1

RESEARCH ARTICLE 12

Phosphorylation of Phosphoenolpyruvate Carboxylase is Essential for 3Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock 4Operation in the Obligate Crassulacean Acid Metabolism Species 5Kalanchoë fedtschenkoi 6

7Susanna F. Boxall, Louisa V. Dever, Jana Kneřová1, Peter D. Gould, and James 8Hartwell9Department of Functional and Comparative Genomics, Institute of Integrative Biology, Crown 10Street, University of Liverpool, Liverpool L69 7ZB, UK. 111Present address: Department of Plant Sciences, Downing Street, University of Cambridge, 12Cambridge CB2 3EA, UK 13

14Corresponding author: James Hartwell, e-mail: james.hartwell@liverpool.ac.uk 15

16Short Title: Silencing PPC phosphorylation in a CAM plant 17

18One-Sentence Summary: Silencing phosphoenolpyruvate carboxylase kinase in a CAM 19species more than halves dark period CO2 fixation, and causes arrhythmia in some 20components of the central circadian clock. 21

22The author responsible for distribution of materials integral to the findings presented in this 23article in accordance with the policy described in the Instructions for Authors 24(www.plantcell.org) is: James Hartwell (james.hartwell@liverpool.ac.uk). 25

26ABSTRACT 27

28Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 29fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated post-30translationally by a circadian clock controlled protein kinase called phosphoenolpyruvate 31carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its 32sensitivity to feedback inhibition by malate, and thus enhancing nocturnal CO2 fixation to 33stored malate. Here, we report the generation and characterization of transgenic RNAi lines of 34the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 35transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to 36a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced 37malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in 38the transcript abundance of KfPPCK1 was accompanied by a loss of oscillations in the 39transcript abundance of many core circadian clock genes, suggesting that perturbing the only 40known link between CAM and the circadian clock feeds back to perturb the central circadian 41clock itself. This work shows that clock control of KfPPCK1 prolongs the activity of PPC 42throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, 43malate accumulation, CAM productivity and core circadian clock robustness. 44

45

Plant Cell Advance Publication. Published on September 8, 2017, doi:10.1105/tpc.17.00301

©2017 The authors. All Rights Reserved

2

INTRODUCTION 46

Crassulacean acid metabolism (CAM) is one of several higher plant photosynthetic 47

CO2-concentrating mechanisms. It is particularly noteworthy for its high water-use efficiency 48

(WUE) relative to C3 and C4 photosynthesis (Borland et al., 2009; Cushman et al., 2015). The 49

functional genomics and evolutionary biology of CAM species has recently received a 50

dramatic upsurge in interest due to the potential of CAM crops, such as agaves and opuntias, 51

as water-wise sources of biomass for biofuels, platform chemicals and food (Cushman et al., 52

2015; Yang et al., 2015). In addition, efforts are underway to engineer CAM into C3 species 53

using synthetic biology approaches (Borland et al., 2014; Borland et al., 2015). CAM into C3 54

aims to improve the WUE of key C3 crops, thereby adapting them to the increased frequency 55

and intensity of droughts and extreme high temperatures predicted under future climate 56

change scenarios. The further exploitation of CAM in these ways will require detailed 57

characterization of CAM gene function, especially with respect to their daily regulation by the 58

circadian clock (Yang et al., 2015; Hartwell et al., 2016). 59

CAM is a classic example of convergent evolution that is known from 35 families of 60

plants spanning monocots, dicots and ferns (Smith and Winter, 1996; Holtum et al., 2007; 61

Tsen and Holtum, 2012; Christin et al., 2014; Silvera et al., 2014). Based on our currently 62

incomplete understanding of the molecular-genetic blueprint for CAM (Hartwell et al., 2016), 63

C3 species are believed to possess ancestral copies of all of the genes required for CAM 64

(Cushman and Bohnert, 1999; Aubry et al., 2011; Ming et al., 2015). In principle, for CAM to 65

evolve from the C3 ancestral state in a leaf succulent CAM species such as Kalanchoë 66

fedtschenkoi, the corresponding metabolic enzymes, metabolite transporters and their 67

cognate regulatory proteins must increase in abundance in leaf mesophyll cells and come 68

under tight temporal control such that certain metabolic steps are confined to the dark period, 69

and others are confined to the light (Cushman and Bohnert, 1999; Borland et al., 2009; 70

Abraham et al., 2016; Brilhaus et al., 2016; Hartwell et al., 2016). However, only limited work 71

has been reported testing this principle using a genetic approach, which represents a 72

powerful method for in planta functional testing of candidate CAM genes when combined with 73

detailed characterization of CAM-associated phenotypes in the resulting transgenic lines 74

(Hartwell et al., 2016; Dever et al., 2015). 75

During the daily CAM cycle, CO2 fixation occurs via two temporally separated steps. In 76

3

the dark, atmospheric CO2 enters through open stomata and is converted to HCO3- either 77

passively or via the reaction catalyzed by a b-carbonic anhydrase. Primary CO2-fixation is 78

catalyzed by phosphoenolpyruvate carboxylase (PPC), which converts PEP and HCO3- to 79

OAA and inorganic phosphate (Chollet et al., 1996). Malate dehydrogenase (MDH) reduces 80

OAA to malate, which is transported to and stored in the vacuole as malic acid (Borland et al., 81

2009). In the light, malate decarboxylation generates CO2 that is refixed by Rubisco in the 82

Calvin cycle; referred to as secondary CO2 fixation. Both primary and secondary CO2-fixation 83

occur in each leaf mesophyll cell, and therefore strict temporal regulation is required to 84

prevent futile cycling between carboxylation and decarboxylation (Hartwell, 2005, 2006). 85

In the model obligate CAM species K. fedtschenkoi (Hartwell et al., 2016), both PPC 86

abundance and specific activity are stable over the light/ dark cycle (Nimmo et al., 1984; 87

Dever et al., 2015), but its allosteric properties are subject to tight temporal control (Nimmo et 88

al., 1986; Nimmo et al., 1987). Several primary metabolites have been reported to influence 89

the activity of PPC in vitro, including malate (allosteric inhibitor) and glucose 6-phosphate 90

(allosteric activator) (Doncaster and Leegood, 1987; Vidal and Chollet, 1997). For a CAM leaf, 91

as the dark period progresses, the vacuolar storage capacity for malic acid reaches its limit, 92

and malate begins to accumulate in the cytosol where it feeds back to inhibit PPC activity. In 93

K. fedtschenkoi, the Ki of PPC for malate was found to be increased by dark period 94

phosphorylation of the enzyme on an N-terminal serine residue catalyzed by the activity of a 95

specific, Ca2+-independent protein kinase, named PPC kinase (PPCK) (Carter et al., 1991; 96

Hartwell et al., 1999; Nimmo, 2000, 2003). The circadian clock controls PPCK transcript 97

levels and activity, such that they peak during the dark period (Hartwell et al., 1996; Hartwell 98

et al., 1999; Taybi et al., 2000; Boxall et al., 2005; Theng et al., 2008). In the light, a protein 99

phosphatase type 2A dephosphorylates PPC, leaving it up to 10 times more sensitive to 100

malate inhibition (Carter et al., 1990). This phosphatase was found to be active throughout 101

the light/ dark cycle (Carter et al., 1991), supporting the proposal that the light/ dark regulation 102

of PPC is mainly a result of the circadian rhythm of PPCK levels (Hartwell et al., 1999), 103

although this has yet to be demonstrated in planta. 104

PPCK remains the only well-defined, temporal/ circadian control point for CAM 105

(Hartwell, 2006; Hartwell et al., 2016). To further elucidate the role of PPC phosphorylation in 106

the temporal control of CO2 fixation associated with CAM, and the extent to which this 107

4

phospho-regulation step helps to prevent metabolic futile cycling during CAM, we generated 108

stable transformants of K. fedtschenkoi expressing RNA interference (RNAi) constructs that 109

targeted the degradation of endogenous KfPPCK1 transcripts. We recovered independent 110

transgenic lines with reduced KfPPCK1 transcripts and reduced phosphorylation of PPC in 111

the dark, and found that phosphorylation was vital for optimizing and sustaining dark CO2 112

fixation associated with CAM. Furthermore, the loss of dark phosphorylation of PPC led to a 113

loss of robust rhythmicity for certain components of the central molecular circadian clock, and 114

for the regulation of several distinct circadian clock outputs. Surprisingly, several key 115

components of the central oscillator were induced in the KfPPCK1 RNAi lines and these 116

genes maintained robust transcript oscillations under constant light and temperature 117

conditions. 118

119

RESULTS 120

Initial screening and characterisation of KfPPCK1 RNAi lines of K. fedtschenkoi 121

As K. fedtschenkoi does not produce viable seed (Garces et al., 2007; Hartwell et al., 122

2016), data is presented for primary transformants that were propagated clonally via leaf 123

plantlets and/ or stem cuttings. Primary transformants were screened initially using high-124

throughput leaf disc tests for starch and acidity (Cushman et al., 2008; Dever et al., 2015). 125

Leaf disc screening identified KfPPCK1 RNAi lines because they had higher leaf pH at dawn 126

than the wild type due to reduced malic acid accumulation during the dark period. Transgenic 127

lines that acidified less during the dark period were screened for the steady-state abundance 128

of KfPPCK1 transcript levels with RT-qPCR (Fig. 1). Comparison of KfPPCK1 transcript levels 129

over a 12-h-light/ 12-h-dark cycle revealed that, relative to wild type, the two best RNAi lines 130

(rPPCK1-1 and rPPCK1-3) had a large reduction in the transcript abundance of the target 131

transcript (Fig. 1A). Whilst both lines displayed reduced levels of KfPPCK1 transcripts relative 132

to the wild type, line rPPCK1-1 had a higher level of KfPPCK1 transcripts in the middle of the 133

dark period than line rPPCK1-3, and neither line displayed a complete loss of KfPPCK1 134

transcript accumulation in the dark (Fig. 1A). These results indicated that we had recovered 135

independent lines with different degrees of silencing of the endogenous transcript level; with 136

line rPPCK1-3 showing the greatest reduction (Fig. 1A). 137

138

PPC phosphorylation and the apparent Ki of PPC for feedback inhibition by L-malate 139

5

In K. fedtschenkoi, PPC has been shown to be subject to a circadian rhythm of dark 140

period phosphorylation/ light period dephosphorylation as a result of rhythmic synthesis and 141

degradation of PPCK (Hartwell et al., 1999). Immunoblotting using an anti-phospho-PPC 142

antibody was used to investigate the phosphorylation state of PPC over a 12-h-light/ 12-h-143

dark cycle in CAM leaves of both the wild type and the rPPCK1 lines (Fig. 1B, left panel). The 144

abundance of PPC itself was measured on replicate immunoblots to check for even protein 145

loading of the blots (Fig. 1B, right panel). In the wild type CAM leaves, PPC was 146

phosphorylated to a high level both at 18:00 and 22:00, 6 and 10 h into the 12 h dark period 147

(Fig. 1B, left panel). Wild type PPC phosphorylation declined rapidly in the light, with a low 148

level of phospho-PPC detected at 2 h into the 12 h light period, but virtually no 149

phosphorylation detected at both 6 and 10 h into the light period (Fig. 1B, left panel). The 150

phosphorylation of PPC in the dark was reduced in both of the rPPCK1 lines (Fig. 1B, left 151

panel). Relative to wild type, line rPPCK1-1 had a reduced peak of phospho-PPC in the 152

middle of the dark period (18:00), but there was a dramatic reduction relative to wild type at 153

the end of the dark period (22:00) (Fig. 1B, left panel). Line rPPCK1-3, failed to achieve a 154

detectable level of PPC phosphorylation above background at any of the sampled light/ dark 155

time points. Thus, rPPCK1-1 had intermediate levels of PPC phosphorylation in the dark, and 156

rPPCK1-3 had the strongest phenotype, as it lacked detectable levels of dark period 157

phosphorylation of PPC (Fig. 1B, left panel). These perturbations of dark phosphorylation of 158

PPC correlated well with the level of KfPPCK1 transcripts in the dark in the two lines, with 159

rPPCK1-3 showing the greatest reduction in KfPPCK1 transcript abundance (Fig. 1A), 160

consistent with the loss of PPC phosphorylation at the same time points (Fig. 1B, left panel). 161

The relative transcript abundance of two other detectable KfPPCK genes, namely KfPPCK2 162

and KfPPCK3, was higher in the rPPCK1-3 lines relative to the wild type (Fig. 1C and 1D). 163

Interestingly, the increased transcript levels for KfPPCK2 and KfPPCK3 in the rPPCK1-3 line 164

(Fig. 1C and 1D) did not rescue the dark period phosphorylation of PPC (Fig. 1B, left panel). 165

The fourth PPCK gene (KfPPCK4) in the K. fedtschenkoi genome could not be detected 166

reliably using RT-qPCR, even after 40 cycles. 167

As the phosphorylation of PPC in the dark results in an increase in its apparent Ki for 168

its feedback inhibitor L-malate (Nimmo et al., 1984, Carter et al., 1991), we also investigated 169

the apparent Ki of PPC for L-malate using rapidly desalted extracts of LP6 sampled in the 170

light at 10:00 (2 h before dusk) and in the dark at 18:00 (middle of the 12 h dark period) (Fig. 171

1B, Ki values below corresponding time points on the left panel, and Supplemental Fig. S1). 172

6

The wild type displayed the well-documented large increase in the apparent Ki of PPC for L-173

malate in the dark period, with the apparent Ki L-malate increasing from 0.5 mM for leaves 174

sampled 2 h before the end of the light period up to 8 mM in the middle of the dark period; the 175

latter coincident with high PPC phosphorylation detected at the same time point (Fig. 1B, left 176

panel, and Supplemental Fig. S1). Line rPPCK1-1 displayed a large reduction in the light to 177

dark differential for the apparent Ki of PPC for L-malate, with the measured values being 178

reduced to an average of 0.6 mM in the light samples, and 2.7 mM for the dark samples (Fig. 179

1B and Supplemental Fig. 1). This was consistent with the reduced level of PPC 180

phosphorylation in rPPCK1-1 in the middle of the dark period (Fig. 1B, left panel). In line 181

rPPCK1-3, which displayed the largest reduction in PPC phosphorylation in the dark period, 182

the apparent Ki of PPC for L-malate was 1 mM in both the light and the dark, confirming that 183

the lack of measurable PPC phosphorylation in the dark period in line rPPCK1-3 resulted in a 184

failure of the leaf mesophyll cells to convert the major, CAM-associated PPC isoform into the 185

phosphorylated, malate-insensitive form that occurs in wild type leaves in the dark period (Fig. 186

1B, left panel, and Supplemental Fig. 1). 187

188

Gas exchange characteristics in light/ dark cycles 189

In K. fedtschenkoi, CAM is induced developmentally along the growing shoot, with the 190

youngest leaves performing only daytime CO2 fixation via Rubisco, and older leaves 191

developing nocturnal CO2 fixation associated with increasing CAM (Jones, 1975; Borland et 192

al., 2009; Dever et al., 2015; Hartwell et al., 2016). Using a multi-cuvette, gas switching, infra-193

red gas analyzer (IRGA) system (described in detail by Dever et al., 2015), we investigated 194

the 24 h pattern of CO2 exchange for detached, full-CAM leaves (leaf pair 6; LP6) from 195

rPPCK1-1, rPPCK1-3 and the wild type that had been entrained in 12-h-light/ 12-h-dark 196

cycles (Fig. 2). Both the wild type and the transgenic lines with reduced KfPPCK1 fixed the 197

majority of their CO2 at night (phase I of CAM: stomata open, primary atmospheric CO2 198

fixation via PPC; Fig. 2A) (Osmond, 1978). Lines rPPCK1-1 and rPPCK1-3 fixed, 199

respectively, only 53% and 34% of wild type CO2 uptake in the dark (Fig. 2A). rPPCK1-1 and 200

rPPCK1-3 also fixed a small amount of CO2 at dawn (phase II; early hours of the light period, 201

stomata remain open briefly, CO2 fixation via PPC and Rubisco), and this phase II peak of 202

CO2 fixation was greater in rPPCK1-3 than in the wild type (Fig. 2A). The wild type reached a 203

maximum CO2 fixation rate in the dark period of 4.4 µmol m-2 s-1, whereas lines rPPCK1-1 204

and rPPCK1-3 reached a peak dark CO2 fixation rate of only 2.4 µmol m-2 s-1. It was 205

7

noteworthy that the rPPCK1-3 line that had the greatest reduction in dark phosphorylation of 206

PPC (Fig. 1B) reached its peak dark CO2 fixation rate 1 h earlier in the dark period than either 207

the wild type or rPPCK1-1 line (Fig. 2A). Furthermore, the CO2 fixation of line rPPCK1-3 208

began to decline after only 4.5 h of the dark period, to such an extent that, from 2 h before 209

dawn, this line was respiring an increasing amount of CO2 throughout the remainder of the 210

dark period (Fig. 2A). By contrast, the wild type and intermediate rPPCK1-1 line continued net 211

atmospheric CO2 fixation for the whole of the dark period (Fig. 2A). 212

Intact small plants (8-leaf-pair stage) with their root system in soil performed all 4 213

phases of CAM (labelled I – IV on Fig. 2B). rPPCK1-1 and rPPCK1-3 fixed respectively 72 % 214

and 62 % of wild type light period CO2 fixation. In the dark, rPPCK1-1 fixed only 12 %, and 215

rPPCK1-3 respired -76 %, of wild type CO2 fixation (Fig. 2B). Whole young plants of rPPCK1-216

3 respired CO2 throughout the dark period, but respiratory CO2 loss from the plants declined 217

from an initial peak straight after the lights went off to a minimum 4 h into the dark period, 218

before rising gently throughout the remainder of the dark (Fig. 2B). However, whole plants of 219

all three lines showed a clear phase III of CAM in the light, evidenced by the pronounced dip 220

in CO2 fixation in the middle of the light period (Fig. 2B). 221

222

Malate, starch and soluble sugar levels 223

Malate and starch levels reciprocate over the light/ dark cycle in starch accumulating 224

CAM species such as K. fedtschenkoi (Borland et al., 2016). Starch breakdown in the dark 225

generates the substrate for glycolysis, which in turn supplies PEP as a substrate for PPC to 226

use in dark CO2 fixation. As we had observed such large changes in the magnitude and 227

duration of dark CO2 fixation in the rPPCK1 lines (Fig. 2A and 2B), it was important to 228

measure the levels and daily light/ dark dynamics of the total leaf malate and starch pools. 229

Wild type, rPPCK1-1 and rPPCK1-3 all accumulated malate in the dark (Fig. 2C). Wild 230

type plants accumulated the most malate by dawn (75.76 µmol gFW-1 S.E. 4.65). Line 231

rPPCK1-1 accumulated 78 %, and rPPCK1-3 accumulated 64 %, of the wild type level of 232

malate by dawn (Fig. 2C). At the end of the light period, approximately 90 % of the dark 233

accumulated malate had been decarboxylated in the wild type and rPPCK1 lines. WT had a Δ 234

malate of 59.19 ± 8.82 µmol gFW-1, rPPCK1-1 had a Δ malate of 47.16 ± 5.23 µmol gFW-1 235

and PPCK1-3 had a Δ malate of 38.54 ± 4.89 µmol gFW-1. 236

At the end of the light period, wild type and rPPCK1 CAM leaves (LP6) had 237

synthesized similar amounts of starch (Fig. 2D). Wild type and rPPCK1-1 leaves broke down 238

8

94% of the accumulated starch during the dark period, whereas rPPCK1-3 only broke down 239

82% of its starch, consistent with the reduced CO2 fixation and malate accumulation achieved 240

by this line (Fig. 2A and 2C). Wild type had a Δ starch of 5.63 ± 0.47 mg gFW-1, PPCK1-1 had 241

a Δ starch of 5.37 ± 0.38 mg gFW-1 and PPCK1-3 had a Δ starch of 4.23 ± 0.67 mg gFW-1. 242

CAM leaves of the closely related obligate species Kalanchoë daigremontiana have 243

been reported previously to display a characteristic post-dawn peak of sucrose peaking at the 244

end of phase II of CAM (Wild et al., 2010). At the beginning of the light period, wild type K. 245

fedtschenkoi had a peak of sucrose phased to 2 h after dawn that was reduced to around half 246

the wild type level in line rPPCK1-3 (Fig. 2E). Fructose accumulation peaked slightly later 247

than the wild type in rPPCK1-3 at 2 h into the dark period (Fig. 2G), whereas the temporal 248

pattern of glucose accumulation was similar between rPPCK1-3 and the wild type (Fig. 2F). 249

250

Gas exchange characteristics under constant light and temperature free-running 251

conditions 252

In constant light, free-running circadian conditions (LL; constant light 100 µmol m-2 s-1, 253

and temperature, 15˚C), detached LP6 from wild type plants displayed the characteristic, 254

robust, and persistent CAM circadian rhythm, with oscillating peaks and troughs of CO2 255

fixation (Fig. 3) (Wilkins, 1992; Hartwell, 2006). rPPCK1-1 and rPPCK1-3 showed a 256

dampening of the CO2 fixation rhythm in constant conditions, with a dramatic reduction in 257

rhythm amplitude (Fig. 3A). The rPPCK1-1 CO2 fixation rhythm maintained clear oscillations 258

throughout the 6.5-day experiment, but the period of the rhythm was shorter than the wild 259

type such that, after 6.5-days, the wild type CO2 fixation trough or minimum coincided with the 260

rPPCK1-1 peak of CO2 fixation (Fig. 3A). The rPPCK1-3 CO2 fixation rhythm dampened 261

more rapidly than rPPCK1-1, so much so that it was virtually arrhythmic after 6.5 days. The 262

weak LL rhythm in line rPPCK1-3 also had an even shorter period than rPPCK1-1 (Fig. 3A). 263

When whole young plants at the 8-leaf-pair stage were measured in the gas exchange 264

system under LL free-running conditions, the CO2 exchange rhythms in the rPPCK1-1 and 265

rPPCK1-3 lines both collapsed to arrhythmia after two to three days (Fig. 3B). The amplitude 266

of the rPPCK1-1 and rPPCK1-3 CO2 fixation rhythms was even lower for whole plants than 267

for detached LP6 (cf. Fig. 3B with 3A). In particular, comparing the first peak and trough of its 268

CO2 fixation rhythm, rPPCK1-3 achieved a maximum amplitude of ~0.25 µmol m-2 s-1. This 269

contrasted strongly with the amplitude of the wild type rhythm for whole plants during the first 270

peak and trough when the amplitude reached over 1.5 µmol m-2 s-1 (Fig. 3B). 271

9

272

Phenotypic characterization under well-watered and drought-stressed conditions 273

CAM is widely recognized and characterized as a water-conserving adaptation to 274

seasonally dry environments. It was therefore important to investigate the physiological 275

responses and performance of the rPPCK1 lines under drought-stress conditions. CO2 276

exchange was measured for drought-stressed whole plants. Wild type and rPPCK1 plants 277

were grown from plantlets under identical conditions to the LP6 stage. Water was then 278

withheld for 25 days. Wild type drought-stressed plants displayed strong CAM in light/ dark 279

cycles, with a large nocturnal phase I period of dark CO2 fixation, and a net loss of CO2 in the 280

light period (Fig. 4). Phase II CO2 uptake at dawn was present in both wild type and RNAi 281

lines, but phase IV CO2 fixation at the end of the light period, which is characteristically 282

observed in well-watered wild type plants (Fig. 2B), was absent in both the RNAi lines and the 283

wild type. 284

As observed in well-watered conditions (Fig. 3B), the LL circadian rhythm of CO2 285

exchange persisted robustly in the wild type drought-stressed plants, but collapsed rapidly 286

towards arrhythmia in both rPPCK1 lines (Fig. 4A). In particular, after 2.5 days of LL, drought-287

stressed rPPCK1-3 moved into net respiratory loss of CO2 and this continued throughout the 288

remaining 4.5 days of the LL experimental run (Fig. 4A). This contrasted strongly with the LL 289

gas exchange for line rPPCK1-3 under well-watered conditions, where it maintained positive 290

net CO2 fixation throughout the LL experiment, both for detached LP6 and young whole plants 291

(Fig. 3A and 3B). 292

293

Growth performance under well-watered and drought-stressed conditions 294

The high WUE of CAM plants allows them to grow productively in arid and semi-arid 295

environments (Borland et al., 2009; Davis et al., 2017). Multiple clones of all lines were grown 296

from plantlets under identical conditions to the LP6 stage. Half of the plants were maintained 297

well-watered as controls, and half were subjected to drought-stress for 138-days. Both sets of 298

plants were maintained in the same growth cabinet such that the only environmental variable 299

was water supply. 300

In well-watered conditions, all plants grew well. However, weighing above-ground 301

shoots and below-ground roots, both as fresh weight and dry weight, revealed that the 302

average fresh and dry weight of the shoots and roots was significantly less than the wild type 303

for line rPPCK1-3 under well-watered conditions (Fig. 4B, 4C, 4D and 4E). The intermediate 304

10

line rPPCK1-1 also had a lower average fresh and dry weight for its shoots and roots under 305

well-watered conditions, although the reductions were not significantly different from the wild 306

type based on Student t-tests (Fig. 4B and 4D). The wet weight of the shoots of rPPCK1-1 307

weighed 91 % of the wild type, whereas the shoots of rPPCK1-3 weighed 55 % of the wild 308

type (n = 7). Differences in shoot dry weight were smaller, with rPPCK1-1 shoots weighing 96 309

% of wild type, and rPPCK1-3 weighing 76% of the wild type shoot dry weight (n = 7). Below-310

ground, root growth differences were less pronounced, although line rPPCK1-3 achieved an 311

average root wet weight that was only 71 % of the wild type (Fig. 4C). 312

The replicate plants subjected to 138-days of drought-stress had fresh weights for both 313

shoots and roots that were much lower than the fresh weights for the well-watered plants. For 314

example, the average fresh weight of wild type shoots from well-watered plants was 188 g, 315

whereas the average fresh weight for the drought-stressed wild type shoots was only 11 g. 316

This demonstrated that the 138-day drought-stress treatment was a severe drought-stress 317

regime, as might be experienced by K. fedtschenkoi during the dry season in its native 318

Madagascan environment. After drought-stress, the fresh and dry weights of both the shoots 319

and roots of the rPPCK1 lines did not differ significantly from the wild type values. The very 320

low dry weights of the drought-stressed shoots of all lines relative to the dry weights achieved 321

by all of the well-watered plants indicated that all lines performed very little shoot growth 322

under such a long-term drought-stress regime (Fig. 4D). However, the reduction in the dry 323

weight of drought-stressed roots relative to the well-watered values was less dramatic (Fig. 324

4E). Drought-stressed root dry weights were between 76 and 89 % of the corresponding well-325

watered values, whereas drought-stressed shoots weighed less than 22 - 26 % of the well-326

watered shoots for all lines. It is clear that all plants put more of their available fixed carbon 327

into below-ground root growth during the prolonged drought-stress treatment used in this 328

experiment. Furthermore, the root fresh weights were almost the same as the root dry weights 329

for the drought-stressed plants, suggesting that, by the end of the 138-day drought treatment, 330

the roots of all lines were almost completely dry, even before being placed in a drying oven. 331

332

Diurnal regulation of the transcript abundance of circadian clock genes 333

Having established that plants lacking KfPPCK1 had lost the rhythm of CO2 uptake in 334

free-running conditions (Fig. 3A and 3B), it was also important to investigate whether the 335

regulation of genes in the core molecular circadian clock was affected under both light/ dark 336

cycles (Fig. 5), and free-running LL conditions (Fig. 6). K. fedtschenkoi has two copies of the 337

11

circadian clock gene TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (KfTOC1-1 and 338

KfTOC1-2), and two CIRCADIAN CLOCK-ASSOCIATED1/ LATE ELONGATED 339

HYPOCOTYL-related genes (KfCCA1-1 and KfCCA1-2). Both KfTOC1 genes were up-340

regulated under light/ dark conditions in the rPPCK1-3 line, with the phasing of the KfTOC1-2 341

peak occurring 4 h earlier in rPPCK1-3 (Fig. 5A and 5B). In rPPCK1-3, both KfCCA1-1 and 342

KfCCA1-2 were slightly up-regulated at the time of their daily peak 2h after dawn, whereas 343

transcript levels of these genes in line rPPCK1-1 were similar to or slightly lower than those in 344

the wild type (Fig. 5C and 5D). A K. fedtschenkoi PSEUDO RESPONSE REGULATOR7 345

ortholog (KfPRR7) was markedly up-regulated at 2 h after dawn in the rPPCK1-3 line (Fig. 346

5E), and a KfPRR37 gene was also up-regulated and peaked 4 h earlier than the wild type at 347

10:00 (2 h before dusk) in line rPPCK1-3 (Fig. 5F). 348

349

Regulation of circadian clock genes under free-running conditions 350

The robust circadian rhythm of CAM CO2 exchange in LL conditions for the wild type 351

(Fig. 3A and 3B) has been shown to correlate tightly with circadian clock control of PPCK and 352

the phosphorylation state of PPC and its Ki for malate (Hartwell, 2005, 2006; Dever et al., 353

2015). Since CO2 exchange rhythms in LL conditions for rPPCK1-1 and rPPCK1-3 showed, at 354

best, low-amplitude rhythmicity in well-watered plants (Fig. 3A & 3B), it was important to also 355

investigate the circadian regulation of the transcript abundance of KfPPCK1 under LL 356

conditions. In wild type plants, KfPPCK1 transcript levels oscillated with a robust circadian 357

rhythm throughout 3 days under LL (Fig. 6A). The period of the rhythmic peaks and troughs 358

matched the well-documented, temperature-dependent, short-period rhythm of gas exchange 359

observed in K. fedtschenkoi (Anderson and Wilkins, 1989; Dever et al., 2015). Consistent with 360

the RNAi-mediated silencing of the target gene, KfPPCK1 transcript abundance was very low 361

in rPPCK1-3 in free running conditions and did not oscillate (Fig. 6A). The up-regulation of 362

KfPPCK2 and KfPPCK3 in plants lacking KfPPCK1 under LD conditions (Fig. 1C and 1D) led 363

us to investigate whether these genes remained rhythmic in constant conditions in line 364

rPPCK1-3. Both KfPPCK2 and KfPPCK3 transcript oscillations remained rhythmic and were 365

dramatically up-regulated at the start of the LL timecouse in rPPCK1-3 (Fig. 6B and 6C). 366

As these results revealed that certain circadian clock controlled output genes were 367

subject to robust oscillations under LL free-running conditions, it was also important to 368

investigate the rhythmicity of core circadian oscillator genes. The rhythmicity of both KfCCA1-369

1 and KfCCA1-2 was dampened in rPPCK1-3 (Fig. 6D and 6E). Interestingly, the two 370

12

KfTOC1 genes were differentially regulated in rPPCK1-3 under constant conditions with 371

KfTOC1-1 being up-regulated and rhythmic, whereas KfTOC1-2 was down-regulated and its 372

rhythm collapsed towards arrythmia (Fig. 6F and 6G). This was surprising as it indicated that 373

the two KfTOC1 genes may be involved in distinct circadian clocks having potentially 374

divergent roles and responding to different entrainment signals, possibly in different tissues/ 375

cell types of the CAM leaf. 376

KfPRR7, KfPRR37 and KfPRR9 all displayed dampening of their transcript rhythms 377

towards arrhythmia in rPPCK1-3 under constant conditions (Fig. 6H, 6I, and 6J). K. 378

fedtschenkoi FLAVIN-BINDING KELCH-REPEAT F-BOX PROTEIN (KfFKF1) and Kf 379

GIGANTEA (KfGI) were both up-regulated and rhythmic in LL (Fig. 6K and 6L), whereas Kf 380

JUMONJI C-DOMAIN CONTAINING PROTEIN30 (KfJMJ30), Kf NIGHT LIGHT-INDUCIBLE 381

AND CLOCK-REGULATED3 (KfLNK3-like) and Kf CYCLING DOF-TRANSCRIPTION 382

FACTOR2 (KfCDF2) were all down regulated but rhythmic in rPPCK1-3 (Fig. 6M, 6N, and 383

6O). KfGI was particularly noteworthy for the fact that the phasing of its transcript peaks and 384

troughs shifted late relative to the wild type in line rPPCK1-3 on the second and third 24 h 385

period of the LL experimental run (Fig. 6L), which contrasted strikingly with the earlier phase/ 386

shorter period of the LL CO2 fixation rhythm for rPPCK1-3 (Fig. 3A). 387

388

Delayed fluorescence rhythms revealed that a further, distinct circadian clock output 389

inside the chloroplast was perturbed in the absence of PPCK activity 390

The level of delayed fluorescence (DF) has previously been reported to be under 391

robust circadian control in K. fedtschenkoi and provides a simple high-throughput assay for 392

measuring circadian period, robustness and accuracy (Gould et al., 2009). DF was measured 393

in wild type, rPPCK1-1 and rPPCK1-3 under LL free running conditions (Fig. 7), and the data 394

were analysed to calculate key circadian rhythm statistics using Biodare (Moore et al., 2014; 395

Zielinski et al., 2014). The wild type had a robust DF oscillation (Fig. 7A). The wild type 396

relative amplitude error (RAE) plot (Fig. 7B), and summary bar chart for the computed period 397

length for the wild type (Fig. 7C, top panel), demonstrated a short period (mean 17.8 h) from 398

both fast Fourier transform (non-linear least squares method) (FFT-NLLS) and spectral 399

resampling (SR) analysis. The mean RAE plot revealed that the values were below the 0.6 400

cut-off (Fig. 7C, bottom panel), supporting statistically the visibly robust and high amplitude 401

wild type short period DF rhythm (Fig. 7A). Biodare analysis demonstrated that both rPPCK1-402

1 (Fig. 7D and 7E) and rPPCK1-3 (Fig. 7F and 7G) lost rhythm robustness through time, 403

13

particularly in the later stages of the LL free-running conditions. Line rPPCK1-1 had an 404

intermediate effect with dampening of the rhythm occurring over several cycles (Fig. 7D). The 405

period length plot for rPPCK1-1 suggested that its average free-running period was slightly 406

longer than that of the wild type, but the larger error suggested this period estimate was not 407

reliable (Fig. 7C, top panel). The corresponding RAE values showed a much higher average 408

for rPPCK1-1 than wild type due to its dampening, reflected in the wide spread of period 409

lengths from short to long (Fig. 7E). The analysis outputs for the DF results from rPPCK1-3 410

revealed a slightly more severe rhythm phenotype than rPPCK1-1 (Fig. 7F and 7G). Although 411

the Biodare graph summarising period length for DF rhythms in rPPCK1-1 and rPPCK1-3 412

indicated longer DF period length for the transgenic lines relative to the wild type (Fig. 7C, top 413

panel), which was at odds with the shorter period for the gas exchange LL rhythms (Fig. 3A 414

and 3 B), visual inspection of the DF rhythms revealed that there may have been a shorter 415

period length for the very low amplitude DF rhythms in rPPCK1-1 and rPPCK1-3 that were 416

observed throughout the latter stages of the LL time course (Fig. 7D and 7F). The RAE values 417

above the 0.6 cut-off for rPPCK1-1 and rPPCK1-3 also indicated that the rhythms were not 418

robust and thus that the period estimates in Fig. 7C should not be over-interpreted due to 419

their high associated RAE values. 420

421

DISCUSSION 422

Physiological consequences of loss of clock controlled PPC phosphorylation in an 423

obligate CAM plant 424

Previous work had shown that the PPCK gene responsible for light-period 425

phosphorylation and in vivo activation of photosynthetic PPC in the C4 species Flaveria 426

bidentis was not essential for high C4 photosynthetic rates in this species (Furumoto et al., 427

2007). This work on a C4 species surprised the wider community, because decades of 428

preceding work had argued that PPC phosphorylation by PPCK was vital for alleviating 429

malate/ aspartate inhibition of PPC in planta, and thus for optimizing photosynthetic CO2 430

fixation in C4 and CAM species (Vidal and Chollet, 1997; Nimmo, 2003). These findings have 431

become all the more surprising in the light of recent work on the evolution of the PPCK gene 432

family in C3 and C4 species of Flaveria (Aldous et al., 2014). A specific, light-induced, C4-433

recruited PPCKA gene was found in each C4 species of Flaveria, and was found to have 434

evolved rapidly since its recruitment to a C4-specific function. All Flaverias also possessed a 435

PPCKB gene that displayed peak transcript abundance in the dark period and that was down-436

14

regulated in the C4 species relative to the C3 species. Aldous et al. (2014) suggested that the 437

C4-recruited ppcA and PPCKA formed a co-evolving substrate-kinase pair, and yet the 438

findings of Furumoto et al. (2007) argued that PPCKA in F. bidentis was dispensable for 439

optimal CO2 fixation via the C4-pathway. Why would there be strong evolutionary selection 440

acting on the C4 ppcA/ PPCKA substrate-kinase pair if the kinase and its phosphorylation of 441

PPC were dispensable for optimal CO2 fixation via the C4 pathway? It was thus vital to 442

investigate the physiological role, if any, of PPCK and PPC phosphorylation in relation to dark 443

period CO2 fixation through to malate in a CAM plant. 444

Our findings revealed that, in the CAM species K. fedtschenkoi, phosphorylation of 445

PPC during the dark period by the circadian clock-controlled KfPPCK1 both maximized and 446

sustained primary atmospheric CO2 fixation via PPC. Overall, our data showed that dark 447

phosphorylation of PPC during CAM in K. fedtschenkoi is not a pre-requisite for dark CO2 448

fixation, as 34 % of dark CO2 fixation and 64 % of nocturnal malate accumulation happened 449

even in the absence of detectable levels of dark PPC phosphorylation in rPPCK1-3 (Fig. 2). 450

However, KfPPCK1 does play a critical role in fine-tuning the amount and duration of dark 451

CO2 fixation, leading to greater malate accumulation and growth in the wild type relative to 452

rPPCK1-3. 453

The high WUE of CAM plants is regarded as one of their key adaptive strengths and 454

the main trait of interest for future exploitation in CAM crops and CAM biodesign for climate-455

resilient agriculture (Borland et al., 2009; Borland et al., 2014). It was thus important to 456

compare the performance of the rPPCK1 lines between well-watered and drought-stressed 457

conditions. Gas exchange analysis of the rPPCK1 lines subjected to drought stress 458

demonstrated that both lines showed similar reductions in dark CO2 fixation relative to the 459

wild type in comparison to the differences observed under well-watered conditions (Fig. 4A). 460

However, the rPPCK1 lines did continue to fix atmospheric CO2 in the dark after drought 461

stress, which contrasted strikingly with our previous findings for transgenic K. fedtschenkoi 462

with reduced CAM and dark CO2 fixation due to reduced activity of NAD-ME or PPDK, both of 463

which failed to fix CO2 for the majority of the light/ dark cycle following drought-stress (Dever 464

et al., 2015). 465

Drought-stress had a dramatic impact on both the wet and dry weight of plants for both 466

the wild type and the rPPCK1 lines, but there was no significant difference between the fresh 467

or dry weight of both the shoots and roots of wild type and rPPCK1 lines following drought-468

stress (Fig. 4). Thus, under our growth conditions, the increased dark CO2 fixation during 469

15

drought stress achieved by the wild type relative to the rPPCK1 lines (Fig. 4A) did not lead to 470

a measurable increase in yield for the wild type. Instead, all lines achieved little if any net CO2 471

fixation after prolonged drought-stress, as their respiratory loss of CO2 in the light period 472

largely balanced their fixation of CO2 in the dark period. This was reflected in the very low dry 473

weight yields, which were greater than 3-fold lower for drought-stressed shoots relative to the 474

well-watered shoots (Fig. 4D). It would be interesting to monitor the gas exchange of the wild 475

type and rPPCK1 lines continuously throughout the drought-stress treatment, so that their gas 476

exchange during the transition from fully hydrated to severely drought-stressed could be 477

investigated in detail. This may reveal whether or not the wild type is able to maintain net daily 478

fixation of CO2 for longer into the drought-stress period. However, if this does occur, it was 479

clearly not sufficient to generate a measurable difference in growth performance after the 480

138-days of drought treatment (Fig. 4). It may be that all lines were locked-down into a CAM-481

idling survival mode after 138-days of drought-stress, and that their wet and dry weights were 482

indistinguishable because they had, by that time, respired away the small but significant 483

differences in their shoot and root dry biomass that were evident under well-watered 484

conditions. 485

486

Molecular and biochemical phenotypes of rPPCK1 lines 487

Both rPPCK1 lines possessed lower KfPPCK1 transcript levels than the wild type 488

throughout the dark period (Fig. 1A). rPPCK1-1 and rPPCK1-3 had similar KfPPCK1 489

transcript levels at both the beginning and end of the light period, but possessed a large 490

difference in transcript abundance in the middle of the dark period, when line rPPCK1-1 491

achieved a significantly higher transcript level than line rPPCK1-3 (18:00, 6 h into the 12 h 492

dark period, Fig. 1A). This was consistent with the higher PPC phosphorylation and apparent 493

Ki of PPC for L-malate detected in line rPPCK1-1 relative to line rPPCK1-3, specifically in the 494

middle of the dark period at 18:00 (Fig. 1B), and the greater dark period CO2 fixation, malate 495

accumulation and starch turnover achieved by this line (Fig. 2A, 2B, 2C, and 2D). Overall, our 496

biochemical measurements revealed a dosage dependent impact of the level of KfPPCK1 497

transcripts each night on the biochemical correlates of CAM. 498

499

Impact of silencing KfPPCK1 on circadian rhythms of CAM CO2 fixation and the 500

operation of the central circadian oscillator 501

16

Since the first discovery of PPCK activity and its circadian control in 1991 (Carter et al., 502

1991), through the subsequent cloning and characterization of the gene encoding this 503

remarkable protein kinase in 1999 (Hartwell et al., 1999), there has been a long-held 504

assumption that this circadian clock controlled kinase is crucial for the temporal coordination 505

and optimization of nocturnal atmospheric CO2 fixation in CAM species, and the associated 506

persistent circadian rhythm of CO2 exchange observed under constant conditions in CAM 507

species such as K. fedtschenkoi and K. daigremontiana (Wilkins, 1992; Nimmo, 2003; Wyka 508

et al., 2004; Hartwell, 2006). However, it has not been possible previously to perturb 509

genetically the level of PPCK activity and dark period PPC phosphorylation in a CAM species, 510

and thus it has not been possible to test the validity of this long held assumption about the 511

pivotal role of PPCK in the circadian control of CAM. 512

By silencing the CAM-associated KfPPCK1 gene with an RNAi construct, we have 513

been able to test in planta the importance of KfPPCK1 for the circadian rhythm of CO2 514

exchange. The loss of dark period PPC phosphorylation in line rPPCK1-3 led to a rapid 515

decline in the circadian rhythm of CO2 exchange when leaves or whole plants pre-entrained 516

under 12-h-light/ 12-h-dark cycles were released into LL free-running conditions (Fig. 3A and 517

3B, Fig. 4A). After 25-days of drought-stress, the weak CO2 exchange rhythms displayed by 518

the well-watered rPPCK1 lines (Fig. 3B) collapsed to arrhythmia more rapidly (Fig. 4A). In 519

particular, line rPPCK1-3 became arrhythmic and continued a steady rate of respiratory loss 520

of CO2 for the majority of the LL time course (Fig. 4A), whereas the drought-stressed wild 521

type continued with a persistent circadian rhythm of CO2 fixation throughout the LL 522

experiment (Fig. 4A). Furthermore, silencing KfPPCK1 also led to DF rhythms collapsing 523

towards arrhythmia, revealing that a distinct circadian clock output that originates inside the 524

chloroplast was also perturbed in the rPPCK1 lines (Fig. 7). These results revealed that wild 525

type levels of circadian clock controlled PPCK are essential for the amplitude, robustness and 526

persistence of the CO2 fixation rhythm associated with CAM and the DF rhythms in the 527

chloroplast in K. fedtschenkoi. 528

In light of the weakened rhythmicity of CO2 exchange in the rPPCK1 lines, it was 529

important to investigate the robustness of transcript abundance oscillations for components of 530

the central molecular oscillator that underpin plant circadian rhythms (Fig. 5 and Fig. 6). Wild 531

type K. fedtschenkoi displayed robust oscillations in the transcript abundance of KfCCA1-1, 532

KfCCA1-2, KfTOC1-1 and KfTOC1-2, but rhythms in the transcript abundance of KfCCA1-1, 533

KfCCA1-2 and KfTOC1-2 collapsed rapidly towards arrhythmia under LL in the rPPCK1-3 line 534

17

(Fig. 6D, 6E and 6G). This revealed that multiple components of the core molecular oscillator 535

failed to maintain wild type levels of robust rhythmicity in the absence of KfPPCK1. In 536

contrast, KfTOC1-1, KfPRR7, KfFKF1 and KfGI were induced and displayed a rhythm with a 537

greater amplitude than the wild type under LL in line rPPCK1-3 (Fig. 6F, 6H, 6K and 6L), 538

revealing that certain sub-components of the core molecular circadian oscillator continued to 539

function with a robust circadian rhythm in the absence of KfPPCK1 activity. These are 540

paradigm-changing findings because KfPPCK1 has previously been regarded as a circadian 541

clock controlled output pathway component that is vital for coupling the central clock to CAM. 542

However, our current findings reveal that some components of the central clock itself stop 543

working correctly in the absence of KfPPCK1. This suggests either that KfPPCK1 is itself part 544

of the central oscillator in K. fedtschenkoi, although this seems highly unlikely considering the 545

known function of PPCK1 in PPC phosphorylation, or, more likely, that the failure to 546

phosphorylate PPC in the dark leads to metabolic changes that influence the operation of the 547

core oscillator, perhaps via a mechanism not dissimilar to that proposed for sugars acting as 548

signals from photosynthesis that can entrain the central clock in Arabidopsis (Haydon et al., 549

2013). 550

It will be important to investigate the wider metabolic and/ or gene expression changes 551

associated with the loss of PPCK and PPC phosphorylation in these lines. Such 552

investigations should provide valuable insights into the types of signals that could link CAM-553

associated metabolites to the central clock during the daily operation of the CAM pathway. It 554

is particularly noteworthy in this respect that we also saw a loss of central clock gene TOC1 555

transcript oscillations in K. fedtschenkoi transgenic RNAi lines with reduced activity of NAD-556

ME (Dever et al., 2015). Those lines turned over only a small proportion of their malate each 557

day, and had a much greater reduction in starch accumulation and turnover than the rPPCK1 558

lines reported here. This suggests that the metabolic and/ or gene regulatory changes that led 559

to dampening of oscillations for a subset of central clock genes in these two distinct sets of K. 560

fedtschenkoi transgenic lines may be more complex than a direct impact of malate or 561

carbohydrate levels on clock operation. However, when we examined sucrose, fructose and 562

glucose levels over the 12-h-light/ 12-h-dark cycle in the rPPCK1 lines reported here, we 563

discovered that line rPPCK1-3 displayed an almost 50% reduction in the 2 h post dawn-564

phased peak of sucrose detected in the wild type (Fig. 2E). 565

Taking into consideration the fact that sucrose has been demonstrated as a central 566

clock-entraining metabolite in the C3 species Arabidopsis (Haydon et al., 2013), our findings 567

18

suggest that sucrose is a strong candidate for a metabolite linking the perturbations in CAM 568

and core clock rhythms reported here for the rPPCK1 lines (Fig. 2E). It is particularly 569

noteworthy in this context that KfPRR7 and KfPRR37 transcript levels were increased in 570

rPPCK1-3 at 2 h and 10 h after dawn, respectively, relative to the wild type (Fig. 5E and 5F). 571

For KfPRR7, this change in transcript level was coincident with the decrease in the post-dawn 572

sucrose peak (Fig. 2E). Haydon et al. (2013) proposed that PRR7 could be a key clock gene 573

functioning in the entrainment of the central oscillator by sucrose signals. It is however 574

noteworthy that our KfPRR7 ortholog displayed a transcript peak phased to 2 h after dawn, 575

coincident with the daily sucrose peak, whereas the Arabidopsis PRR7 transcript level peaked 576

at 8 h into the 12 h light period and was also strongly correlated with the daily sucrose peak, 577

which occurs in the latter part of the light period in Arabidopsis (Haydon et al., 2013). 578

Transcript levels of our KfPRR37 transcript did peak at an equivalent time (10:00, 2 h prior to 579

dusk) to PRR7 in Arabidopsis, and reached its daily peak in rPPCK1-3 some 4 h earlier than 580

the wild type, which peaked at 14:00, 2 h into the dark (Fig. 5F). These striking similarities in 581

the timing and strong correlation of peak sucrose and peak PRR7 transcript levels in both K. 582

fedtschenkoi and Arabidopsis provide support for the proposal that sucrose is a key clock 583

entraining signal in K. fedtschenkoi and that the decrease in the 2 h post-dawn sucrose peak 584

in rPPCK1-3 (Fig. 2E) feeds back to perturb core clock gene regulation (Fig. 6), potentially via 585

KfPRR7 and KfPRR37 (Fig. 5E and 5F). 586

Our data support the proposal that, in the CAM leaf, the circadian clock is set by both 587

light and sucrose. Sucrose has been demonstrated to be a product of CAM-derived starch 588

degradation during the hours at the end of the phase I dark period, although sucrose was also 589

shown to accumulate further during phase II after dawn as a direct result of Rubisco-mediated 590

fixation of atmospheric CO2, as is classically observed in the late afternoon in C3 plants such 591

as Arabidopsis (Wild et al., 2010; Haydon et al., 2013). Both of the KfCCA1 genes and one of 592

the two KfTOC1 genes were down-regulated and displayed dampening rhythmicity in the 593

rPPCK1-3 plants under LL conditions (Fig. 6), and this may be due to the rPPCK1-3 plants 594

being in a state of metabolic starvation after dawn. In C3 leaves of Arabidopsis during 595

starvation, PRR7 represses CCA1 and derepresses the TOC1/GI loop (Dalchau et al., 2011; 596

Haydon et al., 2013; Haydon et al., 2015). The rPPCK1-3 transgenic lines of K. fedtschenkoi 597

displayed de-repression and rhythmicity for KfPRR7, KfTOC1-1, KfGI, and KfFKF1, 598

supporting the proposal that the reduction in the post-dawn sucrose peak and associated 599

increase in KfPRR7 and KfPRR37 may lead to the de-repression of a TOC1-1/ GI loop in K. 600

19

fedtschenkoi. However, in K. fedtschenkoi, whilst KfTOC1-1 was de-repressed, which is 601

similar to the TOC1 regulation reported in Arabidopsis, KfTOC1-2 was repressed, suggesting 602

that in CAM leaves of Kalanchoë there are distinct sub-oscillators of the central circadian 603

clock involving KfTOC1-1 and KfTOC1-2. We cannot at this stage rule out the possibility that 604

these striking differences in the regulation of core circadian clock genes in line rPPCK1-3 605

were a result of differential regulation of the two TOC1 genes occurring in different cell types 606

of the leaf. 607

We have previously demonstrated the utility of K. fedtschenkoi as a model system for 608

elucidating the in planta functioning of candidate core metabolic enzymes associated with 609

CAM (Dever et al., 2015; Hartwell et al., 2016). Our findings here provide in planta 610

confirmation of the importance of the regulatory protein kinase PPCK and its circadian rhythm 611

for optimal and maximal dark CO2 fixation. Plants lacking KfPPCK1 also failed to maintain 612

robust rhythmicity of the transcript oscillations of several core clock components, including 613

both isogenes of KfCCA1 and one isogene of KfTOC1. A key criterion for a gene to be 614

classified as a core clock component is that mis-regulation of the gene should cause 615

arrhythmia within the oscillator itself. Our data fulfill this criterion to some extent for KfPPCK1, 616

and thus our data could be interpreted as evidence that KfPPCK1 operates as a core 617

component of the central circadian clock in K. fedtschenkoi. However, we do not favor that 618

explanation. Instead, our data for daily sucrose oscillations and KfPRR7, KfPRR37, KfTOC1-1 619

and KfGI transcript regulation support the proposal that the pathway of metabolic regulation 620

suggested to integrate photosynthetic sugar metabolism with the clock in Arabidopsis plays a 621

role in the partial loss of rhythmicity observed in this K. fedtschenkoi rPPCK1-3 line (Haydon 622

et al., 2013). Alternatively, malate changes in the rPPCK1-3 line may influence the clock, as 623

malate has been demonstrated to function to influence gene regulation (Finkemeier et al., 624

2013). 625

This work extends the categories of CAM-associated genes for which valuable 626

functional insights have been gained using transgenic K. fedtschenkoi, moving beyond core 627

CAM enzymes to include the circadian clock-controlled post-translational regulatory step 628

catalyzed by PPCK1. Based on these findings, we predict that transgenic approaches applied 629

in K. fedtschenkoi will also prove useful for demonstrating the functional role of other 630

proposed CAM regulators, such as PPDK-regulatory protein (Dever et al., 2015), or pyruvate 631

dehydrogenase kinase (Thelen et al., 2000), or as yet unknown transcription factors and other 632

regulatory proteins that likely function in the circadian clock output pathway that couples the 633

20

core molecular oscillator to the regulation of CAM enzymes via proteins such as PPCK 634

(Hartwell, 2006; Borland et al., 2009; Hartwell et al., 2016). 635

636

Relevance of these findings to efforts to engineer CAM into C3 species 637

Our findings are vitally important to current and ongoing efforts to engineer the CAM 638

pathway into C3 crops using plant synthetic biology approaches because they demonstrate 639

that expressing high levels of PPC alone in leaf mesophyll cells will not be sufficient to 640

achieve optimized operation of CAM dark CO2 fixation. Expressing elevated levels of PPC 641

may yield plants that can fix some atmospheric CO2 in the dark and accumulate a certain 642

level of malate by dawn, which would be a significant advance in and of itself. However, our 643

data for the KfPPCK1 RNAi lines demonstrate that in order for an engineered CAM system to 644

work with the full efficiency demonstrated by existing CAM species, C3 crops will need to be 645

engineered to express both high levels of PPC in leaf mesophyll cells, and a circadian clock-646

controlled PPCK that peaks in the dark period and achieves a 10-fold increase in the Ki of the 647

PPC for malate. 648

We propose that the PPCK and PPC used for this forward engineering work should 649

ideally be a pre-evolved substrate-kinase pair, as has been suggested to have co-evolved in 650

the C4 Flaveria system (Aldous et al., 2014). For example, KfPPC1 (GenBank KM078709) 651

and KfPPCK1 (GenBank AF162662) from K. fedtschenkoi would be an ideal substrate-kinase 652

pair for the forward engineering of CAM into C3 species, although care would need to be 653

taken with promoter choice for the transgenes in order to ensure that the KfPPC1 gene was 654

expressed to very high levels in the leaf mesophyll cells, and KfPPCK1 was expressed under 655

circadian clock control to peak in the middle of the dark in leaf mesophyll cells. 656

657

METHODS 658

659

Plant materials 660

Unless otherwise stated, Kalanchoë fedtschenkoi Hamet et Perrier plants were propagated 661

clonally from stem cuttings or leaf margin adventitious plantlets using the same clonal stock 662

originally obtained from the Royal Botanic Gardens, Kew by Prof. Malcolm Wilkins (Wilkins, 663

1958). Plants were grown and entrained to light/ dark cycles as described in Dever et al. 664

(2015). 665

666

21

Time course experiments 667

For light/dark time course experiments, opposite pairs of LP6 (where leaf pair 1/ LP1 are the 668

youngest leaf pair flanking the shoot apical meristem and leaf pairs are counted down the 669

stem) were collected every 4 h over a 12-h-light (450 µmoles m-2 s-1, 25˚C, 60 % humidity)/ 12-670

h-dark (15˚C, 70 % humidity) cycle, starting 2 h (02:00) after the lights came on at 00:00 h 671

using plants raised and entrained as described by Dever et al. (2015). For constant light, 672

constant temperature, constant humidity (LL) free-running circadian time course experiments, 673

plants were entrained in 12-h-light/ 12-h-dark conditions for 7 days and switched to LL after a 674

dark period as described by Dever et al. (2015). For LL, the constant conditions were: light 100 675

µmol m-2 s-1, temperature 15˚C and humidity 70 %. LP6 were sampled every 4 h from three 676

individual (clonal) plants, starting at 02:00 (2 h after lights on). All sampling involved immediate 677

freezing of leaves in liquid nitrogen. Samples were stored at -80˚C until use. 678

679

Generation of transgenic K. fedtschenkoi lines 680

An intron containing hairpin RNAi construct was designed to target the silencing of the CAM-681

induced and clock-controlled KfPPCK1 gene (GenBank accession: AF162661) (Hartwell et 682

al., 1999). A 359 bp fragment was amplified from CAM leaf cDNA using high fidelity PCR with 683

KOD Hot Start DNA Polymerase (Merck, Germany) and the primers KfPPCK1 RNAi F 5’-684

CACCGCGCAGGACATATTTGAGG-3’ and KfPPCK1 RNAi R 5’-685

GGCCGAATTCCCTCACTCTGCTTC-3’. The amplified fragment spanned the 3' end of the 686

coding sequence and extended into the 3' untranslated region to ensure specificity of the 687

silencing to a single member of the K. fedtschenkoi PPCK gene family. Alignment of the 359 688

bp region with the homologous region from the three other PPCK genes in the K. 689

fedtschenkoi genome demonstrated that none of the other PPCK genes shared any 21 690

nucleotide stretches that were an exact match for KfPPCK1. PCR products were cloned into 691

the pENTR/D Gateway-compatible entry vector via directional TOPO cloning (Life 692

Technologies, UK), and recombined into the intron containing hairpin RNAi binary vector 693

pK7GWIWG2(II) (Karimi et al., 2002) using LR Clonase II enzyme mix (Life Technologies, 694

UK). Constructs were confirmed by DNA sequencing and introduced into Agrobacterium 695

tumefaciens strain GV3101 using the freeze-thaw method (Hofgen and Willmitzer, 1988). 696

Agrobacterium-mediated stable transformation of K. fedtschenkoi was achieved using the 697

tissue culture based method described by Dever et al. (2015). 698

22

699

High-throughput leaf acidity and starch content screens 700

Leaf acidity (as a proxy for leaf malate content) and leaf starch content were screened with 701

leaf disc stains using chlorophenol red and iodine solution, respectively, at both dawn and 702

dusk, as described by Cushman et al. (2008). For each transgenic line, leaf discs were 703

sampled in triplicate at 1 hour before dawn and 1 hour before dusk and stained in a 96 well 704

plate format. 705

706

Net CO2 exchange 707

Gas exchange measurements were performed using a 6-channel, custom-built IRGA system 708

(PP Systems, Hitchin, UK), which allowed the individual environmental control (CO2/H2O) and 709

measurement of rates of CO2 uptake for each of 6 gas exchange cuvettes with measurements 710

collected every 10 min (described in full in Dever et al., 2015). All experiments were repeated 711

at least 3 times using 3 separate individual young plants (8 leaf pairs), or detached LP6 from 712

three separate clonal plants, and representative gas exchange traces are shown. Wild type and 713

rPPCK1 lines were compared in neighboring gas exchange cuvettes during each run, such that 714

the data are directly comparable between each line. For example, many runs included two 715

cuvettes containing wild type, two cuvettes containing rPPCK1-1 and two with rPPCK1-3. 716

717

Drought-stress experiments 718

For the drought-treatment gas exchange experiments, clonal leaf margin plantlets from wild 719

type and the rPPCK1 lines were grown to the 8-leaf-pair stage using 150 mL of our standard 720

compost/ perlite mix (see Dever et al., 2015), and maintained well-watered throughout their 721

development. Water was withheld for 25 days under our standard 12:12 LD growth conditions 722

according to Dever et al. (2015). 723

724

Leaf malate, starch and sucrose content 725

Leaf pair 6 (LP6; full CAM in wild type) from mature plants were sampled into liquid nitrogen at 726

the indicated times, and stored at -80˚C until use. The frozen leaf samples were prepared and 727

assayed for malate and starch as described by Dever et al. (2015) using the standard enzyme-728

linked spectrophotometric methods for assaying malate (Möllering, 1985) and starch (Smith 729

and Zeeman, 2006). 730

23

The concentrations of sucrose, D-fructose and D-glucose were determined using enzyme-731

linked spectrophotometric assays according to the manufacturer’s instructions (Sucrose/ D-732

Fructose/ D-Glucose Assay Kit K-SUFRG, Megazyme International, Ireland). This involved 733

the step-wise enzymatic conversion of each sugar to glucose-6-phosphate (G6P), and 734

subsequent quantification by measuring NADPH production at 340 nm by following oxidation 735

of the G6P in the presence of NADP+ and G6P-dehydrogenase. 736

737

Total RNA isolation and RT-qPCR 738

Total RNA was isolated from 100 mg of frozen, ground leaf tissue using the Qiagen RNeasy 739

kit (Qiagen, Germany) following the manufacturer’s protocol with the addition of 13.5 µL 50 740

mg mL-1 PEG 20000 to the 450 µL RLC buffer used for each extraction. cDNA was 741

synthesized from the total RNA using the Qiagen Quantitect RT kit according to the 742

manufacturer’s instructions (Qiagen, Germany). The resulting cDNA was diluted 1:4 with 743

molecular biology grade water prior to use in RT-qPCR. Transcript levels were determined 744

using 3 technical replicates and 3 biological replicates with SensiFAST SYBR No Rox kit 745

(Bioline, UK) in an Agilent MX3005P qPCR System Cycler using the following program: 95˚C 746

for 2 min, 40 cycles of 95˚C for 5 s, 60˚C for 10 s and 72˚C for 10 s, and primers designed 747

with Primer3. Primer specificity was checked using BLAST searches, and primer efficiency 748

was checked using a melting curve profile standard curve. In every PCR plate an inter-plate 749

calibrator (made from a pool of RNAs assembled from K. fedtschenkoi wild type leaf pair 6 750

samples taken every 4 hours over a light/ dark cycle) was run in triplicate in order to correct 751

any inter-plate variation. In addition, non-template controls were included in every plate to 752

confirm the absence of contamination. Each biological replicate was assayed in triplicate, and 753

the relative abundance of each gene was determined using Agilent MxPRO QPCR software 754

using the manufacturer’s instructions (Agilent Technologies). The results were normalized to 755

a K. fedtschenkoi orthologue of a Thioesterase/thiol ester dehydrase-isomerase (TEDI) 756

superfamily protein (Phytozome Kaladp0068s0118.1; Arabidopsis ortholog AT2G30720.1). 757

Primers for RT-qPCR analyses are listed in Supplemental Table I. 758

759

Immunoblotting 760

Total protein extracts of K. fedtschenkoi leaves were prepared according to Dever et al. (2015). 761

One-dimensional SDS-PAGE and immunoblotting of leaf proteins was carried out following 762

standard methods. Blots were developed using the ECL system (GE Healthcare, UK). 763

24

Immunoblot analysis was carried out using antisera to PPC (raised against PPC from K. 764

fedtschenkoi, kindly supplied by Prof. H.G. Nimmo, University of Glasgow) (Nimmo et al., 765

1986), and the phosphorylated form of PPC (raised against a phospho-PPC peptide from 766

barley, and kindly supplied by Prof. Cristina Echevarría, Universidad de Sevilla, Spain) 767

(González et al., 2002; Feria et al., 2008). 768

769

Determination of the apparent Ki of PPC for L-malate in rapidly desalted leaf extracts 770

Rapidly desalted leaf extracts were prepared as described by Carter et al. (1991) using LP6 771

sampled from three biological replicates of wild type, rPPCK1-1 and rPPCK1-3 at 10:00 (2 h 772

before the end of the 12 h light period) and 18:00 (middle of the 12 h dark period). The 773

apparent Ki of PPC activity for feedback inhibition by L-malate was determined for each rapidly 774

desalted extract according to the method described by Nimmo et al. (1984), except that the 775

range of L-malate concentrations added to the different PPC assays was 0, 0.2, 0.5, 0.7, 1.0, 776

2.0, 3.0, 5.0, 8.0 and 10.0 mM. The concentration of malate needed to achieve 50 % inhibition 777

of the initial PPC activity (apparent Ki) was determined using the plots shown in Supplemental 778

Fig. S1. 779

780

Dry weight growth measurements 781

Mature plants were grown from developmentally synchronized clonal leaf plantlets in 782

greenhouse conditions for 138 days, harvested as separated above-ground and below-783

ground tissues, and dried in an oven at 60˚C until they reached a constant weight. Weights 784

were measured to 2 decimal places in grams using a fine balance. Seven individual clonal 785

plants were grown for each line (Dever et al, 2015). 786

787Delayed fluorescence (DF) measurements 788

The imaging system for DF was identical to the luciferase and delayed fluorescence imaging 789

system described previously (Gould et al., 2009). DF was quantified using Imaris (Bitplane) to 790

measure mean intensity for specific regions within an image. Background intensities were 791

calculated for each image and subtracted, to give a final DF measurement (Gould et al., 792

2009). 793

794

DF rhythm analysis 795

K. fedtschenkoi plants were grown in greenhouse conditions for 4 months and then entrained 796

25

in 12-h light / 25 ˚C : 12-h dark/ 15 ˚C cycles in a Snijders Microclima MC-1000 (Snijders 797

Scientific) growth cabinet for 7 days as described previously (Dever et al., 2015). At dawn on 798

the 8th day, 1.5 cm leaf discs were punched from each of leaf pair 6 for three biological 799

replicates (i.e. two leaf discs from each biological replicate, totalling 6 leaf discs per line) and 800

placed with a few drops of distilled water in one of twenty-five 2 x 2 cm wells in a 10 x 10 cm 801

square petri dish. The drops of distilled water were sufficient to keep the leaf discs hydrated, 802

but were not enough for the discs to float and thus move, which would make the DF image 803

analysis extremely challenging. The petri dish was placed in the imaging system at 22˚C in 804

constant red-blue light (LL). DF images were collected every hour for 108 h as described 805

previously (Gould et al., 2009). The DF images were processed as described by Gould et al. 806

(2009). The luminescence was normalized by subtracting the Y value of the best straight line 807

from the raw Y value. Biodare was used to carry out fast Fourier transform (non-linear least-808

square) analysis and spectral resampling on each DF time course series using the time 809

window from 24 to 108 h in order to generate period estimates and calculate the associated 810

relative amplitude error (RAE) (Moore et al., 2014; Zielinski et al., 2014). The first 24 h of the 811

data were excluded as this represents a bedding-in period for the leaves when transition 812

effects, resulting from the leaves moving from light/ dark cycles to constant light and 813

temperature (LL), may be influencing the data. 814

815

Accession numbers 816

Sequence data associated with this article have been deposited in the GenBank/ EMBL data 817

libraries under the following accession numbers: KfPPCK1, AF162662; KfTOC1-1, 818

KM078716; KfCCA1-1, KM078717; KfCCA1-2, KM078718; KfPPCK2, KM078720; KfGI, 819

KM078724; KfTOC1-2, KM078726. Other accession numbers and gene IDs are presented in 820

Supplemental Table 1. 821

822

Supplemental Data 823

Supplemental Figure 1. Impact of silencing KfPPCK1 on the apparent Ki of PPC for L-malate 824in rapidly desalted leaf extracts 825Supplemental Table 1. Primers used for reverse transcription-quantitative PCR 826

827

ACKNOWLEDGEMENTS 828

26

We thank Prof. Hugh Nimmo (University of Glasgow, UK) and Prof. Cristina Echevarria 829

(Universidad de Sevilla, Spain) for providing the antibodies used in this study. This work was 830

supported in part by the Biotechnology and Biological Sciences Research Council, UK 831

(BBSRC grant no. BB/F009313/1 awarded to J.H.), and in part by US Department of Energy 832

(DOE) Office of Science, Genomic Science Program under Award Number DE-SC0008834. 833

The contents of this article are solely the responsibility of the authors and do not necessarily 834

represent the official views of the DOE. 835

836

AUTHOR CONTRIBUTIONS 837

JH, SFB, LVD and PDG designed the research. SFB performed all experiments except the 838

stable transformations, immunoblots, starch determinations, growth yield drought experiment, 839

and delayed fluorescence measurements. LVD performed the immunoblots, starch 840

determinations and growth yield drought experiment. JK generated the binary constructs and 841

carried out the stable transformation, regeneration and initial screening of the K. fedtschenkoi 842

rPPCK1 transgenic lines, and also collaborated with LVD on the growth yield drought 843

experiment. PDG carried out the delayed fluorescence experiments in collaboration with SFB. 844

SFB, LVD, PDG and JH analyzed the data. SFB and JH wrote the manuscript. 845

846

FIGURE LEGENDS 847

Figure 1. Confirmation of target gene silencing in transgenic K. fedtschenkoi RNAi 848

lines rPPCK1-1 and rPPCK1-3. Down-regulation of the target endogenous KfPPCK1 gene 849

was confirmed at the level of KfPPCK1 transcript abundance (A), and target protein PPC 850

phosphorylation and the apparent Ki of PPC for L-malate in rapidly desalted leaf extracts; 851

measured as L-malate (mM) required for a 50% reduction in extractable PPC activity (B). 852

Gene transcript abundance was measured using RT-qPCR for wild type, rPPCK1-1 and 853

rPPCK1-3 for target genes: A, KfPPCK1; C, KfPPCK2; and D, KfPPCK3. Mature leaves (leaf 854

pair 6) were sampled from three individual, clonal, biological replicate plants collected every 4 855

h across the 12-h-light/ 12-h-dark cycle. A thioesterase/thiol ester dehydrase-isomerase 856

superfamily gene (KfTEDI) was amplified from the same cDNAs as a reference gene. Gene 857

transcript abundance data represents the mean of 3 technical replicates of each of three 858

biological replicates, and was normalized to reference gene (KfTEDI); error bars represent the 859

standard error of the mean calculated for each biological replicate. In all cases, plants were 860

27

entrained under 12-h-light/ 12-h-dark cycles for 7 days prior to sampling. B, Protein 861

abundance (right panel) and the phosphorylation state of PPC (left panel) was determined by 862

immunoblot analyses, and the apparent Ki of PPC for L-malate was measured using rapidly 863

desalted leaf extracts (Ki values in mM below the corresponding left panel immunoblot time 864

points). Total leaf protein (leaf pair 6) was isolated from leaves sampled every 4 h across the 865

12-h-light/ 12-h-dark cycle, separated using SDS-PAGE and used for immunoblot analyses 866

with antibodies raised to a phospho-PPC peptide (left panel). Sample loading was normalized 867

according to total protein and confirmed using the immunoblot for total PPC protein, which is 868

stable over the 12-h-light/ 12-h-dark cycle (right panel). The white bar below each panel 869

represents the 12-h-light period and the black bar below each panel represents the 12-h-dark 870

period. For A, C and D: black data are for the wild type, blue rPPCK1-1 and red rPPCK1-3. 871

872

Figure 2. Impact of silencing KfPPCK1 on 24 h light/ dark gas exchange profiles, and 873

malate, starch and soluble sugar levels for wild type, rPPCK1-1 and rPPCK1-3 under 874

well-watered conditions. A, Gas exchange profile for CAM leaves (leaf pair 6) using plants 875

pre-entrained for 7 days under 12-h-light/ 12-h-dark cycles. B, Gas exchange profile for well-876

watered whole young plants (8-leaf-pairs stage) using plants entrained for 7 days under 12-h-877

light/ 12-h-dark cycles. C, Malate content was determined from leaf pair 6 samples collected 878

every 4 h using plants entrained under 12-h-light/ 12-h-dark cycles using methanol extracts of 879

leaves from wild type, rPPCK1-1 and rPPCK1-3. D, Starch content was determined from leaf 880

pair 6 samples collected at 1 h before dawn and 1 h before dusk. E, F, G, Soluble sugars 881

levels were determined separately for sucrose, glucose and fructose using three biological 882

replicates of leaf pair 6 sampled every 4 h using plants entrained under 12-h-light/ 12-h-dark 883

cycles. In C, D, E, F and G, the error bars represent the standard error of the mean calculated 884

for the three biological replicates measured at each time point. Black data traces represent 885

wild type, blue data rPPCK1-1 and red data rPPCK1-3. 886

887

Figure 3. Effects of silencing KfPPCK1 on CAM CO2 exchange rhythms measured 888

under constant light and temperature (LL) conditions. A, Gas exchange profile for CAM 889

leaves (leaf pair 6) was measured using leaves entrained under a 12-h-light/ 12-h-dark cycle 890

followed by release into constant LL conditions (100 µmol m-2 s-1 light at 15 ˚C). B, Gas 891

exchange profile for well-watered whole young plants (8-leaf-pairs stage) using plants 892

entrained under 12-h-light/ 12-h-dark cycles followed by release into constant LL conditions 893

28

(100 µmol m-2 s-1 light at 15 ˚C). Black data traces represent wild type, blue data rPPCK1-1 894

and red data rPPCK1-3. 895

896

Figure 4. Impact of loss of KfPPCK1 activity on gas exchange profiles (A) and 897

vegetative yield (B) for plants subjected to drought-stress. A, Drought-stressed whole 898

plants (8-leaf-pair stage) were entrained under 12-h-light/ 12-h-dark cycles followed by 899

release into constant LL conditions (100 µmol m-2s-1 light at 15 ˚C). The black data trace 900

represents wild type, blue rPPCK1-1 and red rPPCK1-3. B, C, D and E, Fresh (B, C) and dry 901

(D, E) weight of above-ground biomass (shoot; B, D) and below-ground tissues (roots; C, E) 902

at maturity (138 d under greenhouse conditions) for wild type (WT), rPPCK1-1 and rPPCK1-3 903

under well-watered and drought-stressed conditions. n = 7 plants; error bars represent 904

standard error of the mean. * or *** = significantly different to wild type based on Student’s t-905

test, *P < 0.05 and ***P < 0.001. 906

907

Figure 5. Impact of the loss of KfPPCK1 activity on the light/ dark regulation of the 908

transcript abundance of central circadian clock genes KfTOC1-1, KfTOC1-2, KfCCA1-1, 909

KfCCA1-2, KfPRR7 and KfPRR37. Mature leaves (leaf pair 6) were sampled from three 910

biological replicates every 4 h across the 12-h-light/ 12-h-dark cycle, RNA isolated and used 911

for real-time RT-qPCR. A, TOC1-1. B, TOC1-2. C, CCA1-1. D, CCA1-2. E, PRR7. F, PRR37. 912

A thioesterase/thiol ester dehydrase-isomerase superfamily (KfTEDI) gene was amplified 913

from the same cDNAs as a reference gene. Gene transcript abundance data represents the 914

mean of 3 technical replicates for each of three biological replicates and was normalized to 915

the reference gene (KfTEDI); error bars represent the standard error of the mean calculated 916

for the three biological replicates. In all cases, plants were entrained under 12-h-light/ 12-h-917

dark cycles for 7 days prior to sampling. Black data are for the wild type, blue data rPPCK1-1, 918

and red data rPPCK1-3. 919

920

Figure 6. Impact of the loss of KfPPCK1 activity on circadian clock controlled gene 921

transcript abundance during constant light and temperature free running conditions. A, 922

Circadian rhythm of KfPPCK1 transcript abundance under constant LL conditions (100 µmol 923

m-2 s-1 light at 15 ˚C) for wild type (black line) and rPPCK1-3 (red line). B, KfPPCK2. C, 924

KfPPCK3. D, KfCCA1-1. E, KfCCA1-2. F, KfTOC1-1. G, KfTOC1-2. H, KfPRR7. I, KfPRR37. 925

J, KfPRR9. K, KfFKF1. L, KfGIGANTEA. M, KfJMJD30. N, KfLNK3-like. O, KfCDF2. Mature 926

29

leaves (leaf pair 6) were sampled from three biological replicates every 4 h under constant 927

conditions (100 µmol m-2 s-1 light at 15˚C) for wild type and rPPCK1-3. RNA was isolated and 928

used for real-time RT-qPCR. A thioesterase/thiol ester dehydrase-isomerase superfamily 929

gene (KfTEDI) was amplified as a reference gene from the same cDNAs. Gene transcript 930

abundance data represents the mean of 3 technical replicates for each of three biological 931

replicates, and was normalized to the reference gene (KfTEDI); error bars represent the 932

standard error of the mean calculated for the three biological replicates. In all cases, plants 933

were entrained under 12-h-light/ 12-h-dark cycles prior to release into LL free-running 934

conditions. Black data are for the wild type and red data rPPCK1-3. 935

936

Figure 7. Delayed fluorescence (DF) rhythms collapsed towards arrythmia in lines 937

rPPCK1-1 and rPPCK1-3. Plants were entrained under 12-h-light/12-h-dark cycles before 938

being transferred to constant red/ blue light under the CCD imaging camera (35 µmol m-2 s-1). 939

DF was assayed with a 1-h time resolution for 108 h. The plots represent normalized 940

averages for DF measured for 6 leaf discs sampled from three biological replicates of leaf pair 941

6 for each line. A, Wild type DF rhythm under LL. B, Relative amplitude error (RAE) plot for 942

wild type DF rhythm. C, Mean period length (upper graph) and RAE plots (lower graph) for 943

wild type (black/ grey), rPPCK1-1 (blue/ pale blue) and rPPCK1-3 (red/ pale red); the plotted 944

values were calculated using using Biodare package for circadian rhythm analysis. In both 945

graphs, the dark shade of the colour represents data from Fast Fourier Transform-Non Linear 946

Least Squares (FFT-NLLS) analysis and the paler shade represents data from Spectral 947

Resampling (SR) analysis. D, rPPCK1-1 DF rhythm. E, RAE plot for rPPCK1-1. F, rPPCK1-3 948

DF rhythm. G, RAE plot for rPPCK1-3. Error bars indicate standard error of the mean 949

calculated from three biological replicates. Black data are for the wild type, blue data rPPCK1-950

1 and red data rPPCK1-3. 951

952

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1152

Figure 1. Confirmation of target gene silencing in transgenic K. fedtschenkoi RNAi lines

rPPCK1-1 and rPPCK1-3. Down-regulation of the target endogenous KfPPCK1 gene was confirmed at the level of KfPPCK1 transcript abundance (A), and target protein PPC phosphorylation and the apparent Ki of PPC for L-malate in rapidly desalted leaf extracts; measured as L-malate (mM) required for a 50 % reduction in extractable PPC activity (B). Gene transcript abundance was measured using RT-qPCR for wild type, rPPCK1-1 and rPPCK1-3 for target genes: A, KfPPCK1; C, KfPPCK2; and D, KfPPCK3. Mature leaves (leaf pair 6) were sampled from three individual, clonal, biological replicate plants collected every 4 h across the 12-h-light/ 12-h-dark cycle. A thioesterase/thiol ester dehydrase-isomerase superfamily gene (KfTEDI) was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represents the mean of 3 technical replicates of each of three biological replicates, and was normalized to reference gene (KfTEDI); error bars represent the standard error of the mean calculated for each biological replicate. In all cases, plants were entrained under 12-h-light/ 12-h-dark cycles for 7 days prior to sampling. B, Protein abundance (right panel) and the phosphorylation state of PPC (left panel) was determined by immunoblot analyses, and the apparent Ki of PPC for L-malate was measured using rapidly desalted leaf extracts (Ki values in mM below the corresponding left panel immunoblot time points). Total leaf protein (leaf pair 6) was isolated from leaves sampled every 4 h across the 12-h-light/ 12-h-dark cycle, separated using SDS-PAGE and used for immunoblot analyses with antibodies raised to a phospho-PPC peptide (left panel). Sample loading was normalized according to total protein and confirmed using the immunoblot for total PPC protein, which is stable over the 12-h-light/ 12-h-dark cycle (right panel). The white bar below each panel represents the 12-h-light period and the black bar below each panel represents the 12-h-dark period. For A, C and D: black data are for the wild type, blue rPPCK1-1 and red rPPCK1-3.

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Figure 2. Impact of silencing KfPPCK1 on 24 h light/ dark gas exchange profiles, and

malate, starch and soluble sugar levels for wild type, rPPCK1-1 and rPPCK1-3 under

well-watered conditions. A, Gas exchange profile for CAM leaves (leaf pair 6) using plants pre-entrained for 7 days under 12-h-light/ 12-h-dark cycles. B, Gas exchange profile for well-watered whole young plants (8-leaf-pairs stage) using plants entrained for 7 days under 12-h-light/ 12-h-dark cycles. C, Malate content was determined from leaf pair 6 samples collected every 4 h using plants entrained under 12-h-light/ 12-h-dark cycles using methanol extracts of leaves from wild type, rPPCK1-1 and rPPCK1-3. D, Starch content was determined from leaf pair 6 samples collected at 1 h before dawn and 1 h before dusk. E, F, G, Soluble sugars levels were determined separately for sucrose, glucose and fructose using three biological replicates of leaf pair 6 sampled every 4 h using plants entrained under 12-h-light/ 12-h-dark cycles. In C, D, E, F and G, the error bars represent the standard error of the mean calculated for the three biological replicates measured at each time point. Black data traces represent wild type, blue data rPPCK1-1 and red data rPPCK1-3.

B A

Figure 3. Effects of silencing KfPPCK1 on CAM CO2 exchange rhythms measured under constant light and temperature (LL) conditions. A, Gas exchange profile for CAM leaves (leaf pair 6) was measured using leaves entrained under a 12-h-light/ 12-h-dark cycle followed by release into constant LL conditions (100 µmol m-2 s-1 light at 15 ˚C). B, Gas exchange profile for well-watered whole young plants (8-leaf-pairs stage) using plants entrained under 12-h-light/ 12-h-dark cycles followed by release into constant LL conditions (100 µmol m-2 s-1 light at 15 ˚C). Black data traces represent wild type, blue data rPPCK1-1 and red data rPPCK1-3.

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Figure 4. Impact of loss of KfPPCK1 activity on gas exchange profiles (A) and vegetative yield (B) for plants subjected to drought-stress. A, Drought-stressed whole plants (8-leaf-pair stage) were entrained under 12-h-light/ 12-h-dark cycles followed by release into constant LL conditions (100 µmol m-2s-1 light at 15 ˚C). The black data trace represents wild type, blue rPPCK1-1 and red rPPCK1-3. B, C, D and E, Fresh (B, C) and dry (D, E) weight of above-ground biomass (shoot; B, D) and below-ground tissues (roots; C, E) at maturity (138 d under greenhouse conditions) for wild type (WT), rPPCK1-1 and rPPCK1-3 under well-watered and drought-stressed conditions. n = 7 plants; error bars represent standard error of the mean. * or *** = significantly different to wild type based on Student’s t-test, *P < 0.05 and ***P < 0.001.

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Figure 5. Impact of the loss of KfPPCK1 activity on the light/ dark regulation of the

transcript abundance of central circadian clock genes KfTOC1-1, KfTOC1-2, KfCCA1-

1, KfCCA1-2, KfPRR7 and KfPRR37. Mature leaves (leaf pair 6) were sampled from three biological replicates every 4 h across the 12-h-light/ 12-h-dark cycle, RNA isolated and used for real-time RT-qPCR. A, TOC1-1. B, TOC1-2. C, CCA1-1. D, CCA1-2. E, PRR7. F, PRR37. A thioesterase/thiol ester dehydrase-isomerase superfamily (KfTEDI) gene was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represents the mean of 3 technical replicates for each of three biological replicates and was normalized to the reference gene (KfTEDI); error bars represent the standard error of the mean calculated for the three biological replicates. In all cases, plants were entrained under 12-h-light/ 12-h-dark cycles for 7 days prior to sampling. Black data are for the wild type, blue data rPPCK1-1, and red data rPPCK1-3.

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Figure 6. Impact of the loss of KfPPCK1 activity on circadian clock controlled genetranscript abundance during constant light and temperature free runningconditions. A, Circadian rhythm of KfPPCK1 transcript abundance under constant LLconditions (100 µmol m-2 s-1 light at 15 ˚C) for wild type (black line) and rPPCK1-3 (redline). B, KfPPCK2. C, KfPPCK3. D, KfCCA1-1. E, KfCCA1-2. F, KfTOC1-1. G, KfTOC1-2.H, KfPRR7. I, KfPPR37. J, KfPRR9. K, KfFKF1. L, KfGIGANTEA. M, KfJMJD30. N,KfLNK3-like. O, KfCDF2. Mature leaves (leaf pair 6) were sampled from three biologicalreplicates every 4 h under constant conditions (100 µmol m-2 s-1 light at 15 ˚C) for wildtype and rPPCK1-3. RNA was isolated and used for real-time RT-qPCR. Athioesterase/thiol ester dehydrase-isomerase superfamily gene (KfTEDI) was amplifiedas a reference gene from the same cDNAs. Gene transcript abundance data representsthe mean of 3 technical replicates for each of three biological replicates, and wasnormalized to loading control gene (KfTEDI); error bars represent the standard error ofthe mean calculated for the three biological replicates. In all cases, plants were entrainedunder 12-h-light/ 12-h-dark cycles prior to release into LL free-running conditions. Blacklines represent wild type and red lines rPPCK1-3.

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Figure 7. Delayed fluorescence (DF) rhythms collapsed towards arrythmia in lines

rPPCK1-1 and rPPCK1-3. Plants were entrained under 12-h-light/12-h-dark cycles before being transferred to constant red/ blue light under the CCD imaging camera (35 µmol m-2 s-

1). DF was assayed with a 1-h time resolution for 108 h. The plots represent normalized averages for DF measured for 6 leaf discs sampled from three biological replicates of leaf pair 6 for each line. A, Wild type DF rhythm under LL. B, Relative amplitude error (RAE) plot for wild type DF rhythm. C, Mean period length (upper graph) and RAE plots (lower graph) for wild type (black/ grey), rPPCK1-1 (blue/ pale blue) and rPPCK1-3 (red/ pale red); the plotted values were calculated using using Biodare package for circadian rhythm analysis. In both graphs, the dark shade of the colour represents data from Fast Fourier Transform-Non Linear Least Squares (FFT-NLLS) analysis and the paler shade represents data from Spectral Resampling (SR) analysis. D, rPPCK1-1 DF rhythm. E, RAE plot for rPPCK1-1. F, rPPCK1-3 DF rhythm. G, RAE plot for rPPCK1-3. Error bars indicate standard error of the mean calculated from three biological replicates. Black data are for the wild type, blue data rPPCK1-1 and red data rPPCK1-3.

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DOI 10.1105/tpc.17.00301; originally published online September 8, 2017;Plant Cell

Susanna F Boxall, Louisa V Dever, Jana Knerova, Peter D Gould and James HartwellSpecies Kalanchoë fedtschenkoi

CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Phosphorylation of Phosphoenolpyruvate Carboxylase is Essential for Maximal and Sustained Dark

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