phosphorylation of phosphoenolpyruvate carboxylase is essential … · 3 phosphorylation of...
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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: [email protected] 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 ([email protected]). 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
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Plant Cell Advance Publication. Published on September 8, 2017, doi:10.1105/tpc.17.00301
©2017 The authors. All Rights Reserved
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
Figure 1
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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 3
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.
A Figure 4
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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.
Figure 5
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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.
Figure 7
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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
This information is current as of July 6, 2018
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