such activity in the Y- segment has not yet been reported (Martin et al., 1993).

The so-called charged peptide is found in some dehydrins that contain the S- segment, and it pursues this sequence (Mouillon et al., 2006). The charged peptide shares similarities with the mammal chaperon HSP90, and it might have a possible chaperon effect (not yet proven).

K-segment: EKKGIMDKIKEKLPG

S-segment: SSSSSSSSDS

Y-segment: (V/T)D(E/Q)YGNP

Charged peptide: EEDEDGEKKKKEKKKKKI

Figure 4. The amino-acid sequences of the conserved segments: the K- segment, the S-segment, the Y-segment and the charged peptide.

5.2 Functions of dehydrins Although several characteristic features have been established for the role of dehydrins in stress defense, the in vivo function is not fully known. Howev- er, several studies have shown that they bind and stabilize membranes, acting as molecular shields or scavenging metals and reactive oxygen species.

25 5.2.1 Membrane interaction by dehydrins One of the most commonly proposed functions for dehydrins is to stabilize membranes. In line with this, several dehydrins have been observed to interact with membranes. For example, DHN1 from Zea maize interacts with PA lipids, and upon binding it forms an α-helical structure (Koag et al., 2003). The Thellungiella salsuginea dehydrin 1 (TsDHN-1) was also observed to interact with liposomes and upon binding it increased in its secondary structure (Rahman et al., 2010). An extended section about dehydrins and membrane interaction can be found in the section “Result and discussion”.

5.2.2 Enzyme cryoprotection activity by dehydrins Several in vitro studies have examined a possible role of cryoprotective activity by the dehydrins. For example, one dehydrin from Birch was observed to protect the activity of α-amylase, an enzyme involved in starch degrada- tion, from cold (Rinne et al., 1999). Numerous dehydrins have been shown to protect the enzyme lactate dehydrogenase (LDH) in freeze-thaw cycles (Kazuoka and Oeda 1994; Wisniewski et al., 1999; Hara et al., 2001; Bravo et al., 2003; Reyes et al., 2008). This is also true for a K2 dehydrin from Vitis riparia. In this case no clear interaction between dehydrin and the enzyme could be detected, and the authors therefore suggested that the dehydrin acts more as a molecular shield than with classical chaperone activity (Hughes and Graether 2011).

5.2.3 Post-translational phosphorylation of dehydrins Many proteins undergo phosphorylation, and this modification can affect binding sites and by that act as an on and off regulator. Several dehydrins are phosphorylated both in vivo and in vitro, such as the SK3 dehydrin ERD14 from Arabidopsis thaliana (Alsheikh et al., 2003). Also, the dehydrin Rab17 from maize is phosphorylated both in vitro and in vivo and this have been proposed to regulate its localization within the cell (Jiang and Wang 2004). A stressed plant cell produces different kinases, and phosphorylation might regulate the dehydrin activity. In vitro studies show that different types of kinases seem to be more correlated to dehydrin net charge and not to the conserved segments (Paper II, Paper III).

5.2.4 The dehydrins as metal scavengers Dehydrins interact with various metals, and a suggested function for them is to scavenge metals that may be released upon stress conditions to avoid production of hydroxyl radicals (Graether and Boddington 2014). The four de-

26 hydrins in this thesis – Cor47, Lti29, Lti30 and Rab18 – all bind different metal ions with more or less the same affinity, an interaction that is coordinated by histidines (Svensson et al., 2000). There are also examples of more specific metal binding among dehydrins, such as the citrus dehydrin CuCOR15 that binds strongly to Fe3+, Ni2+, Cu2+ and Zn2+ but more weakly to Mg2+, Ca2+ and Mn2+ (Hara et al., 2005).

5.3 The dehydrins in this thesis This thesis contains studies of the four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47, Lti29, Lti30 and Rab18. They are all disordered in solution and structurally unaffected by the presence of metals or by the increase of protein concentration (Mouillon et al., 2006). Temperature changes do not promote any classical structural transitions (Mouillon et al., 2006). Although it has been shown that they contain PPII helix regions (Mouillon et al., 2006), the PPII helix is a secondary structure that lacks internal hydrogen bonds and may instead be stabilized by water molecules (Rucker and Creamer 2002). This might be a way for the cell to store water.

DHN YnSnKn Stress Localization Localization Unstressed Stressed

Cor47 SK3 L.T. V.T., R.T. G.S.

Lti29 SK3 L.T., ABA, Salt V.T., R.T. V.T., G.S., R.T.

Lti30 K6 L.T. N.D. V.T., G.S.,

Rab18 Y2SK2 ABA, Drought Nucleus in S.G.C. S.G.C., V.T., R.T., V.T.

Table 1. The accumulation of the dehydrins Cor47, Lti29, Lti30 and Rab18 in stressed and unstressed Arabidopsis thaliana. V.T.: Vascular tissue, S.G.C.: Stoma- ta guard cells. R.T: Root tip, G.S.: General staining L.T.: Low temperature, N.D.: Not detected. Data from Nylander et al., 2001.

Cor47 and Lti29 are acidic SK3 dehydrins. Cor47 contains 265 amino acids, has a pI of 4.8 and its expression is highly induced by low temperature (Nylander et al., 2001). Lti29 contains 260 amino acids and has a pI of 5.1. It accumulates in cells stressed by low temperature but also by ABA and high salt concentration. The accumulation of both Cor47 and Lti29 is low in unstressed plants (Nylander et al., 2001), see Table 1.

Lti30 is a highly positively charged protein with 6 K-segments, equally dis- tributed in the amino-acid sequence. It consists of 193 amino acids and has a

6. Results and discussion

6.1 Are dehydrins disordered in vivo? –Structural investigation and the effect of macromolecular crowding Why do plants express the disordered dehydrins in such high concentration and for what purpose? To answer these questions we first have to ask: Are dehydrins disordered and fully flexible proteins in vivo? The environment in a test tube differs markedly from the milieu in a living cell that normally contains a high concentration of different macromolecules and metabolites. This includes proteins, carbohydrates and nucleic acids, and the concentration within a cell (under normal conditions) can be as high as 400 g L-1 (Zimmerman and Trach 1991). Such high concentration of molecules leads to a reduced volume of solvent in the cytosol, a phenomenon that is called macromolecular crowding, and this can have an effect on protein structure (Ellis 2001; Minton 2005). Under stress conditions cellular concentrations are higher than normal and a concentration of more than 900 g L-1 has been measured (Ellis 2001; Bryant et al., 2005). Dehydrins are induced and have to be functional during stress. This usually means extreme crowded cellular conditions. These crowded conditions are known to influence protein structure, and even though dehydrins are disordered in the test tube, there structure should be investigated under conditions that mimic those inside stressed cells.

Two classes of crowding molecules are frequently used: proteins and synthetic polymers. These two classes have different effects on protein structure. High concentrations of proteins are often shown to destabilize interactions within the protein secondary structure (Qu et al., 1998). To mimic cellular crowding, Miklos et al., 2011 used BSA and lysozyme. This affected the globular protein CI2 by destabilizing electrostatic interactions and other nonspecific interactions (Sarkar et al., 2014). This is probably due to lysozyme being highly positively charged, which proteins in the cytoplasm typically are not. In a recent study by Danielsson et al., super oxide dismutase (SOD1) was destabilized in the presence of lysozyme, while the neutral BSA had a slightly stabilizing effect. This was all due to electrostatic interactions (Danielsson et al., 2015). Proteins with a negative net charge act as stabi-

Disordered proteins can be divided into two classes according to crowding: foldable and non-foldable, see Figure 6. Foldable IDPs adopt structure in the presence of crowders and possibly within cells. Non-foldable IDPs retain most of the disordered character even after addition of crowding agents (and possibly within the cell) (Uversky 2009). In this classification system the dehydrins in this study belong to the latter group.

6.1.2 Mimicking cellular dehydration Structural response of dehydrins under dehydration was tested with molecules that coordinate water, such as PEG, glycerol and in plant naturally occurring sugars (sucrose and glucose), see summary of results in Table 2. Again, only minor structural changes were detected, with one exception: a high concentration of glycerol and PEG induced the α-helical structure of all three dehydrins tested, and in particular Cor47. Both PEG and glycerol coordinate high proportions of water molecules and these results were compared to actually completely dehydrated dehydrins, see Figure 7. When the four dehydrins, Cor47, Lti29, Rab18 and Lti30, were dried under air they all adopted helical structure, see Paper IV. Interestingly, after adding solvent to the dried dehydrins they all retrieved their disordered character, see Figure 7 (Paper IV). Dehydrins in this study display similarities with other Lea-proteins that induce helical structure after drying, for example COR15A (group 3 Lea) (Thalhammer et al., 2010). In direct con- trast to this, on behalf of globular proteins, dehydration is connected to loss of structure (Dong et al., 1995). The disordered protein Sml1 was dried under the same condition as the dehydrins. Interestingly, Sml1 adopted β- structure when water was removed, but when the rehydrated protein did not regain its native form and was found to be more similar to folded proteins (Paper IV). This phenomenon might be unique for Lea-proteins and phys- iologically relevant since they are induced at low water levels.

Dehydration agents Cor47 Lti29 Lti30 Glycerol (80 %) +++ ++ ++ PEG 40 (50 %) ++ + + PEG 60 (50 %) ++ + + Glucose (80 %) + + n.t. Sucrose (80 %) - - - Table 2. Summary of the structural changes in the dehydrins affected by the dehydration agents: glycerol, PEG 40, PEG 60 or in the presence of the sugars, glucose or sucrose. +++ strong helical transition, ++ helical transition, + minor helical transition, - no structural change, n.t.: not tested

The four dehydrins were tested with four different lipids with various net charges: Phosphatidic acid (PA), Phosphatidylcholine (PC), Phosphatidyl- glycerol (PG) and Phosphatidylserine (PS), see Figure 8. PS and PG are both monoacidic phospholipids that have one negatively charged phosphate bound to the headgroup that is linked to the lipid backbone. PA has one negatively charged phosphate but no headgroup. PC is zwitterionic with a positively charged headgroup attached to the negative phosphate, see Figure 8. The results show that the lipid binding dehydrins bind stronger to negatively charged lipids PG, PS and PA, see Table 4.

The conclusion is that the membrane binding of dehydrins is driven by electrostatics controlled by positive protein net charge in combination with negatively charged lipids.

Both lipid-binding dehydrins in this thesis (Lti30 and Rab18) assemble vesicles into macro-sized aggregates as they bind, and sometimes these aggregates are detectable with the naked eye. This reaction is reversible, and the catalytic breakdown of Lti30 with trypsin dissolves the aggregates (Paper II). To observe leakage of the vesicles in the presence of Lti30, vesicles were filled with the fluorescent dye calcein. The results show that vesicles are intact and Lti30 does not cause leakage of vesicles. The aggregation propensity is used to study what determines the interaction between dehydrin and phospholipid vesicles (Paper II, Paper III, Paper IV and Paper V) and further how the binding can be modified by tuning the conditions (Paper II, Paper IV and Paper V). Stopped-flow scattering was used to monitor lipid binding. In addition to this, light microscopic pictures were taken of vesicle aggregates.

Lipid Lti30 (K6) Rab18 (Y2SK2) Lti29 (SK3) Cor47 (SK3)

PC (zwitterionic) + - - -

PG (-1) ++++ + - - PA (-1) +++ + - - PS (-1) ++++ + - -

Table 4. Summary of lipid binding dehydrins. Biacore results, measured relative response unit (R.U.). – 0-500 R.U., + 500-1000 R.U., ++ 1000-1500 R.U., +++ 1500- 2000 R.U., ++++ 2000-2500 R.U.

35 6.3 Lipid binding of Lti30 is driven by protonation of histidines Since neither the K-segment alone nor all dehydrins interact with lipids, what is required for a dehydrin to bind? In Lti30, histidine (His) pairs flank all K-segments, see Figure 5. His is an amino acid that can undergo protonation and deprotonation at physiological pH. The pKa value of His side-chain is approximately 6.0, and if His are essential in the interaction binding will be sensitive to changes in pH. Results from both Biacore and vesicle aggregation experiments demonstrate that the interaction is indeed pH dependent. Lti30 aggregates vesicles heavily at pH 4, while at pH 9 aggregates are di- minished.

In Paper II a model demonstrates the pH dependent lipid binding. The model shows that when pH is low and His in Lti30 is protonated the lipid binding affinity is higher compared to when the His are not protonated. In the model, a titration point with a pKa value of 6.5 can be estimated, a value very close to the pKa for a free His in solution, which tells of the importance of His in Lti30 membrane binding.

Also, the K-segment as a short peptide with the two pairs of flanking His binds and aggregates vesicles, while the peptide without His fails to interact. It should be noted that the amino-acid sequences of the K-segments in Lti30 differ from the general consensus concerning K-segments that have an overall higher positive net charge (Graether and Boddington 2014). In Paper III, the role of net charges of the different K-segments in membrane binding was tested. The result shows that the positive net charge of the segment is crucial for binding to phospholipid vesicles. Hence, the low net charge of the Lti30 K-segments actually allows for His to work as an on/off switch for membrane binding.

This behavior shares similarities with the FYVE zinc finger domain that takes part in several signaling pathways and interacts with phosphatidylinositol-3-phosphate (Wywial and Singh 2010). The binding of the FYVE domain to membranes is regulated by the protonation of His pairs due to an acidic intracellular milieu (Lee et al., 2005). Local low pH can be generated at the plasma membrane by the negative electrostatic potential that attracts protons from the cytosol, leading to a local decrease in pH at the membrane surface and a significant pH gradient in the surroundings (Uversky 2009).

By comparing the net charge of different dehydrins, one can predict which dehydrins bind membranes, based on the hypothesis that electrostatic attrac- tion is the major determinant for membrane binding (Paper III). In short, the more positively charged dehydrin, the better membrane binder.

36 6.3.1 Lti30 is a potent lipid binder with a high vesicle aggregation propensity By monitoring binding between Lti30 and vesicles with various negative net charge, the lowest vesicle charge needed to aggregate vesicles to an observ- able amount was found to be only 2 % DOPG (DOPC:DOPG 98:2) (Paper III). Binding, measured by stopped flow scattering, shows an optimum at 6 % of negatively charged phospholipids. In vivo, membranes consist of a substantially higher concentration of negatively charged phospholipids (Furt et al., 2011). Notably, the ratio is even increasing during stress (Uemura et al., 1995; Furt et al., 2011). The inner leaflet of the plasma membrane contains the highest negative net charge, but acidic phospholipids are present in all cell membranes (van Meer 1998; Tjellstrom et al., 2010). Since the experiments monitor the aggregation of vesicles, a reaction that is secondary to binding, it is likely that binding as the initial reaction takes place at an even lower net charge than 2 % PG. Another interesting detail is that Lti30 can cluster vesicles even upon increasing the DOPG content to 40 % at a point where the inter-vesicular repulsion must be substantial (Paper III).

6.3.2 Lti30 changes the lipid phase transition temperature when binding to phospholipid vesicles. What is the effect on the membranes of Lti30 binding? To test if Lti30 trig- gers leakage of membranes after the binding and aggregation of vesicles, vesicles were filled with the fluorescent dye calcein to monitor any leakage. The result shows that the binding of Lti30 does not cause any leakage and that vesicles are intact even after aggregation (Paper II). The catalytic breakdown of Lti30 with trypsin also shows that the vesicle aggregation can be dissolved and is reversible (Paper II).

In Paper II, the role of Lti30 as a membrane stabilizer is studied. Scanning calorimetric studies show that Lti30 decreases the phase transition temperature (Tm) of phospholipid vesicles by 3 °C. Similar results were later also shown for the Vitis riparia K2 dehydrin that decreased the transition temperature of a membrane by 3 °C (Clarke et al., 2015). The question then arises, how can a decrease in Tm protect the membrane at lower temperature? i: The decrease of the Tm increases the motion of the lipid bilayer at lower temperatures and by this protects membranes from leakage. The decrease of Tm also keeps the biological functional liquid phase at lower temperatures. ii: During dehydration, a state developed by low temperature and freezing there will be an increase of the Tm in the phospholipids, and this event will lead to leakage during rehydration (see section 2.1 “how water stress affects

37 the cell membrane”). By decreasing the Tm, Lti30 might possibly stabilize the bilayer during rehydration. This effect has in several trials been demon- strated with different sugars. For example, trehalose interacts with the phospholipid headgroups and decreases the Tm during drying by keeping the headgroups separated and decreasing the van der Waals interactions between the acyl chains of the lipids (Crowe 2002).

6.3.3 Lti30 induce structure upon membrane binding Lti30 is a fully disordered protein in solution in the absence of vesicles (Mouillon et al., 2006). The helical prediction program AGADIR shows that Lti30 possess low helical tendency, although it shows 6 maxima in the helical propensity which corresponds precisely to the K-segments (Mouillon et al., 2006). FTIR data of Lti30 as it binds vesicles displays a change from random coil to α-helical structure, a so-called disorder-to-order transition (Paper III). The formation of structure upon binding is pH dependent, i.e. depending on His protonation. In Paper III, the structure of the membrane binding K-segment with flanking His in the presence of bicells (PC:PG 4:1) is investigated with high resolution NMR. When the K-segment interacts with the membrane surface a structural transition from random coil to an α- helix is observed. The hydrophobic residues of the K-segment line up on one side of the helix and probably anchors into the membrane.

In line with other K-segment containing IDPs, e.g. Vitis riparia K2 dehydrin (Clarke et al., 2015; Atkinson et al., 2016), Lti30 shows a clear cut disorder- to-order transition when anchoring into vesicles by the formation of an amphipathic helix.

6.4 Membrane binding of dehydrins can be tuned by phosphorylation and coordination of Ca2+ and Zn2+ ions. Are there additional factors that can tune dehydrin binding? In Paper IV the effect of metal binding (Ca2+ and Zn2+) and phosphorylation on dehydrin membrane binding was studied. Lti30 binds lipids strongly as do protonated Rab18 (at pH 4.3) and both induce vesicle aggregation (PC:PG (1:4)). The two acidic dehydrins Cor47 and Lti29 fail to bind even at pH 4.3. In this case protonation of their His in combination with the three K-segments embraced in their sequence is not enough to turn them into lipid binders, see Table 3 and Table 5.

During plant stress, a multitude of second messengers are released and different kinases will be up-regulated and activated (Xiong et al., 2002). Can additional factors, such as metal ions or phosphate groups alter the overall

38 net charge of the dehydrins and in such way regulate their membrane affinity? In the next section, the possible effects of kinases by post-translational phosphorylation of dehydrins and how the coordination of metals can adjust dehydrin lipid binding are discussed.

6.4.1 Dehydrin phosphorylation tunes the membrane interaction Dehydrins can be phosphorylated in vivo (Alsheikh et al., 2003) and espe- cially the S-segment is a site for phosphorylation (Jiang and Wang 2004). Cor47, Lti29, Lti30 and Rab18 can be phosphorylated in vitro (Paper I, II, IV and V). Interestingly, the in vitro phosphorylation of the four dehydrins is catalyzed by different kinases, whereas Cor47 and Lti29 are phosphorylated by casein kinase II and Lti30 is phosphorylated by protein kinase C. Rab18 can be a substrate of both (Paper I, II and IV). This indicates the possibility that different subclasses of the dehydrins can be regulated by different kinases. Phosphorylation can regulate dehydrins interaction with cellular targets by altering the net charge, and in Paper II it is suggested that phosphorylation is a way to modify Lti30´s membrane binding propensity. This is also true for Rab18, and in Paper IV it is shown that the aggregated vesicle clusters are reduced in size when Rab18 is phosphorylated. Hence, in both cases, the phosphorylation works as an electrostatic off-switch. As a consequence of phosphorylation, Lti30 binds lipid vesicles at lower concentrations in titration absorbance experiments. This would suggest that phosphorylation changes the structure assembling of Lti30 to make the K-segment more open to binding. Phosphorylation does not have any detectable effect for the non-membrane binding dehydrins Lti29 and Cor47. None of the four dehydrins responded structurally to phosphorylation, but remained disordered (Paper I, II and V). The conclusion that phosphorylation decreases membrane affinity is not exclusive for dehydrins, for example the phosphorylated MARCKS protein shows a 20 fold reduction in membrane binding (Kim et al., 1994).

6.4.2 Ca2+ and Zn2+ ions coordinate dehydrins binding to phospholipids Calcium is an important second messenger in plant stress response (Mahajan and Tuteja 2005). Increased calcium concentration due to the reduction of water is sensed by calcium binding proteins, and this starts a cascade of intracellular reactions (Mahajan and Tuteja 2005). Dehydrins are known to bind metals, but can metals compensate for negative net charge and alter the binding propensity of dehydrins to membranes? To test this the role of Ca2+ and Zn2+ coordination in lipid binding of the dehydrins was investigated in Paper IV, see Table 5. The results show that Ca2+ and Zn2+ have markedly

39 different effects. Coordination of calcium mainly augments the membrane affinity of dehydrins that already bind lipids in the absence of metal ions (Lti30 and Rab18), whereas coordination of zinc also induces membrane binding and vesicle assembly of those that fail to associate with membranes in the absence of metal ions, namely Cor47 and Lti29. However, it should be noted that the concentration of Zn2+ needed to induce membrane binding of Cor47 and Lti29 (1 mM) in this membrane mimetic falls outside the physiological range. Zn2+ is an essential metal in plants, but already at low concentrations it is cytotoxic. The concentrations of cytosolic free Zn2+ in Ara- bidopsis thaliana roots have recently been measured to 0.4 nM (Lanquar et al., 2014). Ca2+ concentration in the cytosol is normally in the range of 10-200 nM, but in the cell wall, vacuole, the mitochondria and ER it is normally higher, 1-10 mM (Kader and Lindberg 2010). Stress can generate higher Ca2+ concentration in the cytosol in the μM range (Kader and Lindberg 2010). Neverthe- less, to be noted, cytosolic concentrations hold for free Zn2+ or Ca2+. In the cell it is a dynamic equilibrium in a system of Zn2+ or Ca2+ binding proteins with various affinity. The effective metal concentration is dependent on the affinity and the concentration of metal binding proteins such as dehydrins.

Cor47 (SK3) Lti29 (SK3) Lti30 (K6) Rab18 (Y2SK2)

Vesicle agg. Yes pH 6.3 Lipid bind. Yes Yes Phos. Lipid bind. Yes Yes Ca2+ Lipid binding Yes Yes Yes Yes Zn2+ Lipid bind. Yes Yes Ca2+ + Phos. Lipid bind. Yes Yes Yes Zn2+ + Phos.

Table 5, Summary of experimental result for lipid binding of Cor47, Lti29, Lti30 and Rab18 tuned by phosphorylation, coordination of calcium and zinc or in combination. Yes: Detectable vesicle aggregation, i.e. lipid binding.

40 6.4.3 Phosphorylated dehydrins binding lipids in the presence of Ca2+ and Zn2+ -combined effects? It is known that ion binding of dehydrins in some cases is dependent on phosphorylation (Alsheikh et al., 2003). To test this possible combined effect, membrane binding of phosphorylated Cor47, Lti29, Lti30 and Rab18 in the presence of Zn2+ or Ca2+ was investigated, see Table 5. The observation is that the modest effect of Ca2+ on lipid binding is effectively enhanced by phosphorylation in the cases of lipid binding dehydrins (Lti30 and Rab18). For Cor47, the membrane interaction detected with Zn2+ is abolished when the dehydrin is phosphorylated (Paper IV).

6.5 Rab18 the first specific binding dehydrin? - Interaction with inositol lipids. In addition to the more general electrostatic interaction, we found that Rab18 also exhibited specific binding to certain phosphoinositides (Paper V). Inositol lipids can be found in plant membranes, although the phosphorylated versions exist in lower amounts as compared to other organisms. Nev- ertheless, these lipids fulfill various functions in plant cells (Munnik and Nielsen 2011). The precise role of phosphatidylinositols in plants is very difficult to investigate since their levels are normally low and may also only be transiently present (Lee et al., 1996). It is known that phosphatidylinositol levels change rapidly in response to the stress regulator ABA while other lipids remain unchanged (Lee et al., 1996).

In Paper V binding to inositol lipids was tested for the two lipid binders Lti30 and Rab18. Whereas Lti30 interacts with the inositol lipids as predicted from electrostatics, Rab18 binds with high specificity to both PI(4,5)P2 and PI(5)P (Paper V). This interaction is not purely electrostatic, as shown by the fact that Rab18 cannot bind lipid vesicles with high negative charge (PC:PG, 1:3), but is able to interact with vesicles containing 2 % phosphoinositides (PC:PI(4,5)P2, 96:4).

When investigated in further detail, Rab18 preferably binds to phosphoinositides with a phosphate group in position 5 of the inositol ring. i.e. PI(4,5)P2 and PI(5)P (Paper V). A phosphate group in position 3 prohibits the binding and no interaction is detectable for PI(3,4)P2 and PI(3)P, see Figure 9. To our knowledge, this is the first specific interaction reported for any dehydrin (Paper V).

are the Arg critical in these cases? As shown by Li et al., in comparison with Lys, Arg can retain its charge when inserted into a membrane while Lys becomes deprotonated. Arg are also predicted to attract more phosphate and water in the membrane by H-bonding between Arg and phosphate, which stabilizes the interaction (Li et al., 2013). In this respect, Arg are more suita- ble in protein membrane interactions.

6.5.3 Rab18: a conditional peripheral membrane protein? A model of possible function of Rab18 in the stomata guard cells Certain drought and ABA induced dehydrins are known to be localized in the stomata guard cells, among them Rab18 (Nylander et al., 2001). Since Rab18 specifically binds PI(4,5)P2 lipids, what is the outcome of this in vivo?

The expression of dehydrins in guard cells has been shown to be regulated by ABA activated protein kinase (AAPK) (Li et al., 2002). A nucleus localized dehydrin, Rab17 (Y2SK2) from maize was shown to be translocated from nucleus to cytosol upon phosphorylation of the S-segment by Casein kinase II (Riera et al., 2004). Maize Rab17 contains, like Rab18, the basic cluster RRKK before a K-segment. The initial RRKK sequence also shares similarities with a nuclear localization signal (NLS) binding phosphoprotein. NLS mediates the transport of proteins into the nucleus (Tinland et al., 1992; Moede et al., 1999). Rab17 was shown to bind to NLS and the authors suggested that the RRKK sequence participated in nuclear protein transport (Goday et al., 1994).

ABA increases the concentration of calcium in the guard cells, a process that is still not fully understood (Webb et al., 2001). The increased calcium levels are further known to work as an off-switch for the binding of disordered proteins to PI(4,5)P2 (McLaughlin and Murray 2005). This is also true for Rab18, where PIP2 binding is strongly inhibited in the presence of calcium. Interestingly, phosphorylation of Rab18 slightly decreases the effect of calcium (Paper V). On the other hand phosphorylation of MARCKS and MBP inhibits interaction with PI(4,5)P2, while for Rab18 it is changing the binding pattern by reducing the binding to a somewhat lower level.

When plants are stressed by drought they close their guard cells to avoid water loss. The closing of the guard cells induced by ABA is a key response in water related stress in plants. Stomata opening and closing is regulated by a complex series of reactions that are not only under the control of ABA but also a variety of other different factors, for example light (Daszkowska- Golec and Szarejko 2013). One route to guard cell closure involves PI(4,5)P2. The function of PI(4,5)P2 seems to be to bind anion channels,

44 phosphorylated by ABA induced kinases in a way similar to Rab17. That Rab18 can be phosphorylated in vitro is shown in papers IV and V. If so, the hypothesis is that phosphorylated Rab18 is translocated into the cytosol. This is partly confirmed by a more cytosolic localization of Rab18 in ABA treated plants (Nylander et al., 2001). Released Rab18 then scavenges PI(4,5)P2 and by this prevents the lipid from binding to anion channels in the membrane. The ion channels then pumps ions out of the guard cell, and as a consequence, the guard cell will close and the loss of water will be reduced, see Figure 11. This proposition has yet to be tested but if true - Rab18 would be a conditional peripheral membrane protein. Moreover, this reveals a new function of dehydrins as a regulator of cellular response.

46 Concluding remarks

During my doctoral studies I have studied the intrinsically disordered plant dehydrins. The most important results in this thesis are summarized here: We show that the crowding agents ficoll and dextran have low influence on the structure of the three dehydrins Cor47, Lti29 and Lti30 and that these are highly flexible proteins. Dehydrins are disordered proteins and stay disordered and flexible even in highly crowded environments. Since only sugars and polymers were tested, what remains is how dehydrins react upon a mix of the high background of proteins that are usually charged and create an electrostatic active milieu. All dehydrins seem to fold into an α-helix when completely dried out and also in the presence of dehydration agents. However, a plant cell is rarely completely dry and if similar structural transitions take place in vivo needs to be further investigated. The interaction of positively charged dehydrin Lti30 to phospholipid vesicles is compiled by the K-segments together with flanking His. pH dependent protonation switches of the histidines turn the interaction on and off. Upon binding lipid vesicles, the structure of the K-segments transfer from random coil to α-helix with hydrophobic amino-acid residues penetrating the polar lipid headgroups of the membrane surface. The K-segment plays a crucial role in binding of dehydrins’ to membrane. For Lti30 lipid binding is coordinated by the K-segment together with flanking histidines. In Paper III we also see that the overall net charge of the K-segment affects the affinity of the K-segment to membranes. By this, the model makes it possible for us to predict which dehydrins are membrane binders and which are not. By calculating the charge of the K-segment, and in combination with the overall net charge of a dehydrin, it is possible to predict which dehydrins could make electrostatic interaction with membranes and which could not. Lti30 aggregates phospholipid vesicles in vitro, and the interaction is modulated by the negative charges of the membrane with an optimum of 6 % negative charge regarding the phospholipid vesicles. In our system, as little as 2 % of the negatively charged lipids were needed for vesicle aggregation. Since most cell membranes have a much higher amount of negatively charged lipids, it is highly likely that Lti30 can bind membranes in vivo.

47 The regulation of dehydrins during stress is sensitive and multifaceted. The phosphorylation of the different subclasses of dehydrins is controlled by different kinases. Factors like phosphorylation and coordination of metal ions tune dehydrin membrane binding. Calcium and zinc have different effects, as zinc promotes binding of all dehydrins, even the acidic ones (Lti29 and Cor47). Calcium only affects the lipid binding dehydrins (Lti30 and Rab18) by enhancing the lipid binding affinity. We showed, for the first time, that a dehydrin exhibits specific lipid binding: Rab18 specifically and functionally binds to PI(4,5)P2. The binding of Rab18 is not driven by electrostatics as in the case of Lti30, but the interaction is coordinated by two Arg contiguous ones of the K-segments with phosphate groups on a PI(4,5)P2 lipid. We report that Rab18 binds and interacts specifically to phosphoinositides with the phosphate group in position 5: PI(4,5)P2>PI(3,5)P2>PI(3,4)P2. Our conclusion is that Rab18 is a conditional peripheral membrane protein involved in stress regulation.

48 Svensk sammanfattning

Växter utsätts dagligen för värme, kyla, höga salthalter och torka. Dessa faktorer leder till en låg mängd vatten i växtens celler och vid en snabb förändring kallas det för torkstress. Alla levande organismer består av en eller flera celler. En cell definieras av ett yttre membran som skyddar cellen mot dess omgivning. Det är viktigt att membranet hålls intakt och att ämnen transporteras kontrollerat från den ena sidan till den andra. Vid låga vattenhalter riskerar membranet att destabiliseras med bland annat läckage som följd. Vid torkstress sker också skador på proteiner som finns i cellen. Proteiner har många funktioner i en cell såsom enzymaktivitet för att katalysera andra reaktioner eller att reglera t.ex genuttryck. De flesta proteiner behöver omge sig av vatten för att inte förlora sin struktur och vid torkstress riskerar därför proteiner att bli dysfunctionella. Växter har inte ben och måste därför stå kvar där fröet har satt sin grodd. Hur kan de då klara av att växa under ibland extrema förhållanden? För att överleva har växter utvecklat ett effektivt och omfattande försvarssystem som bland annat består av: olika skyddsproteiner såsom dehydriner och chaperoner, samt produktion av olika sockerarter som hjälper att bibehålla cellstrukturen. Vid torkstress uttrycks en grupp av proteiner som kallas för Dehydriner. Denna proteingrupp har en speciell aminosyrasammansättning och tillhör klassen av strukturlösa proteiner. Dehydriner uttrycks i hög koncentration vid olika typer av torkstress men den faktiska funktionen har varit okänd. I dehydriners aminosyrasekvens finns vissa typiska sekvenser som kallas för K-segmentet, Y-segmentet och S-segmentet. För att ingå i gruppen dehydriner ska minst ett K-segment finnas i proteinsekvensen. K-segmentet har tidigare predicerats till att bilda en helix, en typ av proteinstruktur, och binda till membraner för att skydda dessa vid torkstress. Under min doktorandperiod har jag studerat fyra stycken dehydriner från växten Arabidopsis thaliana Lti30, Rab18, Cor47 och Lti29 och har framförallt undersökt om dehydrinerna binder till membraner. Att behålla ett intakt membran är en av de viktigast uppgifterna för en växt under stressade förhållanden. Dehydrinen Lti30 har visat sig binda elektrostatiskt till negativt laddade membran. Vi har även sett att interaktionen sker med hjälp av K-segmenten tillsammans med par av intillägande histidiner. Då K-segmentet binder till

49 membraner går det från strukturlöst till att forma en helix. Lti30 ändrar även karaktären av membranet då det binder och gör det troligtvis mer köldtåligt genom att hjälpa det förhindra läckage vilket i sin tur hjälper växten att överleva vid lägre temperaturer.

Vi har även visat att dehydrinen Rab18 binder specifikt till phosphoinositiden PI(4,5)P2. PI(4,5)P2 är en lipid som har många strängar på sin lyra bl.a aktiverar den jonkanaler i klyvöppningarna vilket i sin tur leder till att cellerna öppnas och att en aktiv fotosyntes kan ske. Vid aktiv fotosyntes utsöndrar klyvöppningar vattenånga och syre. Detta är självklart ödestigert för en växt som är utsatt för torka och det är viktigt att de då stängs. Då Rab18 binder till PI(4,5)P2 är vår hypotes att Rab18 binder till lipiden PI(4,5)P2 och att då jonkanalerna stängs. Stängda jonkanaler leder till stängda klyvöppningar, reducerad vattenförlust och överlevnad av växten.

50 Acknowledgement

First of all, I am very grateful to you, Pia, for being a fantastic supervisor. You are always enthusiastic and positive, which is truly encouraging. I have learnt a lot. Mikael I am grateful for your support and the interest you took in my projects. Jens, you always have great ideas and good answers to my questions. All of you creates an inspiring work place.

The Oliveberg group (past and present), without good colleagues this would not have been possible. Ellinor, you are always helpful and we shared lots of laugher, both when waited for the column to finish or outside the lab (“på en torsdag”). Lisa, what would I have done if you weren’t in the lab? We`ve talked about most. Linda, Ylva, Martin, Ann-Sofie, Micke for all nice lunches. Therese, Xin, Fan, Huabing, Seongil and Sarah for all the important and not so important discussions in the office, the lab or at the coffee maker.

Thanks to all co-authors and collaborators: Jean-Marie Mouillon, Michael Kutzer, Jan Procek, Gerhard Gröbner, Andreas Barth, Nadejda Eremina, Jens Danielsson, Dorothea Bartels, Jan Petersen, Horst Röhrig.

During the time I have been at DBB I met some fantastic people. Fatemeh, thank you for sharing your knowledge of vesicle preparation. Beata Kmiec, for antibody guidance. Emelie, Linda, Scarlett, Patricia, Salome for nice discussions at DBB and outside. All former and present people at DBB: Thanks for making the department such a nice place to work at.

Stefan, Maria, Alex, Lotta, Ann, Malin, Elisabeth. Tack för att ni alltid hjälper till när jag kommit förbi med frågor (eller när jag missat nåt).

Håkan, Matthew, Torbjörn, Peter, utan er skulle DBB helt enkelt inte funka.

Ann-Louise, vi som följts åt på hela “resan” från första terminen i Karlstad till DBB. Vi har skrattat, gråtit och haft så mycket kul!

Karin och Janne, för all hjälp med barnpassning, våra barn är ytterst privi- ligerade som har er.

51 Mamma pappa för att ni alltid finns där oavsett vad. Det betyder mer än ni tror.

All övrig familj och vänner.

Henrik och Moa. Ni har för alltid ändrat mitt liv. Ni är helt underbara och har verkligen lärt mig sätta perspektiv på saker och ting.

Patrik, tack för att du finns i mitt liv. Vi klarar vad som helst tillsammans. Vi är ett team!

52 References

Alsheikh MK, Heyen BJ, Randall SK (2003) Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation. J Biol Chem 278(42): 40882-40889 Arakawa T, Timasheff SN (1984) Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry 23(25): 5912-5923 Atkinson J, Clarke MW, Warnica JM, Boddington KF, Graether SP (2016) Structure of an Intrinsically Disordered Stress Protein Alone and Bound to a Membrane Surface. Biophys J 111(3): 480-491 Battaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias AA (2008) The enigmatic LEA proteins and other hydrophilins. Plant Physiol 148(1): 6-24 Blok MC, van der Neut-Kok EC, van Deenen LL, de Gier J (1975) The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes. Biochim Biophys Acta 406(2): 187- 196 Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptations to Environmental Stresses. Plant Cell 7(7): 1099-1111 Borg M, Mittag T, Pawson T, Tyers M, Forman-Kay JD, Chan HS (2007) Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity. Proc Natl Acad Sci U S A 104(23): 9650-9655 Bourhis JM, Johansson K, Receveur-Brechot V, Oldfield CJ, Dunker KA, Canard B, Longhi S (2004) The C-terminal domain of measles virus nucleoprotein belongs to the class of intrinsically disordered proteins that fold upon binding to their physiological partner. Virus Res 99(2): 157-167 Bravo LA, Gallardo J, Navarrete A, Olave N, Martinez J, Alberdi M, Close TJ, Corcuera LJ (2003) Cryoprotective activity of a cold-induced dehydrin purified from barley. Physiologia Plantarum 118(2): 262-269 Brini F, Hanin M, Lumbreras V, Amara I, Khoudi H, Hassairi A, Pages M, Masmoudi K (2007) Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana. Plant Cell Rep 26(11): 2017-2026 Bryant JE, Lecomte JT, Lee AL, Young GB, Pielak GJ (2005) Protein dynamics in living cells. Biochemistry 44(26): 9275-9279 Caroni P (2001) New EMBO members' review: actin cytoskeleton regulation through modulation of PI(4,5)P(2) rafts. EMBO J 20(16): 4332- 4336

53 Chakrabortee S, Boschetti C, Walton LJ, Sarkar S, Rubinsztein DC, Tunnacliffe A (2007) Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc Natl Acad Sci U S A 104(46): 18073-18078 Chakrabortee S, Meersman F, Kaminski Schierle GS, Bertoncini CW, McGee B, Kaminski CF, Tunnacliffe A (2010) Catalytic and chaperone- like functions in an intrinsically disordered protein associated with desiccation tolerance. Proc Natl Acad Sci U S A 107(37): 16084-16089 Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM, Sherr CJ (1999) The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 18(6): 1571-1583 Clarke MW, Boddington KF, Warnica JM, Atkinson J, McKenna S, Madge J, Barker CH, Graether SP (2015) Structural and Functional Insights into the Cryoprotection of Membranes by the Intrinsically Disordered Dehydrins. J Biol Chem 290(45): 26900-26913 Close TJ (1996) Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins. Physiologia Plantarum 97(4): 795-803 Cooke RJ, Oliver J, Davies DD (1979) Stress and Protein Turnover in Lemna minor. Plant Physiol 64(6): 1109-1113 Crowe JH, Hoekstra FA, Crowe LM (1992) Anhydrobiosis. Annu Rev Physiol 54: 579-599 Crowe LM (2002) Lessons from nature: the role of sugars in anhydrobiosis. Comp Biochem Physiol A Mol Integr Physiol 131(3): 505-513 Csermely P (1997) Proteins, RNAs and chaperones in enzyme evolution: a folding perspective. Trends Biochem Sci 22(5): 147-149 Danielsson J, Mu X, Lang L, Wang HB, Binolfi A, Theillet FX, Bekei B, Logan DT, Selenko P, Wennerstrom H, Oliveberg M (2015) Thermodynamics of protein destabilization in live cells. Proceedings of the National Academy of Sciences of the United States of America 112(40): 12402-12407 Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, Sarhan F (1998) Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 10(4): 623- 638 Daszkowska-Golec A, Szarejko I (2013) Open or close the gate - stomata action under the control of phytohormones in drought stress conditions. Front Plant Sci 4: 138 Davidson WS, Jonas A, Clayton DF, George JM (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 273(16): 9443-9449 Dedmon MM, Patel CN, Young GB, Pielak GJ (2002) FlgM gains structure in living cells. Proc Natl Acad Sci U S A 99(20): 12681-12684 Dinakar C, Bartels D (2013) Desiccation tolerance in resurrection plants: new insights from transcriptome, proteome and metabolome analysis. Front Plant Sci 4: 482

54 Dong A, Prestrelski SJ, Allison SD, Carpenter JF (1995) Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation. J Pharm Sci 84(4): 415-424 Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z (2001) Intrinsically disordered protein. J Mol Graph Model 19(1): 26-59 Dunker AK, Obradovic Z (2001) The protein trinity--linking function and disorder. Nat Biotechnol 19(9): 805-806 Dunker AK, Obradovic Z, Romero P, Garner EC, Brown CJ (2000) Intrinsic protein disorder in complete genomes. Genome Inform Ser Workshop Genome Inform 11: 161-171 Dunker AK, Silman I, Uversky VN, Sussman JL (2008) Function and structure of inherently disordered proteins. Curr Opin Struct Biol 18(6): 756- 764 Dure L, 3rd (1993) A repeating 11-mer amino acid motif and plant desiccation. Plant J 3(3): 363-369 Dure L, 3rd, Crouch M, Harada J, Ho TH, Mundy J, Quatrano R, Thomas T, Sung ZR (1989) Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol Biol 12(5): 475-486 Dure L, 3rd, Greenway SC, Galau GA (1981) Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. Biochemistry 20(14): 4162-4168 Dure L, Chlan C (1981) Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XII. PURIFICATION AND PROPERTIES OF PRINCIPAL STORAGE PROTEINS. Plant Physiol 68(1): 180-186 Dure L, Galau GA (1981) Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XIII. REGULATION OF BIOSYNTHESIS OF PRINCIPAL STORAGE PROTEINS. Plant Physiol 68(1): 187-194 Dyson HJ, Wright PE (2002) Coupling of folding and binding for unstructured proteins. Curr Opin Struct Biol 12(1): 54-60 Ellis RJ (2001) Macromolecular crowding: an important but neglected aspect of the intracellular environment. Curr Opin Struct Biol 11(1): 114- 119 Furt F, Simon-Plas F, Mongrand S (2011) Lipids of the Plant Plasma Membrane. Plant Plasma Membrane 19: 3-30 Galau GA, Hughes DW, Dure L, 3rd (1986) Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs. Plant Mol Biol 7(3): 155-170 Galau GA, Wang HY, Hughes DW (1992) Cotton Lea4 (D19) and LeaA2 (D132) Group 1 Lea Genes Encoding Water Stress-Related Proteins Containing a 20-Amino Acid Motif. Plant Physiol 99(2): 783-788

55 Goday A, Jensen AB, Culianez-Macia FA, Mar Alba M, Figueras M, Serratosa J, Torrent M, Pages M (1994) The maize abscisic acid- responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals. Plant Cell 6(3): 351-360 Goyal K, Walton LJ, Tunnacliffe A (2005) LEA proteins prevent protein aggregation due to water stress. Biochem J 388(Pt 1): 151-157 Graether SP, Boddington KF (2014) Disorder and function: a review of the dehydrin protein family. Front Plant Sci 5: 576 Guerrero FD, Jones JT, Mullet JE (1990) Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol 15(1): 11- 26 Guy C, Haskell D, Li QB (1998) Association of proteins with the stress 70 molecular chaperones at low temperature: Evidence for the existence of cold labile proteins in spinach. Cryobiology 36(4): 301-314 Hansen SB, Tao X, MacKinnon R (2011) Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2. Nature 477(7365): 495-498 Hara M, Fujinaga M, Kuboi T (2005) Metal binding by citrus dehydrin with histidine-rich domains. J Exp Bot 56(420): 2695-2703 Hara M, Terashima S, Kuboi T (2001) Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu. Journal of Plant Physiology 158(10): 1333-1339 Hatters DM, Minton AP, Howlett GJ (2002) Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II. J Biol Chem 277(10): 7824-7830 Houde M, Dallaire S, N'Dong D, Sarhan F (2004) Overexpression of the acidic dehydrin WCOR410 improves freezing tolerance in transgenic strawberry leaves. Plant Biotechnol J 2(5): 381-387 Huang CL, Feng S, Hilgemann DW (1998) Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. Nature 391(6669): 803-806 Hughes S, Graether SP (2011) Cryoprotective mechanism of a small intrinsically disordered dehydrin protein. Protein Sci 20(1): 42-50 Ingram J, Bartels D (1996) The Molecular Basis of Dehydration Tolerance in Plants. Annu Rev Plant Physiol Plant Mol Biol 47: 377-403 Jao CC, Der-Sarkissian A, Chen J, Langen R (2004) Structure of membrane-bound alpha-synuclein studied by site-directed spin labeling. Proc Natl Acad Sci U S A 101(22): 8331-8336 Jiang X, Wang Y (2004) Beta-elimination coupled with tandem mass spectrometry for the identification of in vivo and in vitro phosphorylation sites in maize dehydrin DHN1 protein. Biochemistry 43(49): 15567-15576 Kader MA, Lindberg S (2010) Cytosolic calcium and pH signaling in plants under salinity stress. Plant Signal Behav 5(3): 233-238 Kaplan F, Guy CL (2004) beta-Amylase induction and the protective role of maltose during temperature shock. Plant Physiol 135(3): 1674-1684

56 Kazuoka T, Oeda K (1994) Purification and Characterization of Cor85- Oligomeric Complex from Cold-Acclimated Spinach. Plant and Cell Physiology 35(4): 601-611 Kim J, Shishido T, Jiang X, Aderem A, McLaughlin S (1994) Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles. J Biol Chem 269(45): 28214-28219 Kim TH, Bohmer M, Hu H, Nishimura N, Schroeder JI (2010) Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling. Annu Rev Plant Biol 61: 561-591 Koag MC, Fenton RD, Wilkens S, Close TJ (2003) The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity. Plant Physiol 131(1): 309-316 Koag MC, Wilkens S, Fenton RD, Resnik J, Vo E, Close TJ (2009) The K-segment of maize DHN1 mediates binding to anionic phospholipid vesicles and concomitant structural changes. Plant Physiol 150(3): 1503- 1514 Kovacs D, Kalmar E, Torok Z, Tompa P (2008) Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins. Plant Physiol 147(1): 381-390 Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63(4): 1593- 1608 Kriwacki RW, Hengst L, Tennant L, Reed SI, Wright PE (1996) Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc Natl Acad Sci U S A 93(21): 11504-11509 Kurnik M, Hedberg L, Danielsson J, Oliveberg M (2012) Folding without charges. Proceedings of the National Academy of Sciences of the United States of America 109(15): 5705-5710 Labhilili M, Joudrier P, Gautier MF (1995) Characterization of cDNAs encoding Triticum durum dehydrins and their expression patterns in cultivars that differ in drought tolerance. Plant Science 112(2): 219-230 Lanquar V, Grossmann G, Vinkenborg JL, Merkx M, Thomine S, Frommer WB (2014) Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology. New Phytol 202(1): 198-208 Lee HJ, Choi C, Lee SJ (2002) Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form. J Biol Chem 277(1): 671-678 Lee SA, Eyeson R, Cheever ML, Geng J, Verkhusha VV, Burd C, Overduin M, Kutateladze TG (2005) Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch. Proc Natl Acad Sci U S A 102(37): 13052-13057 Lee Y, Choi YB, Suh S, Lee J, Assmann SM, Joe CO, Kelleher JF, Crain RC (1996) Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba. Plant Physiol 110(3): 987-996

57 Lee Y, Kim YW, Jeon BW, Park KY, Suh SJ, Seo J, Kwak JM, Martinoia E, Hwang I, Lee Y (2007) Phosphatidylinositol 4,5- bisphosphate is important for stomatal opening. Plant J 52(5): 803-816 Li J, Kinoshita T, Pandey S, Ng CK, Gygi SP, Shimazaki K, Assmann SM (2002) Modulation of an RNA-binding protein by abscisic-acid- activated protein kinase. Nature 418(6899): 793-797 Li L, Vorobyov I, Allen TW (2013) The different interactions of lysine and arginine side chains with lipid membranes. J Phys Chem B 117(40): 11906- 11920 Love JJ, Li X, Chung J, Dyson HJ, Wright PE (2004) The LEF-1 high- mobility group domain undergoes a disorder-to-order transition upon formation of a complex with cognate DNA. Biochemistry 43(27): 8725- 8734 Madden TD, Bally MB, Hope MJ, Cullis PR, Schieren HP, Janoff AS (1985) Protection of large unilamellar vesicles by trehalose during dehydration: retention of vesicle contents. Biochim Biophys Acta 817(1): 67-74 Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444(2): 139-158 Manenti S, Sorokine O, Van Dorsselaer A, Taniguchi H (1992) Affinity purification and characterization of myristoylated alanine-rich protein kinase C substrate (MARCKS) from bovine brain. Comparison of the cytoplasmic and the membrane-bound forms. J Biol Chem 267(31): 22310-22315 Martin J, Geromanos S, Tempst P, Hartl FU (1993) Identification of nucleotide-binding regions in the chaperonin proteins GroEL and GroES. Nature 366(6452): 279-282 McLaughlin S, Aderem A (1995) The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem Sci 20(7): 272-276 McLaughlin S, Murray D (2005) Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438(7068): 605-611 Minton AP (2001) The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J Biol Chem 276(14): 10577-10580 Minton AP (2005) Models for excluded volume interaction between an unfolded protein and rigid macromolecular cosolutes: macromolecular crowding and protein stability revisited. Biophys J 88(2): 971-985 Moede T, Leibiger B, Pour HG, Berggren PO, Leibiger IB (1999) Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1. Febs Letters 461(3): 229-234 Mori MX, Inoue R (2014) New experimental trends for phosphoinositides research on ion transporter/channel regulation. J Pharmacol Sci 126(3): 186- 197 Mouillon JM, Gustafsson P, Harryson P (2006) Structural investigation of disordered stress proteins. Comparison of full-length dehydrins with isolated peptides of their conserved segments. Plant Physiol 141(2): 638-650

58 Munnik T, Nielsen E (2011) Green light for polyphosphoinositide signals in plants. Curr Opin Plant Biol 14(5): 489-497 Musse AA, Gao W, Homchaudhuri L, Boggs JM, Harauz G (2008) Myelin basic protein as a "PI(4,5)P2-modulin": a new biological function for a major central nervous system protein. Biochemistry 47(39): 10372-10382 Neven LG, Haskell DW, Hofig A, Li QB, Guy CL (1993) Characterization of a spinach gene responsive to low temperature and water stress. Plant Mol Biol 21(2): 291-305 Nylander M, Svensson J, Palva ET, Welin BV (2001) Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant Mol Biol 45(3): 263-279 Pancsa R, Tompa P (2012) Structural disorder in eukaryotes. PLoS One 7(4): e34687 Petersen J, Eriksson SK, Harryson P, Pierog S, Colby T, Bartels D, Rohrig H (2012) The lysine-rich motif of intrinsically disordered stress protein CDeT11-24 from Craterostigma plantagineum is responsible for phosphatidic acid binding and protection of enzymes from damaging effects caused by desiccation. J Exp Bot 63(13): 4919-4929 Plana M, Itarte E, Eritja R, Goday A, Pages M, Martinez MC (1991) Phosphorylation of maize RAB-17 protein by casein kinase 2. J Biol Chem 266(33): 22510-22514 Puhakainen T, Hess MW, Makela P, Svensson J, Heino P, Palva ET (2004) Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol Biol 54(5): 743-753 Qin Z, Cafiso DS (1996) Membrane structure of protein kinase C and calmodulin binding domain of myristoylated alanine rich C kinase substrate determined by site-directed spin labeling. Biochemistry 35(9): 2917-2925 Qu Y, Bolen CL, Bolen DW (1998) Osmolyte-driven contraction of a random coil protein. Proc Natl Acad Sci U S A 95(16): 9268-9273 Rahman LN, Chen L, Nazim S, Bamm VV, Yaish MW, Moffatt BA, Dutcher JR, Harauz G (2010) Interactions of intrinsically disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes - synergistic effects of lipid composition and temperature on secondary structure. Biochem Cell Biol 88(5): 791-807 Ramesh SA, Tyerman SD, Xu B, Bose J, Kaur S, Conn V, Domingos P, Ullah S, Wege S, Shabala S, Feijo JA, Ryan PR, Gilliham M (2015) GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters. Nat Commun 6: 7879 Rawicz W, Olbrich KC, McIntosh T, Needham D, Evans E (2000) Effect of chain length and unsaturation on elasticity of lipid bilayers. Biophys J 79(1): 328-339 Renault H, Roussel V, El Amrani A, Arzel M, Renault D, Bouchereau A, Deleu C (2010) The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance. BMC Plant Biol 10: 20 Reyes JL, Campos F, Wei H, Arora R, Yang Y, Karlson DT, Covarrubias AA (2008) Functional dissection of hydrophilins during in vitro freeze protection. Plant Cell Environ 31(12): 1781-1790

59 Riera M, Figueras M, Lopez C, Goday A, Pages M (2004) Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize. Proc Natl Acad Sci U S A 101(26): 9879-9884 Rinne PL, Kaikuranta PL, van der Plas LH, van der Schoot C (1999) Dehydrins in cold-acclimated apices of birch (Betula pubescens ehrh. ): production, localization and potential role in rescuing enzyme function during dehydration. Planta 209(4): 377-388 Rucker AL, Creamer TP (2002) Polyproline II helical structure in protein unfolded states: lysine peptides revisited. Protein Sci 11(4): 980-985 Sarkar M, Lu J, Pielak GJ (2014) Protein crowder charge and protein stability. Biochemistry 53(10): 1601-1606 Schaffer MA, Fischer RL (1988) Analysis of mRNAs that Accumulate in Response to Low Temperature Identifies a Thiol Protease Gene in Tomato. Plant Physiol 87(2): 431-436 Schroeder JI, Schmidt C, Sheaffer J (1993) Identification of High- Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells. Plant Cell 5(12): 1831- 1841 Schwartz A, Ilan N, Schwarz M, Scheaffer J, Assmann SM, Schroeder JI (1995) Anion-Channel Blockers Inhibit S-Type Anion Channels and Abscisic Acid Responses in Guard Cells. Plant Physiol 109(2): 651-658 Sigalov AB, Hendricks GM (2009) Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition. Biochem Biophys Res Commun 389(2): 388-393 Sirichandra C, Wasilewska A, Vlad F, Valon C, Leung J (2009) The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action. J Exp Bot 60(5): 1439-1463 Stacy RA, Aalen RB (1998) Identification of sequence homology between the internal hydrophilic repeated motifs of group 1 late-embryogenesis- abundant proteins in plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis. Planta 206(3): 476-478 Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci U S A 95(24): 14570-14575 Svensson J, Palva ET, Welin B (2000) Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography. Protein Expr Purif 20(2): 169-178 Szasz CS, Alexa A, Toth K, Rakacs M, Langowski J, Tompa P (2011) Protein disorder prevails under crowded conditions. Biochemistry 50(26): 5834-5844 Thalhammer A, Hundertmark M, Popova AV, Seckler R, Hincha DK (2010) Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state. Biochim Biophys Acta 1798(9): 1812-1820 Tinland B, Koukolikovanicola Z, Hall MN, Hohn B (1992) The T-DNA- Linked Vird2 Protein Contains 2 Distinct Functional Nuclear-Localization

60 Signals. Proceedings of the National Academy of Sciences of the United States of America 89(16): 7442-7446 Tjellstrom H, Hellgren LI, Wieslander A, Sandelius AS (2010) Lipid asymmetry in plant plasma membranes: phosphate deficiency-induced phospholipid replacement is restricted to the cytosolic leaflet. FASEB J 24(4): 1128-1138 Tompa P (2012) Intrinsically disordered proteins: a 10-year recap. Trends Biochem Sci 37(12): 509-516 Tompa P, Csermely P (2004) The role of structural disorder in the function of RNA and protein chaperones. FASEB J 18(11): 1169-1175 Tompa P, Szasz C, Buday L (2005) Structural disorder throws new light on moonlighting. Trends Biochem Sci 30(9): 484-489 Trauble H, Sackmann E (1972) Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. 3. Structure of a steroid-lecithin system below and above the lipid-phase transition. J Am Chem Soc 94(13): 4499-4510 Tunnacliffe A, Hincha DK, Leprince O, Macherel D (2010) LEA Proteins: Versatility of Form and Function. Dormancy and Resistance in Harsh Environments 21: 91-108 Uemura M, Joseph RA, Steponkus PL (1995) Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions). Plant Physiol 109(1): 15-30 Ulmer TS, Bax A (2005) Comparison of structure and dynamics of micelle- bound human alpha-synuclein and Parkinson disease variants. J Biol Chem 280(52): 43179-43187 Ulmer TS, Bax A, Cole NB, Nussbaum RL (2005) Structure and dynamics of micelle-bound human alpha-synuclein. J Biol Chem 280(10): 9595-9603 Uversky VN (2009) Intrinsically disordered proteins and their environment: effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding. Protein J 28(7- 8): 305-325 Uversky VN (2011) Intrinsically disordered proteins from A to Z. Int J Biochem Cell Biol 43(8): 1090-1103 Uversky VN, Gillespie JR, Fink AL (2000) Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins 41(3): 415-427 Uversky VN, Oldfield CJ, Dunker AK (2005) Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. J Mol Recognit 18(5): 343-384 Valluru R, Van den Ende W (2008) Plant fructans in stress environments: emerging concepts and future prospects. J Exp Bot 59(11): 2905-2916 van Meer G (1998) Lipids of the Golgi membrane. Trends Cell Biol 8(1): 29-33 Wang J, Gambhir A, Hangyas-Mihalyne G, Murray D, Golebiewska U, McLaughlin S (2002) Lateral sequestration of phosphatidylinositol 4,5- bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions. J Biol Chem 277(37): 34401-34412

61 Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat- shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9(5): 244-252 Webb AA, Larman MG, Montgomery LT, Taylor JE, Hetherington AM (2001) The role of calcium in ABA-induced gene expression and stomatal movements. Plant J 26(3): 351-362 Wisniewski M, Webb R, Balsamo R, Close TJ, Yu XM, Griffith M (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: A dehydrin from peach (Prunus persica). Physiologia Plantarum 105(4): 600-608 Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re- assessing the protein structure-function paradigm. J Mol Biol 293(2): 321- 331 Wywial E, Singh SM (2010) Identification and structural characterization of FYVE domain-containing proteins of Arabidopsis thaliana. BMC Plant Biol 10: 157 Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14 Suppl: S165-183 Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247-273 Zhu M, Fink AL (2003) Lipid binding inhibits alpha-synuclein fibril formation. J Biol Chem 278(19): 16873-16877 Zimmerman SB, Trach SO (1991) Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol 222(3): 599-620