trehalose 6-phosphate positively regulates fatty acid biosynthesis …€¦ · 24/09/2018 · t6p...

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RESEARCH ARTICLE 1

Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by 2

Stabilizing WRINKLED1 3

Zhiyang Zhai1*

, Jantana Keereetaweep1*

, Hui Liu1, Regina Feil

2, John E. Lunn

2, John Shanklin

1# 4

1Dept of Biology, BNL 463, 50 Bell Ave, Upton NY 11973, USA 5

2Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, 6 Germany 7

#Corresponding author: [email protected] 8

*ZZ and JK contributed equally to this work.9

Short Title: Mechanism of T6P-regulated fatty acid biosynthesis 10

One Sentence Summary: Trehalose 6-phosphate binds to the KIN10 subunit of SnRK1 and inhibits it in 11 a GRIK-dependent manner, positively regulating fatty acid synthesis by stabilizing WRINKLED1. 12

The author responsible for distribution of materials integral to the findings presented in this article in 13 accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: John 14 Shanklin ([email protected]). 15

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ABSTRACT 17 WRINKLED1 (WRI1), the transcriptional activator of fatty acid synthesis, was recently identified as a 18 target of KIN10, a catalytic α-subunit of the SUCROSE-NON-FERMENTING-1-RELATED PROTEIN 19 KINASE-1 (SnRK1). We tested the hypothesis that trehalose 6-phosphate (T6P), a signal of cellular 20 sucrose status, can regulate fatty acid synthesis by inhibiting SnRK1. Incubation of Brassica napus 21 suspension cells in medium containing T6P, or overexpression of the Escherichia coli T6P synthase, 22 OtsA, in Nicotiana benthamiana, significantly increased T6P levels, WRI1 levels and FA synthesis rates. 23 T6P directly bound to purified recombinant KIN10 with an equilibrium dissociation constant (Kd) of 32 ± 24 6 μM based on microscale thermophoresis. GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1) 25 bound to KIN10 (Kd 19 ± 3 μM) and activated it by phosphorylation. In the presence of T6P, the GRIK1-26 KIN10 association was weakened by more than threefold (Kd 67.7 ± 9.8 μM) which reduced both the 27 phosphorylation of KIN10 and its activity. T6P-dependent inhibition of SnRK1 activity was reduced in 28 extracts of individual Arabidopsis grik1 and grik2 mutants relative to wild type, while SnRK1 activity in 29 grik1 grik2 extracts was enhanced by T6P. These results indicate that the T6P sensitivity of SnRK1 in 30 vivo is GRIK1/GRIK2-dependent. Based on our findings, we propose a mechanistic model that links 31 sugar signaling and FA homeostasis. 32

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Plant Cell Advance Publication. Published on September 24, 2018, doi:10.1105/tpc.18.00521

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INTRODUCTION 35

Lipids are primary metabolites in cells, acting as structural components of cell membranes, energy-dense 36

storage compounds and cell signaling molecules. Fatty acids are major components of lipids and 37

triacylglycerols (TAGs) are storage lipids that accumulate mostly in oil bodies in plants (Li-Beisson et al., 38

2013). Imported sugar provides substrates including carbon skeletons in the form of acetyl-CoA, ATP and 39

reductants (NADPH) for de novo fatty acid synthesis in sink organs. Several lines of evidence have 40

identified links between sucrose (Suc) levels and fatty acid synthesis. For example, wrinkled seed (rr), 41

the classical Mendel pea mutant in which a gene encoding a starch-branching enzyme is disrupted by a 42

transposon-like insertion, showed both higher levels of Suc and increased lipid accumulation 43

(Bhattacharyya et al., 1990). A 3-fold increase in Suc and a 30% increase in leaf TAG accumulation are 44

observed in leaves of Arabidopsis (Arabidopsis thaliana) in which starch synthesis is decreased by RNAi-45

mediated suppression of the small subunit of AGPase (ADG1) (Sanjaya et al., 2011). In addition, 46

Arabidopsis exhibits a 4-fold TAG increase in roots when cultured on one-half-strength MS medium 47

supplemented with 5% Suc, compared to medium without Suc (Kelly et al., 2013). To test the influence 48

of endogenous sugar content on FA and TAG accumulation, we generated a high-leaf-sugar mutant by 49

simultaneously restricting sucrose phloem loading and blocking starch synthesis by crossing the suc2 50

(encoding a Suc/H+ symporter that loads Suc into phloem) mutant (Srivastava et al., 2008) and an adg1 51

mutant (Lin et al., 1988). The sugar content (combined glucose (Glc) and Suc) in adg1 suc2 leaves was 52

80-fold higher than that of WT and TAG accumulation increased more than 10-fold with respect to WT to53

1% of dry weight, demonstrating a strong positive relationship between sugar accumulation and TAG 54

accumulation (Zhai et al., 2017b). 55

That sugars play dual roles in providing carbon skeletons and sugar signaling is well established for the 56

synthesis of starch (Nakamura et al., 1991; Harn et al., 2000; Wang et al., 2001; Nagata et al., 2012), 57

fructans (Nagaraj et al., 2001; Noël et al., 2001) and anthocyanins (Tiessen et al., 2002). There is 58

increasing evidence that sugar signaling is also important in regulating lipid synthesis. For example, 59

glucose and fructose are necessary for seedlings ectopically overexpressing WRINKLED 1 (WRI1) to 60

accumulate TAG (Cernac and Benning, 2004). WRI1 is an APETALA2 (AP2) transcriptional factor that 61

induces the expression of more than 20 genes involved in glycolysis and fatty acid synthesis (Cernac and 62

Benning, 2004; Maeo et al., 2009; Ma et al., 2013). WRI1 expression is enhanced by Suc in Arabidopsis 63

leaves (Masaki et al., 2005). Sanjaya et al. (2011) also observed that the expression of WRI1 in seedling 64

of AGP-deficient lines is increased compared to wild type. However, in the Arabidopsis adg1 suc2 65

mutant, WRI1 expression is not significantly different from wild type, but the abundance of the WRI1 66

protein is increased due to its stabilization (Zhai et al., 2017b). Sugar-dependent regulation of WRI1 67

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expression and WRI1 protein stability implies that sugar signaling is involved in regulating lipid (TAG) 68

synthesis. 69

Previously, we reported that a catalytic -subunit of SnRK1, KIN10, can directly phosphorylate WRI1 70

within its two AP2 DNA-binding domains (Zhai et al., 2017a), close to the previously identified 14-3-3 71

binding sites (Ma et al., 2016), initiating its proteasomal degradation. This finding is consistent with 72

observations from adg1 suc2 that high sugar levels post-translationally stabilize WRI1. In the plant cell, 73

SnRK1 is an important metabolic sensor that is activated when sugars are low, resulting in metabolic 74

reprogramming that leads to a general inhibition of catabolism and stimulation of anabolism (Baena-75

González et al., 2007). The catalytic activity of the heterotrimeric (//) SnRK1 complex resides in its 76

subunits, which in Arabidopsis are encoded by KIN10, KIN11 and SnRK1.3. These subunits are 77

activated by phosphorylation of threonine 175 (T175) (or its equivalent) within their T-loops (Estruch et 78

al., 1992; Hawley et al., 1996; Shen et al., 2009). The degree of T-loop phosphorylation reflects the 79

metabolic status of the cell; under low-energy/sugar or stress conditions it becomes phosphorylated and 80

thereby activated. Conversely, under favorable, i.e., high-energy/sugar conditions, T-loop 81

phosphorylation is reversed, leading to a decrease in KIN10 activity. In plants, GEMINIVIRUS REP-82

INTERACTING KINASE1 (GRIK1) and GRIK2 are responsible for phosphorylating T175 in KIN10 83

(Shen et al., 2009; Glab et al., 2017), whereas protein phosphatases ABSCISIC ACID INSENSITIVE1 84

(ABI1) and PROTEIN PHOSPHATASE TYPE 2CA (PP2CA) mediate its dephosphorylation (Rodrigues 85

et al., 2013). It has also been reported that activated SnRK1 can phosphorylate and moderate the activity 86

of GRIKs, providing a potential feedback control mechanism to regulate SnRK1 activation (Crozet et al., 87

2010). 88

Trehalose 6-phosphate (T6P) acts as a signal of sucrose availability connecting plant growth and 89

development to its metabolic status (Schluepmann et al., 2003; Lunn et al., 2006; Yadav et al., 2014; 90

Figueroa and Lunn, 2016). T6P is synthesized by the action of T6P synthase (TPS) on two activated 91

forms of Glc, UDP-Glc (UDPG) and Glc 6-phosphate (G6P), both of which are central to plant 92

metabolism (Cabib and Leloir, 1958). SnRK1 activity in crude extracts from developing Arabidopsis 93

tissues is inhibited by T6P, and this inhibition depends on unknown protein factor(s) principally 94

expressed in young tissues (Zhang et al., 2009; Martínez-Barajas et al., 2011; Griffiths et al., 2016). T6P 95

levels are quite variable with respect to organism, tissue types, developmental states, and can exhibit 96

dynamic responses to environmental cues. Several reports from Arabidopsis and maize (Zea mays) have 97

estimated T6P to be present in the low (4-47) micromolar range (Martins et al., 2013; Nuccio et al., 98

2015). However, as T6P is probably not distributed homogeneously in the tissues analyzed, these values 99

should be regarded as minimum estimates of the in vivo concentrations of T6P. 100

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Because WRI1 is a direct target of KIN10 and T6P is a potent inhibitor of SnRK1, we tested the 101

hypothesis that T6P can regulate fatty acid synthesis via inhibition of SnRK1 and subsequent stabilization 102

of WRI1. Data from experiments employing exogenous T6P treatment, or overexpression of TPS to 103

elevate T6P levels, provided support for the hypothesis. Further, we observed direct binding between T6P 104

and purified recombinant KIN10 and quantitated their interaction. To do this we used microscale 105

thermophoresis (MST), a biophysical technique which provides precise equilibrium dissociation constants 106

(Kds) for protein-protein and protein-ligand interactions (Wienken et al., 2010; Baaske et al., 2011). 107

Using both MST and genetic approaches employing grik mutants, we demonstrated that T6P weakens the 108

GRIK-KIN10 association i.e., increases the equilibrium dissociation constant by more than threefold, 109

identifying it as a mediator for T6P-dependent inhibition of SnRK1. 110

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RESULTS 112

T6P positively regulates fatty acid biosynthesis by stabilizing WRI1 in Brassica napus suspension 113

cells 114

To test the regulatory effect of T6P on fatty acid biosynthesis, a microspore-derived cell suspension 115

culture of Brassica napus L. cv. Jet Neuf (subsequently referred to as B. napus) (Supplemental Fig. 1) 116

was used because it is a well-established model system for developing oilseeds (Shi et al., 2008; Andre et 117

al., 2012). Because T6P does not efficiently penetrate the cell walls of intact plants (Griffiths et al., 2016), 118

we exposed B. napus suspension cells to relatively high levels (100 µM) of T6P. This led to a rise in 119

intracellular T6P levels after 3 h of T6P exposure, compared to cells incubated in the presence of the 120

same concentration of sucrose or sorbitol (Fig. 1A), indicating that under our experimental conditions B. 121

napus suspension cells are able to take up exogenous T6P. 122

A KIN10 kinase activity assay was performed using a sucrose-phosphate synthase (SPS)-derived target 123

peptide containing the KIN10-phosphorylation-target-sequence (Bhalerao et al., 1999). KIN10 activity 124

was 40% lower in T6P-treated cells relative to sucrose-treated cells after 2 d of treatment (Fig. 1B). We 125

next investigated the effects of T6P treatment on WRI1. As shown in Fig. 1C, WRI1 accumulated to 126

approximately 3-fold higher levels in T6P-treated cells relative to either sucrose- or sorbitol-treated 127

controls after 2 d of treatment. Consistent with the observed increase in WRI1, total fatty acid levels 128

increased by more than 30% in T6P-treated cells relative to sucrose- or sorbitol-treated controls after 2 d 129

(Fig. 1D). 130

It was previously reported that high concentrations of sucrose stabilize WRI1 and favor fatty acid 131

synthesis (Zhai et al., 2017b). We found that incubation in medium containing 100 mM sucrose stabilized 132

WRI1 and increased total fatty acid accumulation in B. napus suspension cells to a similar degree as 133

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incubation with 100 µM T6P (Supplemental Fig. 2), consistent with previous reports that T6P is more 134

potent than sucrose in regulating SnRK1 (Zhang et al., 2009). 135

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Overexpression of a bacterial trehalose 6-phosphate synthase increases de novo fatty acid 137

biosynthesis in Nicotiana benthamiana leaves 138

An alternative approach to increasing T6P content in plants is by heterologous expression of the 139

Escherichia coli trehalose 6-phosphate synthase (TPS), OtsA which converts UDPG and G6P to T6P 140

(Schluepmann et al., 2003). To test whether T6P positively regulates fatty acid synthesis in plant 141

vegetative tissue, OtsA was transiently expressed in leaves of Nicotiana benthamiana. After 3 d, T6P 142

levels were elevated by 1,000-fold relative to empty vector (EV) controls (Fig. 2A). Consistent with the 143

observation in T6P-treated B. napus cells, KIN10 activity in leaves expressing OtsA was significantly 144

lower than EV controls (Fig. 2B). OtsA-expressing leaves also accumulated significantly higher levels of 145

TAG and total fatty acids than EV controls (Fig. 2C and 2D). To test whether higher TAG accumulation 146

in OtsA-expressing leaves resulted from de novo fatty acid biosynthesis, we performed [1-14C] acetate 147

labeling studies in N. benthamiana leaves. The rate of fatty acid synthesis in leaves expressing OtsA was 148

29% higher than in leaves expressing EV (Fig. 2E). 149

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T6P specifically binds to Arabidopsis KIN10 and weakens its association with GRIK1 151

Next, we set out to determine how T6P inhibits SnRK1 to further understand the regulation of fatty acid 152

synthesis by T6P. We tested the hypothesis that T6P directly binds to KIN10 and regulates its activity. 153

As a first step, we used microscale thermophoresis (MST) analysis to evaluate whether T6P, sucrose, 154

sucrose-6ʹ-P, sorbitol or trehalose could bind to purified recombinant KIN10. Only T6P showed evidence 155

of binding to KIN10 when each of the sugars were presented at 50 µM (Table 1, Fig. 3, and Supplemental 156

Fig. 3). We also tested the possibility that T6P competes with ATP in binding to KIN10. The binding of 157

T6P with KIN10 was compared for native KIN10 and a previously described KIN10 ATP-binding 158

mutant, K48A (Shen et al., 2009). As expected, KIN10 (K48A) did not bind ATP; however, it retained 159

the ability to bind T6P, suggesting T6P likely binds at a site distinct from the ATP-binding site on KIN10 160

(Table 1). 161

A detailed MST study was performed to quantitate the binding of T6P to KIN10, in which serial dilutions 162

of T6P were titrated against KIN10. The data were used to calculate an equilibrium dissociation constant 163

(Kd) of 32.0 ± 5.6 µM (Fig. 3 and Table 2), demonstrating a direct interaction between T6P and KIN10 164

similar to that determined for ATP (Table 2). The observation that the Kd for ATP was unchanged in the 165

presence of 50 µM T6P shows that ATP and T6P bind at different sites of KIN10 (Table 2). 166

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In plants, GRIK1 and GRIK2 can bind to KIN10 and phosphorylate it, resulting in its activation (Shen 167

and Hanley-Bowdoin, 2006; Shen et al., 2009), so we tested whether T6P can also bind to purified 168

GRIK1 (Supplemental Fig. 4). MST analysis showed that T6P does not interact directly with GRIK1 169

(Table 2). To investigate whether the inhibition of SnRK1 activity by T6P is related to GRIK1 activation 170

of KIN10, we tested whether the binding of T6P to KIN10 affected the equilibrium dissociation constant 171

between KIN10 and GRIK1. In the absence of T6P, GRIK1 bound to KIN10 with a Kd of 19.6 ± 2.5 µM 172

(Fig. 3 and Table 2), whereas in the presence of 50 µM T6P, the Kd between GRIK1 and KIN10 173

increased significantly (Student’s t-test, P<0.05) by 3.5-fold to 67.7 ± 9.8 µM (Fig. 3 and Table 2). These 174

results demonstrate that binding of T6P to KIN10 significantly weakens the interaction between GRIK1 175

and KIN10. 176

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T6P decreases KIN10 phosphorylation and suppresses its activity in the presence of GRIK1 178

The results showing T6P weakens the interaction between GRIK1 and KIN10 prompted us to quantitate 179

the effects of T6P on the GRIK1-dependent T-loop phosphorylation of KIN10, which in turn mediates its 180

activation. 32P phosphorylation assays were conducted using purified preparations of GRIK1 and KIN10 181

in the presence or absence of various sugars (Fig. 4). Under the assay conditions, phosphorylation of 182

KIN10 was strictly dependent on GRIK1 (Fig. 4A). The inclusion of T6P strongly suppressed GRIK1-183

dependent phosphorylation of KIN10, while sucrose or sorbitol had no discernable effect relative to the 184

sugar-free control (Fig. 4A). In the absence of KIN10, autophosphorylation of GRIK1 appears to be 185

minimally influenced by the presence or absence of sugars (Fig. 4A). 186

As described above, GRIK-dependent phosphorylation of KIN10 results in its activation. We therefore 187

evaluated KIN10 activity in the presence or absence of sugars using the well-established SPS peptide 188

phosphorylation assay (Fig. 4B, in its linear range (Supplemental Fig. 5)). GRIK1-dependent KIN10 189

phosphorylation of the SPS peptide was significantly suppressed (Student’s t-test, P<0.05) by 190

approximately 50% in the presence of T6P (Fig. 4B). In the absence of GRIK1, KIN10 phosphorylation 191

of SPS peptide was reduced by approximately 5-fold, and did not show the T6P-dependent inhibition 192

observed for KIN10 phosphorylation in the presence of GRIK1. Indeed, we made an unexpected 193

observation that KIN10 phosphorylation of the SPS peptide was stimulated approximately 25% upon the 194

inclusion of T6P (Fig. 4B). 195

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T6P treatment decreases KIN10 phosphorylation in Brassica napus suspension cells 197

To test the effect of T6P on KIN10 in vivo, B. napus cells were incubated with 100 µM T6P, sucrose or 198

sorbitol for 1 or 2 days. Total proteins were extracted from cells and specific antibodies were used to 199

detect T-loop phosphorylated KIN10 (P-KIN10) (Nukarinen et al., 2016), KIN10 (Rodrigues et al., 2013), 200

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and GRIK1 (Shen and Hanley-Bowdoin, 2006). As shown in Fig. 5, phosphorylated KIN10 levels were 201

substantially reduced relative to sorbitol-treated cells, while total KIN10 protein levels were quite similar 202

across the treatments. This pattern is consistent with the decreases in GRIK1-dependent KIN10 203

phosphorylation in the presence of T6P observed for in vitro protein phosphorylation assays employing 204

isolated purified enzymes described above (Fig. 4). 205

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In Arabidopsis, T6P inhibition of SnRK1 is GRIK1- and GRIK2-dependent 207

As described above, T6P inhibits GRIK1-dependent KIN10 activation in assays containing recombinant 208

GRIK1and KIN10. To further explore these interactions, Arabidopsis grik1, grik2 and grik1 grik2 null 209

mutants (Glab et al., 2017) were used to test whether T6P inhibition of SnRK1 is GRIK1- and GRIK2-210

dependent in planta (Supplemental Fig. 6). SnRK1 activity in extracts of 12-d-old seedlings of grik1 211

grik2 was only 26% of wild type (Fig. 6), which is consistent with published findings that the 212

phosphorylated form of KIN10 is undetectable in the grik1 grik2 double mutant (Glab et al., 2017). 213

Inclusion of 100 µM T6P (i.e., approximately 3-fold the Kd) significantly inhibited SnRK1 activity in 214

crude extracts of wild type, grik1 and grik2 by 60%, 24% and 39%, respectively, but no inhibition was 215

observed for extracts of the grik1 grik2 double mutant. Indeed, SnRK1 activity was significantly 216

activated (by approx. 2-fold) in the presence of 100 µM T6P, consistent with the in vitro assays described 217

above employing purified KIN10 in the absence of GRIK1. 218

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DISCUSSION 220

Accumulated evidence has established a role for sugar signaling in regulating lipid synthesis. The recent 221

discovery that SnRK1, a key metabolic sensor in plants, phosphorylates WRI1 and promotes its 222

proteasomal degradation connects plant metabolic status with fatty acid synthesis (Zhai et al., 2017a). In 223

developing tissues, SnRK1 is reported to be inhibited by T6P (Zhang et al., 2009), a signal of cellular 224

sucrose status (Lunn et al., 2006), by an unknown mechanism that involves undefined protein factor(s). In 225

this work, we showed that T6P positively regulates fatty acid synthesis in B. napus cells and N. 226

benthamiana leaves by incubation in medium supplemented with T6P, and overexpression of a bacterial 227

TPS, OtsA, respectively. Both treatments significantly elevated tissue T6P levels and stabilization of 228

WRI1. These findings are consistent with previous observations that T6P inhibits SnRK1, and that 229

SnRK1 in turn suppresses WRI1 via phosphorylation that predisposes the WRI1 protein to targeted for 230

degradation (Zhai et al., 2017a). 231

That T6P has been postulated to play a global role in sugar signaling and homeostasis (Lunn et al., 2006; 232

Figueroa and Lunn, 2016) led us to ask how T6P was inhibiting SnRK1 in this study. In a surprise 233

finding, we discovered that T6P binds directly to KIN10 and that this binding weakens the interaction 234

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between KIN10 and GRIK1. We therefore propose a model for T6P-regulated plant fatty acid synthesis in 235

which GRIK is a factor that mediates T6P inhibition of SnRK1 (Fig. 7). According to the model, in cells 236

with low sucrose and T6P levels, GRIK associates with KIN10 and phosphorylates its activation loop. 237

This enhances SnRK1 activity and the phosphorylation of its many targets including WRI1, which 238

predisposes it to proteasomal degradation, thereby downregulating fatty acid synthesis, and reducing 239

cellular demand for energy and reductant. Alternatively, when sugars and T6P are abundant, T6P binds to 240

KIN10, reducing its affinity for GRIK, lowering the likelihood of KIN10 activation loop phosphorylation. 241

SnRK1 activity thus decreases and less WRI1 becomes phosphorylated and degraded, resulting in 242

increased WRI1 accumulation, thereby up-regulating fatty acid synthesis. 243

Several lines of evidence to support this model. 1) T6P feeding to B. napus cells or transient expression of 244

OtsA in N. benthamiana leaves resulted in elevated T6P levels and significantly increased fatty acid 245

content. 2) [1-14C]acetate labeling of N. benthamiana leaves expressing OtsA showed that increased fatty 246

acid and TAG accumulation resulted from higher a higher rates of de novo fatty acid synthesis. 3) 247

Increased WRI1 protein levels were observed in tissues with elevated levels of T6P. 4) T6P bound to 248

KIN10, a catalytic subunit of SnRK1 with a Kd within the published physiological range. 5) There was a 249

3.5-fold reduction in the association between GRIK1 and KIN10 in the presence of physiologically-250

relevant concentrations of T6P. 6) The incubation of B. napus cells in the presence of T6P led to a 251

reduction of T-loop activation of KIN10 and of its activity. 7) The reduction in KIN10 activity in the 252

presence of T6P was strictly GRIK1-dependent in reconstitution experiments employing bacterially-253

expressed and purified KIN10 and GRIK1. 8) Extracts from young Arabidopsis leaves of wild type and 254

grik mutants exhibited GRIK-dependent T6P inhibition of SnRK1 activity, consistent with the purified 255

GRIK1/KIN10 reconstitution experiments. 256

The observed reduction of binding affinity between GRIK1 and KIN10 in the presence of T6P can be 257

attributed to T6P binding to KIN10 because T6P specifically binds to KIN10 but not GRIK1. We initially 258

considered the possibility that T6P inhibited KIN10 activity by competing with its ATP binding site, but 259

rejected the idea because T6P did not change the binding constant for ATP, consistent with previous 260

reports based on assays in crude extracts (Zhang et al., 2009). In principle, the observed T6P-dependent 261

increase in equilibrium dissociation constant for the KIN10 and GRIK1 association could result from a 262

reduction in complex formation or an increase in its propensity to dissociate. How T6P interacts with 263

KIN10 and how that in turn affects the interaction between KIN10 and GRIK1 is presently unknown. It 264

is possible that T6P binds to a domain on KIN10 that overlaps with its GRIK1 interaction interface 265

directly, or alternatively, T6P could bind to a remote site on KIN10 causing a conformational change that 266

allosterically reduces the strength of the GRIK1-KIN10 interaction indirectly. Future structural studies 267

will be required to distinguish between these possibilities. 268

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The discovery that T6P binds to KIN10 initially led us to hypothesize the simplest scenario, i.e., that T6P 269

binding directly inhibits its catalytic activity. To test this hypothesis, assays were performed to measure 270

the phosphorylation activity of KIN10 in the presence or absence of T6P using the well-established SPS 271

peptide substrate. These experiments employing bacterially-expressed and purified KIN10 yielded the 272

apparently paradoxical result that in the absence of GRIK1, T6P reproducibly activated, rather than 273

repressed, KIN10 activity. Subsequent experiments with extracts of young leaves from the grik1 grik2 274

double mutant corroborated the activation of KIN10 by T6P in the absence of GRIK activity. Thus, 275

KIN10 is subject to two apparently opposing effects with respect to T6P: in the presence of GRIK, T6P 276

binding weakened the GRIK-KIN10 interaction decreasing T-loop phosphorylation and activation of 277

KIN10, whereas in the absence of GRIK, T6P binding resulted in a small enhancement of KIN10 activity. 278

Thus, under conditions favoring the accumulation of GRIK proteins, e.g., developing tissues, source 279

tissues or under some stress condition such as viral infection (Shen and Hanley-Bowdoin, 2006), the 280

GRIK-dependent inhibitory effect would predominate, resulting in net inhibition of SnRK1 activity by 281

T6P. 282

The findings presented herein suggest a plant-specific mechanism in which SnRK1 senses cellular 283

metabolic status changes through T6P. The sensitivity of SnRK1 to T6P and other sugar-phosphates 284

(Glc6P and Glc1P; Nunes et al., 2013), and its insensitivity to AMP (Emanuelle et al., 2015), are 285

consistent with SnRK1 acting as a general metabolic sensor, in contrast to its animal homologue, the 286

AMP-activated protein kinase (AMPK), which is primarily a sensor of energy status. Showing how 287

SnRK1 activity is influenced by sucrose status via T6P provides insights into the evolution of the plant-288

specific function of SnRK1 relative to its animal (AMPK) and yeast (SNF1) homologues. The details of 289

WRI1 stabilization by T6P and its consequences for lipid synthesis underscore the complexity of SnRK1 290

regulation, which likely evolved due to its central role in integrating diverse cellular inputs and mediating 291

cellular responses that contribute to metabolic and energy homeostasis (Sheen, 2014). 292

In summary, we recently identified SnRK1 as a regulator of WRI1(Zhai et al., 2017a) and established a 293

key role for sugar beyond simply supplying carbon skeletons for fatty acid synthesis by inhibiting 294

SnRK1-dependent WRI1 phosphorylation, which predisposes it for degradation. The present work adds a 295

new layer of mechanistic detail to this sugar signaling pathway by identifying a role for T6P, a key sugar-296

signaling intermediate. T6P reduces SnRK1 activity by binding to KIN10 and weakening its interaction 297

with GRIK1 (its activating kinase), thus reducing SnRK1 activity, WRI1 phosphorylation and 298

degradation, thereby stimulating fatty acid synthesis. 299

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302

10

METHODS 303

304

Plant Materials and Growth Conditions 305

Arabidopsis (Columbia-0) grik1 (snak2), grik2 (snak1), and grik1 (+/−) grik2 (−/−) sesqui mutants were 306

obtained from Nathalie Glab (Institute of Plant Sciences Paris-Saclay, France) (Glab et al., 2017). For 307

experiments with the grik1 grik2 double mutants, double homozygous null individuals grik1(-/-) grik2(-/-) 308

were selected from the progeny of the grik1(+/-) grik2(-/-) sesqui parental line as described in 309

Supplemental Fig. 6. Arabidopsis seeds were surface-sterilized and selected on agar plates containing 310

half-strength Murashige and Skoog salts. After one week, seedlings were transplanted to moist soil (seed 311

BM2 mix, Berger, Saint-Modeste, Canada). All plants (Arabidopsis and Nicotiana benthamiana) were 312

grown with a 16h-light/8h-dark photoperiod (combination of cool white fluorescent lamps and 313

incandescent lamps, at a photosynthetic photon flux density of 250 μmol m-2 s-1) with a 23°/19°C 314

day/night, 16h/8h temperature regime and approximately 75% relative humidity. 315

316

Genetic Constructs 317

The OtsA coding regions were amplified by PCR from E. coli genomic DNA using primer pairs listed in 318

Supplemental Table 1. The PCR products were then cloned into the Invitrogen GATEWAYM 319

pDONR/Zeo vector (Thermo Fisher Scientific, Waltham, MA; www.thermofisher.com) using the BP 320

reaction and sub-cloned (LR reaction) into the plant GATEWAYTM binary vector pGWB414 (Nakagawa 321

et al., 2007). Expression vectors for the recombinant His6-tagged kinase domain of Arabidopsis KIN10, 322

KIN10 (K48A) and KIN10 (T175A) were obtained from Wei Shen (North Carolina State University, US) 323

(Shen et al., 2009). The GRIK1 expression vector for producing recombinant His6-tagged GRIK1 protein 324

was previously described (Zhai et al., 2017a). 325

326

Expression and Purification of Recombinant KIN10 and GRIK1 327

Recombinant KIN10 (including KIN10 (K48A) and KIN10 (T175A)) and GRIK1 proteins with N- 328

terminal His6-tags were expressed in E. coli BL21(DE3). Protein purification was performed as described 329

by Shen et al. (2009). Briefly, 1 L of LB medium was inoculated with 10 ml of a saturated growth E. coli 330

culture, incubated at 37°C with shaking at 225 RPM until its optical density at 600 nm reached 0.6, at 331

which time the temperature of the culture was reduced by cooling on ice for 30 min before the addition of 332

isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. The culture was 333

incubated for a further 16 h at 16°C. Cells were collected by centrifugation at 6000 ×g for 15 min at 4°C 334

and re-suspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10 mM imidazole 335

and 0.1% (v/v) Triton X-100. The cells were disrupted by triple passage through an EmulsiFlex-C3 336

11

(Avestin, Canada; www.avestin.com) at 1000 PSI (6894 kPa). The cell lysate was clarified by 337

centrifugation at 15,000 ×g for 30 min at 4°C. The supernatant was applied to a 2 mL Ni-NTA resin 338

column (Qiagen, Hilden, Germany; www.qiagen.com) equilibrated with lysis buffer supplemented. The 339

column was washed twice with five column volumes of lysis buffer and once with wash buffer (20 mM 340

Tris-HCl, pH8.0, 0.5 M NaCl, 30 mM imidazole, 0.1% (v/v) Triton-X100), and then bound proteins were 341

eluted with 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10% (v/v) glycerol supplemented with 500 mM 342

imidazole. The eluted protein samples were desalted using an Econo-Pac® 10DG Chromatography 343

Column (Bio-Rad, Hercules, CA; www.bio-rad.com) into 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 50% 344

glycerol, 1 mM DTT. Protein preparations were separated into aliquots and stored at -20°C until use. 345

346

MicroScale Thermophoresis (MST) 347

Sucrose, sucrose-6ʹ-P, trehalose, sorbitol, and ATP were purchased from Sigma-Aldrich. T6P (Purity ≥ 348

95%, CAS#:136632-28-5) was purchased from Santa Cruz Biotechnology. Thermophoretic assays were 349

conducted using a Monolith NT.115 apparatus (NanoTemperTechnologies, South San Francisco, CA; 350

nanotempertech.com). Target proteins (KIN10 and GRIK1) were fluorescently labeled according to the 351

protocol for N-hydroxysuccinimide coupling of the dye NT647 (NanoTemper Technologies) to lysine 352

residues. Target protein and dye were incubated on ice for 30 min. Free dye was separated from labeled 353

protein by size-exclusion chromatography using MST buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 354

10 MgCl2, 1 mM DTT, and 0.05% (v/v) Tween 20). For binding tests, 100 nM target protein 355

(fluorescently labeled) was incubated with 50 μM unlabeled ligand for 10 min prior to the measurement. 356

To determine Kds, 100 nM target protein was incubated with a serial dilution of the ligand. Samples of 357

approximately 10 μl were loaded into capillaries and inserted into the MST instrument loading tray 358

(Monolith NT.115). The thermophoresis experiments were carried out using 40% MST power and 80% 359

LED power at 25 °C. Fifty µM T6P was included to test its effect on the associations of KIN10 with ATP 360

or GRIK1. 361

362

Protein Phosphorylation Assay 363

The in vitro protein phosphorylation assay was performed according to Shen et al. (2009). Briefly, a 25-364

µL assay was performed with 1 µg of KIN10 and 1 µg of GRIK1 in Reaction Buffer: 25 mM Tris-HCl, 365

pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, and 92.5 kBq [γ-32P] ATP (PerkinElmer, Waltham, 366

MA; www.perkinelmer.com) at 30°C for 20 min. The reaction was terminated by addition of 5 µL of 367

6×SDS-poly acrylamide gel electrophoresis loading buffer. A 10-µL aliquot of each assay was separated 368

by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. 32P-labeled proteins were 369

visualized by autoradiography employing a phosphor screen in conjunction with a TyphoonTM FLA 7000 370

12

imager (GE Healthcare Life Sciences, Rahway, NJ; www.gelifesciences.com). For phosphorylation 371

assays supplemented with sucrose, T6P or sorbitol, 1 mM of each was added to the reaction mix 372

containing KIN10, and incubated on ice for 5 min before GRIK1 was added to the reaction. 373

374

SnRK1 (KIN10) Activity Assay 375

Plant soluble protein extraction and SnRK1 activity assays were performed according to the methods 376

described (Ananieva et al., 2008; Zhang et al., 2009). Briefly, 50 mg (fresh weight) of suspension cells or 377

plant tissues were extracted in 100 µL of ice-cold Homogenization Buffer: 100 mM Tricine-NaOH, pH 378

8.0, 25 mM NaF, 5 mM DTT, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM benzamidine, 1 mM 379

phenylmethylsulfonyl fluoride, 1×protease inhibitor cocktail (P9599, Sigma-Aldrich, St Louis, MI; 380

www.sigmaaldrich.com), 1× phosphatase inhibitors (PhosStopTM, Roche Life Science, Penzberg, 381

Germany; lifescience.roche.com), 2% (v/v) [or (w/v)] insoluble polyvinylpolypyrrolidone (PVPP). The 382

homogenate was centrifuged at 13,000 ×g at 4°C for 15 min before spin-desalting of the supernatant (50 383

µL) (IllustraTMMicroSpinTM G-25 micro columns, GE Healthcare Life Science). Protein concentration 384

was measured with Bradford reagent (B6916, Sigma) using bovine serum albumin as standard. A 25-µL 385

assay was assembled with 10 µg total protein per sample in Kinase Reaction buffer: 50 mM HEPES-386

NaOH, pH7.5, 5 mM MgCl2, 200 µM SPS peptide (RDHMPRIRSEMQIWSED), 4 mM DTT, 0.5 µM 387

okadaic acid, 0.2 mM ATP, 12.2 kBq [γ-32P] ATP and incubated at 30°C for 5 min. The assay was 388

stopped by blotting 10 µL per reaction on 4-cm2 squares of Whatman P81 Phosphocellulose paper 389

(Whatman, Maidstone, UK; www.gelifesciences.com/whatman), immersed in 1% (v/v) phosphoric acid, 390

then washed with four 800-mL volumes of 1% phosphoric acid, immersed in acetone, dried, and 391

transferred to liquid scintillation vials. Radioactivity associated with phosphorylated SPS peptide was 392

determined by liquid scintillation counting using a Tri-carb (PerkinElmer). 393

For the purified KIN10 activity assay, 0.05 µg KIN10 or 0.05 µg KIN10 and 0.05 µg GRIK1 were 394

included in Kinase Reaction Buffer. For KIN10 kinase activity assays supplemented with sugars, 1 mM 395

of sucrose, T6P or sorbitol, was added to KIN10 and incubated on ice for 5 min before GRIK1was added 396

to the reaction. 397

398

Cell Culture Growth 399

Brassica napus cv Jet Neuf suspension cell cultures were grown in NLN medium (containing 3% sucrose) 400

as previously described (Shi et al., 2008). Prior to experiments suspension cell cultures were maintained 401

in liquid media for 7 days with media being refreshed every 2 days. For sugar treatments, cell cultures 402

were subsequently cultured on NLN solid media (containing 1% (w/v) sucrose and 8 g/L tissue culture 403

grade agar) supplemented with 100 µM of different types of sugars: sucrose, sorbitol, or T6P. Cells were 404

13

collected after 8 hours, 1, 2, and 4 days, by filtration, rinsed three times with water, flash frozen, and 405

stored at 80°C until use. 406

407

408

409

T6P Quantification 410

Water-soluble metabolites were extracted from aliquots (10-20 mg) of frozen tissue powder using 411

chloroform-methanol (Lunn et al., 2006) and evaporated to dryness using a centrifugal vacuum drier. The 412

dried extract was dissolved in 350 µL purified H2O and filtered through MultiScreen PCR-96 Filter Plate 413

membranes (Merck Millipore; www.merckmillipore.com) to remove high molecular weight compounds. 414

T6P, phosphorylated intermediates and organic acids were measured by high performance anion-415

exchange chromatography coupled to tandem mass spectrometry as described by Lunn et al. (Lunn et al., 416

2006), with modifications as described in Figueroa et al. (2016). 417

418

TAG and Total Fatty Acid Quantification 419

Total lipids (TAG plus polar lipids) were isolated from 100 mg of freshly harvested leaf tissue by the 420

addition of 700 µl of methanol:chloroform:formic acid (2:1:0.1, by volume) by vigorous shaking for 30 421

min, after which 1 ml of 1 M KCl, 0.2 M H3PO4 was added. After mixing, the samples were centrifuged 422

at 1,500 ×g (4°C) and total lipids were collected in the lower phase (chloroform). For TAG quantification, 423

60 µl of total lipid were separated by Silica Gel 60 (Merck Millipore, Billerica, MA; 424

www.emdmillipore.com) TLC developed with hexane:diethyl ether:acetic acid (70:30:1, by volume) and 425

visualized by spraying with 0.05% (w/v) primuline (in 80% (v/v) acetone). TAG fractions identified 426

under UV light were scraped from the plate and transmethylated to fatty acid methyl esters (FAMEs) by 427

incubation in 1 ml 12% (w/w) boron trichloride in methanol at 85°C for 40 min. For total fatty acid 428

quantification, 10 µl of total lipids were directly transmethylated with boron trichloride-methanol as 429

described above. For both assays, 5 µg heptadecanoic acid (C17:0) was added as internal standard prior to 430

transmethylation. FAMEs were extracted into hexane and dried under a nitrogen stream before being 431

dissolved in 100 µl hexane and analyzed by GC-MS with an Agilent Technologies (Santa Clara, CA; 432

www.agilent.com) 7890A GC System equipped with an Agilent 60m DB23 capillary column (ID 250-433

μm) and a 5975C mass selective detector. 434

435

In vivo [1-14

C] Acetate Labeling 436

Labeling experiments were performed essentially as described by Koo et al. (2004). N. benthamiana 437

leaves were incubated in 25 mM MES-NaOH, pH 5.7 buffer containing 0.01% (w/v) Tween-20 as wetting 438

14

agent under illumination (180 µmol m-2 s-1) at 25°C. Labeling was initiated by the addition of 370 kBq of 439

sodium [1-14C] acetate solution (2.15 GBq/mmol, American Radiolabeled Chemicals, St Louis, MO; 440

www.arcincusa.com). Labeling was terminated by removal of the medium from the leaf, and the sample 441

was washed three times with water. Total lipids were extracted and separated as described above. 442

Radioactivity associated with total lipids was determined by liquid scintillation counting using a Tri-carb 443

instrument (PerkinElmer). 444

445

Agroinfiltration of Nicotiana Benthamiana 446

Transient gene expression in N. benthamiana by agroinfiltration was accomplished using a previously 447

described procedure (Ohad and Yalovsky, 2010). Leaves were harvested 3 days after infiltration with 448

different constructs and analyzed for T6P and lipid contents and for SnRK1 kinase activity and in vivo [1-449

14C] acetate labeling. 450

451

Antibodies and Immunoblotting 452

Anti-WRI1 polyclonal antibodies were described in (Zhai et al., 2017a). Anti-KIN10 (Catalog No. 453

AS10919, Lot No. 1610) and -histone H3 (Catalog No. AS10710’ Lot No. 1411) polyclonal antibodies 454

were purchased from Agrisera, Vännäs, Sweden; www.agrisera.com). Anti-phospho-AMPK alpha-1 455

(Thr172) (Catalog No. PA5-17831, Lot No. TG2607252) was purchased from ThermoFisher. Anti-456

GRIK1 (Catalog No. PHY0844S, Lot No. 4480A5) and GRIK2 (Catalog No. PHY0845S, Lot No. 457

5480A7) were purchased from PhytoAB (Redwood City, CA; www.phytoab.com). All primary 458

antibodies were used at a 1:1000 dilution. Proteins were resolved by SDS-PAGE and transferred to PVDF 459

membrane for immunoblot analysis. Immunoblots of targeted proteins were visualized using HRP-460

conjugated secondary antibodies (Catalog No. AP187P, Millipore) with SuperSignalTM West Femto 461

Maximum Sensitivity Substrate (Catalog No. 34095, ThermoFisher). Immunoblot signals were detected 462

and digitalized with Image Quant LAS4000 and quantified with GelAnalyzer2010a. 463

464

Accession Numbers 465

Sequence data from this article can be found in The Arabidopsis Information Resource or UniProtKB 466

under the following accession numbers: KIN10 (At3g01090), GRIK1 (At3g45240), GRIK2 (At5g60550), 467

WRI1 (At3g54320), OtsA (P31677). 468

469

Supplemental Data 470

Supplemental Figure 1. Microspore-derived cell suspension cultures of Brassica napus L. cv. Jet Neuf 471

in liquid culture and plate culture. 472

15

Supplemental Figure 2. Sucrose stabilizes WRI1 and increases fatty acid synthesis in Brassica napus 473

suspension cells, but only if presented at a 1,000-fold higher concentration than T6P. 474

Supplemental Figure 3. Microscale thermophoretic binding tests show a specific association between 475

KIN10 and T6P at a concentration of 50 M. 476

Supplemental Figure 4. Purified recombinant KIN10 and GRIK1 expressed from E. coli. 477

Supplemental Figure 5. Fit of DPM by the amount of purified KIN10 and GRIK1 in a 25-mL reaction. 478

Supplemental Figure 6. The phenotypes of grik mutants. 479

Supplemental Table 1. Oligonucleotide primer sequence pairs. 480

Supplemental File 1. Statistical analysis results for each figure. 481

482

483

Acknowledgements 484

This work was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy 485

Sciences under contract number DE-SC0012704, specifically through the Physical Biosciences program 486

of the Chemical Sciences, Geosciences, and Biosciences Division. (Z.Z., J.K., H.L., J.S.). T6P analysis 487

was supported by the Max Planck Society (R.F. and J.E.L.). 488

489

Author Contributions: JS, ZZ and JK conceived the study. ZZ, JK, HL, RF and JL performed 490 experiments. ZZ, JK, RF, JL and JS analyzed data. ZZ, JK, JL and JS wrote the manuscript. 491

492

493

16

Table 1. T6P specifically binds KIN10. 494

Ligand

(50 µM)

Target

KIN10 KIN10 (K48A)

ATP + ̶

T6P + +

Sucrose ̶ n.d.

Trehalose ̶ n.d.

Sorbitol ̶ n.d.

Suc-6’-P ̶ n.d.

495

Microscale thermophoresis (MST) was performed to evaluate the interactions between target proteins 496

(KIN10 and KIN10 (K48A)) and potential ligands. +, interaction detected; -, lack of interaction; n.d., not 497

determined. 498

499

Table 2. T6P binding to KIN10 weakens the interaction between GRIK1and KIN10. 500

Target Ligand(s) Kd (µM)

KIN10 ATP 12.8±1.1

KIN10 T6P 32.0±5.6

GRIK1 T6P ―

KIN10 GRIK1 19.5±2.6

KIN10 ATP+T6P (50 µM) 12.9±1.2

KIN10 GRIK1+T6P (50 µM) 67.7±9.8

501

Microscale thermophoresis (MST) was performed to determine the equilibrium dissociation constants 502

(Kd) between KIN10, GRIK1 and T6P. Values represent mean±SD, n=3 independent replicates. Kd 503

values for KIN10 and GRIK1 in the presence of 50 M T6P were significantly different (P<0.002 by 504

Student’s t-test of three independent replicates) from those in the absence of T6P. -, lack of observed 505

interaction between GRIK1 and T6P. 506

507

508

509

510

511

17

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513

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glucose pyrophosphorylase a novel regulatory mechanism linking starch synthesis to the sucrose 654 supply. The Plant Cell 14, 2191-2213. 655

Wang, S.-J., Yeh, K.-W., and Tsai, C.-Y. (2001). Regulation of starch granule-bound starch synthase I 656 gene expression by circadian clock and sucrose in the source tissue of sweet potato. Plant 657 Science 161, 635-644. 658

Wienken, C.J., Baaske, P., Rothbauer, U., Braun, D., and Duhr, S. (2010). Protein-binding assays in 659 biological liquids using microscale thermophoresis. Nature communications 1, ncomms1093. 660

Yadav, U.P., Ivakov, A., Feil, R., Duan, G.Y., Walther, D., Giavalisco, P., Piques, M., Carillo, P., 661 Hubberten, H.-M., and Stitt, M. (2014). The sucrose–trehalose 6-phosphate (Tre6P) nexus: 662 specificity and mechanisms of sucrose signalling by Tre6P. Journal of Experimental Botany 65, 663 1051-1068. 664

Zhai, Z., Liu, H., and Shanklin, J. (2017a). Phosphorylation of WRINKLED1 by KIN10 results in its 665 proteasomal degradation, providing a link between energy homeostasis and Lipid biosynthesis. 666 The Plant Cell 29, 871-889. 667

Zhai, Z., Liu, H., Xu, C., and Shanklin, J. (2017b). Sugar potentiation of fatty acid and triacylglycerol 668 accumulation. Plant Physiology 175, 696-707. 669

Zhang, Y., Primavesi, L.F., Jhurreea, D., Andralojc, P.J., Mitchell, R.A., Powers, S.J., Schluepmann, H., 670 Delatte, T., Wingler, A., and Paul, M.J. (2009). Inhibition of SNF1-related protein kinase1 activity 671 and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiology 149, 1860-672 1871. 673

674

675

FIGURE LEGENDS 676

677

Figure 1. The effects of T6P on microspore-derived cell suspension cultures of Brassica napus. (A) T6P 678

contents in B. napus cells cultured on plates supplemented with 100 µM of trehalose 6-phosphate (T6P), 679

sucrose (Suc) or sorbitol (Sor) for 3, 8 hours (h) and 2 days (d). (B) SnRK1 activity in the crude extracts 680

of suspension cells treated for 2 days with sugars as indicated. (C) WRI1 protein levels in (B) are shown 681

by immunoblot with WRI1 specific antibody. Protein loading is shown by histone H3 (H3) from the same 682

sample. A representative immunoblot is shown. The histogram under immunoblots shows relative WRI1 683

protein levels quantified with GelAnalyzer2010 and normalized against corresponding protein levels of 684

histone H3. (D) Total fatty acid at 2-day samples is shown for suspension cells treated with sugars as 685

indicated. In this figure, values represent mean±SD of 3 independent biological replicates. Levels 686

indicated with different letters above histogram bars are significantly different (Student’s t test for all 687

pairs of sugars at the same time point, P<0.05; Supplemental File 1). 688

689

Figure 2. Transient expression of T6P synthase OtsA increases biosynthesis of both TAG and total fatty 690

acid in Nicotiana benthamiana leaves. (A) T6P contents in five-week-old N. benthamiana leaves 691

transformed with empty vector (EV) or expressing OtsA for 3 days. Values represent mean±SD of 4 692

independent biological replicates. (B) SnRK1 activity in crude extracts of infiltrated N. benthamiana 693

21

leaves described in (A). TAG (C) and total fatty acids (D) were quantified in the samples described in 694

(A). (E) [1-14C] acetate incorporation into fatty acyl products by strips of N. benthamiana leaves 695

transiently expressing genes as indicated. Values represent mean incorporation ±SD of 5 independent 696

biological replicates after 30 min of labeling. Asterisks denote statistically significant differences from the 697

EV control material (Student’s t test, *, P<0.05; **, P<0.01; Supplemental File 1). DPM, disintegrations 698

per minute. 699

700

Figure 3. T6P weakens the interaction between GRIK1 and KIN10. Thermophoretic experiments were 701

performed to determine the equilibrium dissociation constants between KIN10, GRIK1 and T6P (the 702

means of three independent replicates are shown in Table 2). In the thermophoresis experiments, T6P was 703

titrated against KIN10 (A), GRIK1 was titrated against KIN10 in the absence of T6P (B) or in the 704

presence of 50 µM T6P (C). Dissociation curves were fit to the data to calculate the Kd values shown. 705

Data shown are the mean±SD, n=3 independent replicates. 706

707

Figure 4. T6P suppresses KIN10 activation by GRIK1 in vitro. (A) In vitro protein phosphorylation 708

assays were performed using purified recombinant KIN10 and GRIK1. Reactions either contained (+) or 709

did not (-) contain 1 µg of GRIK1, 1 µg of KIN10 or 1 mM of trehalose 6-phosphate (T6P), sucrose (Suc) 710

or sorbitol (Sor), as indicated, in the presence of [γ-32P] ATP. After the reaction, proteins were separated 711

using SDS-PAGE and transferred to PVDF membranes. 32P -labeled proteins were visualized by 712

autoradiography. Inactive KIN10 kinase mutants: KIN10 (T175A) and KIN10 (M48A) were also 713

included as negative controls. A representative autoradiograph is shown. (B) KIN10 activity was 714

determined by measuring the incorporation of 32P from [γ-32P] ATP into the SnRK1-target peptide of 715

sucrose-phosphate synthase (SPS). Kinase activity was quantified for 50 ng of KIN10, or for a mixture of 716

50 ng of KIN10 + 50 ng of GRIK1 or for 50 ng of GRIK1, in the absence (-) or presence of 1 mM T6P or 717

Suc. Values represent mean±SD of 3 independent replicates. Levels indicated with different letters above 718

histogram bars are significantly different (Student’s t test for all pairs of the same proteins, P<0.05; 719

Supplemental File 1). DPM, disintegrations per minute. 720

721

Figure 5. T6P treatment decreases KIN10 phosphorylation in microspore-derived cell suspension cultures 722

of Brassica napus. The levels of phosphorylated KIN10, P-KIN10, KIN10 and GRIK1 were quantified 723

from B. napus cells cultured on plates supplemented with 100 µM of trehalose 6-phosphate (T6P), 724

sucrose (Suc) or sorbitol (Sor) for 1 day (1d) or 2 days (2d) as indicated. A representative immunoblot for 725

each protein is shown. Protein loading is shown by histone H3 from the same sample. 726

727

22

Figure 6. Inhibition of SnRK1 by T6P is GRIK-dependent. SnRK1 activity is quantified by the 728

incorporation of 32P from [γ-32P] ATP into the SPS peptide. Activity was measured in crude extracts from729

12-day-old seedlings of Arabidopsis wild type (WT), grik1 and grik2 single mutants and the grik1grik2730

double mutant in the absence (-) or presence (+) of 100 M T6P. Values represent mean±SD of 3 731

independent biological replicates. Asterisks denote statistically significant differences from the respective 732

control samples lacking T6P for each genotype (Student’s t test, *, P<0.05; **, P<0.01; Supplemental File 733

1). 734

735

Figure 7. T6P-regulated plant fatty acid synthesis. High cellular sugar (carbon) is associated with the 736

accumulation of trehalose 6-phosphate (T6P) which binds to KIN10 (small molecule superimposed on 737

KIN10), the catalytic subunit of SnRK1, weakening its affinity for GRIK. This results in decreased 738

phosphorylation of the KIN10 activation loop, thereby reducing the proportion of activated KIN10. 739

Conversely, when the cellular sucrose level is low, levels of T6P decrease and GRIK binds tightly to 740

KIN10, phosphorylating its activation loop and increasing SnRK1 activity. Activated KIN10 741

phosphorylates and inactivates 3-HYDROXY-3-METHYLGLUTARYL COA REDUCTASE 1, 742

NITRATE REDUCTASE, SUCROSE PHOSPHATE SYNTHASE and WRINKLED1 to reduce energy-743

consuming pathways (anabolism) and at the same time, via phosphorylation of other target proteins 744

including several transcription factors, promotes a variety of major catabolic pathways to generate energy. 745

Activated KIN10 can also phosphorylate GRIK and reduce its activity. 746

747

Fig. 1. The effects of T6P on microspore-derived cell suspension cultures of Brassicanapus. (A) T6P contents in B. napus cells cultured on plates supplemented with 100 µMof trehalose 6-phosphate (T6P), sucrose (Suc) or sorbitol (Sor) for 3, 8 hours (h) and 2days (d). (B) SnRK1 activity in the crude extracts of suspension cells treated for 2 dayswith sugars as indicated. (C) WRI1 protein levels in (B) are shown by immunoblot withWRI1 specific antibody. Protein loading is shown by histone H3 (H3) from the samesample. A representative immunoblot is shown. The histogram under immunoblots showsrelative WRI1 protein levels quantified with GelAnalyzer2010 and normalized againstcorresponding protein levels of histone H3. (D) Total fatty acid at 2-day samples is shownfor suspension cells treated with sugars as indicated. In this figure, values representmean±SD of 3 independent biological replicates. Levels indicated with different lettersabove histogram bars are significantly different (Student’s t test for all pairs of sugars atthe same time point, P<0.05).

A

Suc T6P Sor0

5

10

15

20 b

%To

talf

atty

acid

(FW

)

D

T6P

(nm

ole

g-1FW

)

Suc T6P Sor Suc T6P Sor8h 2d

Suc T6P Sor3h

1.5

2.0

2.53.0

1.0

00.5

00.2

0.4

0.6

0.8

1.0

1.2

Suc T6P Sor

B

Rel

ativ

eS

nRK

1ac

tivity

Suc T6P Sor

WRI1

C

H3

MKD

2055

01234

Rel

ativ

epr

otei

nle

vels

ofW

RI1

a a

b

a ab

aa

b

aa b

a a

b

a a

*

0

2.03.04.0

1.0

D

%To

talf

atty

acid

(FW

)

**

0.10.20.30.40.50.6

0

%TA

G(D

W)

C

**

4

8

12

0EV otsA

0

3

4

2

1

T6P

(µm

ole

g-1FW

)

A

*

12345

0

6

DP

M(1

04 )10

0m

gFW

-1

EV otsA

E

*

00.20.50.71.01.2

B

Rel

ativ

eS

nRK

1ac

tivity

Fig. 2. Transient expression of T6P synthase otsAincreases biosynthesis of both TAG and total fatty acidin Nicotiana benthamiana leaves. (A) T6P contents infive-week-old N. benthamiana leaves transformed withempty vector (EV) or expressing otsA for 3 days.Values represent mean±SD of 4 independent biologicalreplicates. (B) SnRK1 activity in crude extracts ofinfiltrated N. benthamiana leaves described in (A). TAG(C) and total fatty (D) were quantified in the samplesdescribed in (A). (E) [1-14C] acetate incorporation intofatty acyl products by strips of N. benthamiana leavestransiently expressing genes as indicated. Valuesrepresent mean incorporation±SD of 5 independentbiological replicates after 30 min of labeling. Asterisksdenote statistically significant differences from the EVcontrol material (Student’s t test, *, P<0.05; **, P<0.01).

EV otsA

EV otsA

EV otsA

EV otsA

852

850

848

846Kd=32.0µM

C

Target=KIN10Ligand=T6P

Kd=19.5µM

Target=KIN10Ligand=GRIK1

Kd=67.7µM

Target=KIN10Ligand=GRIK1Ligand=T6P(50µM)

A

B

844

Fnor

m[0 /

00]

850

845

840

Fnor

m[0 /

00]

832

830

828

826

Fnor

m[0 /

00]

Fig. 3. T6P weakens the interaction between GRIK1 and KIN10. Thermophoreticexperiments to determine the strength of interactions between KIN10, GRIK1 and T6P (themeans of three independent replicates are shown in Table 2). In the thermophoresisexperiments, T6P was titrated against KIN10 (A), GRIK1 was titrated against KIN10 in theabsence of T6P (B) or in the presence of 50 µM T6P (C). Dissociation curves were fitted tothe data for calculation of the Kd values shown. Data shown are the mean±SD, n=3independent repetitions.

Ligand concentration(log[M])

824

-7 -6 -5 -4 -3 -2 -1 0

-9 -8 -7 -6 -5 -4 -3 -2

-9 -8 -7 -6 -5 -4 -3 -2

KIN10 + + + + + – – – –GRIK1 – + + + + + + + +

T6P – – +

pKIN10

pGRIK1

–

KIN10

6

4

1

3

5

2

78

KIN10+GRIK1– T6P Suc – T6P Suc – T6P Suc

Fig. 4. T6P suppresses KIN10 activation by GRIK1 in vitro. (A) In vitro proteinphosphorylation assays were performed using purified recombinant KIN10 and GRIK1.Reactions either contained (+) or did not (–) contain 1 µg of GRIK1, 1 µg of KIN10 or 1mM of trehalose 6-phosphate(T6P), sucrose (Suc) and sorbitol (Sor) as indicated in thepresence of [γ-32P] ATP. After reaction, proteins were separated using SDS-PAGE andtransferred to PVDF membranes. 32P-labeled proteins were visualized by autoradiography.Inactive KIN10 kinase mutants: KIN10 (T175A) and KIN10 (M48A) were also included asnegative controls. A representative autoradiography was shown. (B) KIN10 activity wasdetermined by measuring the incorporation of 32P from [γ-32P] ATP into the SnRK1-targetpeptide of sucrose-phosphate synthase (SPS). Kinase activity was quantified for 50 ng ofKIN10 or for a mixture of 50 ng of KIN10 + 50 ng of GRIK1 or for 50ng of GRIK1, inabsence (–) or presence of 1 mM of T6P or Suc. Values represent mean±SD of 3independent replicates. Levels indicated with different letters above histogram bars aresignificantly different (Student’s t test for all pairs for the same proteins, P<0.05). DPM,disintegrations per minute.

B

A

Phos

phor

ylat

ion

ofSP

Spe

ptid

e(D

PM(1

05 )of

32P)

SucSor

– –+

+

– –– – – – – – –– – – – – – –

++

+T175A

– ––

–

– –M48A

––

GRIK1

ba a

b

aa

aa a

P-KIN10

KIN10

H3

M(KD)

1d 2dSuc T6P Sor Suc T6P Sor

GRIK1

Fig. 5. T6P treatment decreases KIN10 phosphorylation in microspore-derived cellsuspension cultures of Brassica napus. The levels of phosphorylated KIN10, P-KIN10,KIN10 and GRIK1 were quantified from B. napus cells cultured on plates supplementedwith 100 µM of trehalose 6-phosphate (T6P), sucrose (Suc) or sorbitol (Sor) for 1 day (1d)or 2 days (2d) as indicated. A representative immunoblot for each protein is shown.Protein loading is shown by histone H3 from the same sample.

50

50

2015

50

Fig. 6. Inhibition of SnRK1 by T6P is GRIK-dependent. SnRK1 activity isquantified by the incorporation of 32P from [γ-32P] ATP into the SPS peptide.Activity was measured in crude extracts from 12-day old seedlings of Arabidopsiswild type (WT), grik1 and grik2 single mutants and the grik1grik2 double mutant inthe absence (-) or presence (+) of 100 µM T6P. Values represent mean±SD of 3independent biological replicates. Asterisks denote statistically significantdifferences from the respective control samples lacking T6P for each genotype(Student’s t test, *, P<0.05; **, P<0.01).

T6P(100µM)+– +– +– +–

WTgrik1grik2grik1grik2

3.0

2.5

2.0

1.5

1.0

0.5

032P

inco

rpor

atio

nin

toS

PS

pept

ide

DP

M(1

04 )m

in-1

µg-1

prot

ein

** ****

*

Fig. 7. T6P-regulated plant fatty acid synthesis.

High cellular sugar (carbon) is associated with the accumulation of trehalose

6-phosphate (T6P) which binds to KIN10 (small molecule superimposed on

KIN10), the catalytic subunit of SnRK1, weakening its affinity for GRIK. This

results in decreased phosphorylation of the KIN10 activation loop, thereby

reducing the proportion of activated KIN10. Conversely, when the cellular

sucrose level is low, levels of T6P decrease and GRIK binds tightly to KIN10,

phosphorylating its activation loop and increasing SnRK1 activity. Activated

KIN10 phosphorylates WRINKLED1 (and other target proteins), resulting its

proteasomal degradation, tuning down fatty acid to reduce carbon (energy)-

consumption. Activated KIN10 can also phosphorylate GRIK and reduce its

activity. E3, ubiquitin-protein ligase. BCCP2, BIOTIN CARBOXYL CARRIER

PROTEIN. KAS1, KETOACYL SYNTHASE. PKP-β1, PLASTIDIAL

PYRUVATE KINASE. AAs, amino acids.

KIN10

GRIK GRIK

KIN10

(Activated)

T

P

T6P

High sugarLow sugar

: Positive: Negative

KIN10 KIN10

WRI1

175T175

T175

T175

P

P

PP

WRI1

P

WRI1

P

E3

Target proteins

AAs

Proteasome

BCCP2,

KAS1,

PKP-β1…

Fatty acid

synthesis

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http://www.ncbi.nlm.nih.gov/pubmed?term=Ananieva%2C E%2EA%2E%2C Gillaspy%2C G%2EE%2E%2C Ely%2C A%2E%2C Burnette%2C R%2EN%2E%2C and Les Erickson%2C F%2E %282008%29%2E Interaction of the WD40 domain of a myoinositol polyphosphate 5%2Dphosphatase with SnRK1 links inositol%2C sugar%2C and stress signaling%2E Plant Physiology 148%2C 1868%2D1882%2E&dopt=abstract

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ananieva,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Interaction of the WD40 domain of a myoinositol polyphosphate 5-phosphatase with SnRK1 links inositol, sugar, and stress signaling&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Andre%2C C%2E%2C Haslam%2C R%2EP%2E%2C and Shanklin%2C J%2E %282012%29%2E Feedback regulation of plastidic acetyl%2DCoA carboxylase by 18%3A 1%2Dacyl carrier protein in Brassica napus%2E Proceedings of the National Academy of Sciences 109%2C 10107%2D10112%2E&dopt=abstract

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Andre,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Feedback regulation of plastidic acetyl-CoA carboxylase by 18: 1-acyl carrier protein in Brassica napus&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Feedback regulation of plastidic acetyl-CoA carboxylase by 18: 1-acyl carrier protein in Brassica napus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Andre,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Baaske%2C P%2E%2C Wienken%2C C%2E%2C Willemsen%2C M%2E%2C Braun%2C D%2E%2C and Duhr%2C S%2E %282011%29%2E Protein%2Dbinding assays in biological liquids using microscale thermophoresis%2E Journal of Biomolecular Techniques%3A JBT 22%2C S55%2E&dopt=abstract

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baaske,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Protein-binding assays in biological liquids using microscale thermophoresis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Protein-binding assays in biological liquids using microscale thermophoresis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baaske,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Baena%2DGonz�lez%2C E%2E%2C Rolland%2C F%2E%2C Thevelein%2C J%2EM%2E%2C and Sheen%2C J%2E %282007%29%2E A central integrator of transcription networks in plant stress and energy signalling%2E Nature 448%2C 938%2D942%2E&dopt=abstract

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baena-González,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A central integrator of transcription networks in plant stress and energy signalling&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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DOI 10.1105/tpc.18.00521; originally published online September 24, 2018;Plant Cell

Zhiyang Zhai, Jantana Keereetaweep, Hui Liu, Regina Feil, John E. Lunn and John ShanklinTrehalose 6-phosphate positively regulates fatty acid biosynthesis by stabilizing WRINKLED1

This information is current as of April 10, 2021

Supplemental Data /content/suppl/2018/09/24/tpc.18.00521.DC1.html

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