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RESEARCH ARTICLE
Kalanchoë PPC1 is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes
Susanna F. Boxall, Nirja Kadu, Louisa V. Dever, Jana Kneřová, Jade L. Waller,Peter J. D. Gould and James Hartwella
Department of Functional and Comparative Genomics, Institute of Integrative Biology, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK. aCorresponding Author: [email protected]
Short Title: Silencing PPC1 in an obligate CAM plant
One-sentence Summary: Silencing phosphoenolpyruvate carboxylase in a Crassulacean acid metabolism species prevented nocturnal CO2 fixation and malate accumulation and perturbed the circadian clock and guard cell signalling.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: James Hartwell ([email protected]).
ABSTRACT
Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate (PEP) carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO2 (as HCO3
-), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around RuBisCO for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNAi to silence the CAM isogene PPC1 in Kalanchoë laxiflora. Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1. Furthermore, a subset of gene transcripts within the central circadian oscillator were upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B. These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.
INTRODUCTION1 2
Crassulacean acid metabolism (CAM) is a pathway of photosynthetic CO2 fixation 3
found in species adapted to low rainfall and/or periodic drought, such as the 4
Plant Cell Advance Publication. Published on February 12, 2020, doi:10.1105/tpc.19.00481
©2020 The author(s).
2
Madagascan-endemic succulent, Kalanchoë laxiflora Baker (Family: Crassulaceae; 5
Order: Saxifragales) (Hartwell et al., 2016). CAM species open their stomata for 6
primary atmospheric CO2 fixation in the dark, when their environment is cooler and 7
more humid, and close their stomata in the light, when the atmosphere is at its 8
hottest and driest (Osmond, 1978). The increased water use efficiency (WUE) and 9
CO2 fixation efficiency of CAM species have led to the proposal that the use of 10
productive CAM crop species, including certain Agave and Opuntia species, 11
represents a viable approach to generating biomass for biofuels and renewable 12
platform chemicals for industry through their cultivation on seasonally-dry lands that 13
are not well-suited to food crop production (Borland et al., 2009; Cushman et al., 14
2015). Furthermore, efforts are underway to engineer CAM into key C3 crops 15
(Borland et al., 2014; DePaoli et al., 2014; Borland et al., 2015; Lim et al., 2019). 16
Recently, genome, transcriptome, proteome, and metabolome datasets for a 17
phylogenetically diverse range of CAM plants of independent origin have started to 18
open up a large catalogue of putative CAM genes. CAM species represented by 19
published ‘omics datasets span orchids (Phalaenopsis equestris and Erycina pusilla, 20
Orchidaceae, monocots), pineapple (Ananas comosus, Bromeliaceae, monocots), 21
Agave (A. americana, A. deserti, A. tequilana, Agavaceae, monocots), Yucca (Y. 22
aloifolia, Asparagaceae, monocots), Kalanchoë (K. fedtschenkoi and K. laxiflora, 23
Crassulaceae, eudicots), Mesembryanthemum (M. crystallinum, Aizoaceae, 24
eudicots), and waterleaf ( T. triangulare, Portulacaceae, eudicots) (Cushman et al., 25
2008; Gross et al., 2013; Cai et al., 2015; Ming et al., 2015; Abraham et al., 2016; 26
Brilhaus et al., 2016; Hartwell et al., 2016; Yang et al., 2017; Heyduk et al., 2018; 27
Heyduk et al., 2019). The optimal exploitation of CAM will be facilitated through 28
decoding the molecular-genetic blueprint for CAM from these genomes and 29
transcriptomes. In turn, to be fully realised, this opportunity requires functional 30
genomics approaches using transgenic and/or mutant lines of model CAM species to 31
test and define in detail candidate CAM gene functions (Dever et al., 2015; Hartwell 32
et al., 2016; Boxall et al., 2017). 33
During CAM, primary nocturnal CO2 assimilation is catalyzed by 34
phosphoenolpyruvate (PEP) carboxylase (PPC), generating oxaloacetate, which is 35
rapidly converted to malate and stored in the vacuole as malic acid (Borland et al., 36
2009). At dawn, malic acid is transported out of the vacuole and malate is 37
decarboxylated in the mitochondria, chloroplasts and/or cytosol, and the released 38
3
CO2 is re-fixed by RuBisCO behind closed stomata (Borland et al., 2009). Strict 39
temporal control prevents a futile cycle between the enzymes and metabolite 40
transporters driving malate production in the dark and those driving malate 41
decarboxylation in the light (Hartwell, 2006). PPC regulation is central to this 42
temporal control. PPC is activated allosterically by glucose 6-phosphate (G6P) and 43
inhibited by malate, aspartate and glutamate (O’Leary et al., 2011). The circadian 44
clock optimizes the timing of the CAM carboxylation and decarboxylation pathways 45
to prevent futile cycling (Wilkins, 1992; Hartwell, 2005). 46
Temporal optimization of PPC involves protein phosphorylation in the dark 47
period, a process catalysed by the circadian clock-controlled protein kinase, 48
phosphoenolpyruvate carboxylase kinase (PPCK) (Carter et al., 1991; Hartwell et al., 49
1996; Hartwell et al., 1999; Taybi et al., 2000; Boxall et al., 2005). Phosphorylated 50
PPC is less sensitive to feedback inhibition by malate, which in turn ensures 51
sustained CO2 fixation as malic acid accumulates throughout the dark period 52
(Nimmo et al., 1984; Carter et al., 1991; Boxall et al., 2017). In the light, PPC 53
becomes more sensitive to inhibition by malate due to dephosphorylation by a 54
protein phosphatase type 2A (PP2A), which is not known to be subject to circadian 55
control (Carter et al., 1990). 56
In CAM species such as Kalanchoë, the PEP substrate required for nocturnal 57
atmospheric CO2 fixation by PPC is generated through starch breakdown and 58
glycolysis (Borland et al., 2016). In the C3 model species Arabidopsis thaliana, 59
nocturnal degradation of leaf starch begins with the phosphorylation of glucan chains 60
by GLUCAN WATER DIKINASE (GWD) and PHOSPHOGLUCAN WATER 61
DIKINASE (PWD) (Ritte et al., 2006). The phosphorylated glucan chains are then 62
further degraded by ALPHA-AMYLASES (AMYs) and BETA-AMYLASES (BAMs) to 63
maltose and glucose. Nocturnal starch hydrolysis by BAMs is the predominant 64
pathway in C3 leaves, with chloroplastic BAM3 being the major BAM isozyme driving 65
nocturnal starch degradation in photosynthetic leaf mesophyll cells of Arabidopsis 66
(Fulton et al., 2008). BAM1, BAM2 and BAM5-9 are not required for nocturnal starch 67
degradation in Arabidopsis leaf mesophyll cells (Santelia and Lunn, 2017). Maltose 68
and glucose are exported from chloroplasts by MALTOSE EXCESS1 (MEX1) and 69
PLASTIDIC GLUCOSE TRANSPORTER (pGlcT), respectively, with MEX1 being the 70
predominant C3 route for carbon export (Smith et al., 2005). In the facultative CAM 71
4
species M. crystallinum, chloroplasts possess transporters for triose phosphate 72
(TPT), G6P (GPT), glucose (pGlcT) and maltose (MEX1) (Neuhaus and Schulte, 73
1996; Kore-eda et al., 2005; Kore-eda et al., 2013). Chloroplasts from C3 leaves of 74
M. crystallinum exported maltose during starch degradation, whereas chloroplasts 75
isolated from CAM-induced leaves predominantly exported G6P, supporting the 76
proposal that starch is broken down via plastidic starch phosphorylase (PHS1) 77
during nocturnal CO2 fixation (Neuhaus and Schulte, 1996). 78
A further defining characteristic of CAM relates to nocturnal stomatal opening 79
and CO2 uptake, and light period stomatal closure during malate decarboxylation 80
and peak internal CO2 supply (Males and Griffiths, 2017). Stomatal control in CAM 81
species is the inverse of the stomatal regulation observed in C3 species (Borland et 82
al., 2014). The opening and closing of stomata are driven by the turgor of the guard 83
cell (GC) pair that surrounds the stomatal pore. High turgor drives stomatal opening, 84
and a reduction in turgor leads to stomatal closure. The increase in turgor during 85
opening is driven by the accumulation of K+, Cl- and malate2- ions plus sugars in the 86
GCs (Jezek and Blatt, 2017). The closure of stomata is driven by a reversal of GC 87
ion channels and metabolism, with K+ and Cl- being transported out, and metabolites 88
being turned over within the GCs. Stomatal aperture responds to changing light, 89
CO2, abscisic acid (ABA), solutes and water availability (Kim et al., 2010; Daloso et 90
al., 2016; Horrer et al., 2016; Zhang et al., 2018; Yoshida et al., 2019). 91
In addition to its role in CAM and C4 plants, PPC performs an anapleurotic 92
function by replenishing tricarboxylic acid cycle intermediates utilized for amino acid 93
biosynthesis (Chollet et al., 1996). PPC also functions in the formation of malate as a 94
counter anion for light period opening in C3 GCs and supports nitrogen fixation into 95
amino acids in legume root nodules (Chollet et al., 1996). The major leaf PPCs in 96
Arabidopsis, which are encoded by PPC1 (AT1G53310) and PPC2 (AT2G42600), 97
are crucial for leaf carbon and nitrogen metabolism (Shi et al., 2015). Compared to 98
the wild type, the double ppc1 ppc2 null mutant accumulates less starch and sucrose 99
and has reduced malate, citrate, and ammonium assimilation; these metabolic 100
changes lead to a severe, growth-arrested phenotype (Shi et al., 2015). 101
Although PPC catalyses primary CO2 fixation in CAM and C4 plants, the only 102
reported PPC mutants are for the C4 eudicot grain amaranth (Amaranthus edulis) 103
and the C4 monocot green foxtail (Setaria viridis) (Dever et al., 1995; Alonso-104
Cantabrana et al., 2018). C4 PPC catalyzes light period primary CO2 fixation in 105
5
mesophyll cells, generating malate or aspartate, which is shuttled to bundle sheath 106
cells for decarboxylation (von Caemmerer and Furbank, 2003). This leads to the 107
concentration of CO2 around RuBisCO, which is only present in bundle sheath cells. 108
Photorespiration is thus minimised. The loss of the C4 PPC isogene in A. edulis 109
caused a severe and lethal growth phenotype in normal air, with the homozygous 110
mutant plants only managing to reach flowering and set seed when grown at highly 111
elevated CO2 (Dever et al., 1995). In S. viridis, the C4 PPC expression was reduced 112
to very low levels using RNAi in transgenic lines (Alonso-Cantabrana et al., 2018). 113
These lines grew very slowly even at 2% CO2 (normal air is 0.04%) and developed 114
increased numbers of plasmodesmatal pit fields at the mesophyll-bundle sheath 115
interface (Alonso-Cantabrana et al., 2018). By contrast, no CAM mutants lacking 116
PPC have been described, but transgenic lines of Kalanchoë lacking the light-period, 117
decarboxylation pathway enzymes mitochondrial NAD-malic enzyme (NAD-ME) and 118
pyruvate orthophosphate dikinase (PPDK) displayed a near complete loss of dark 119
CO2 fixation and failed to turnover significant malate during the light period (Dever et 120
al., 2015). In Kalanchoë, NAD-ME catalyses the conversion of malate to pyruvate 121
and CO2 during the light period, and PPDK converts the pyruvate to PEP, allowing 122
subsequent recycling of the by-product of malate decarboxylation through 123
gluconeogenesis to stored starch (Dever et al., 2015). Another CAM mutant lacking 124
the starch synthesis pathway enzyme plastidic phosphoglucomutase has been 125
reported in the inducible CAM species M. crystallinum (Cushman et al., 2008). 126
In addition, the CAM-associated PPCK1 gene in Kalanchoë was silenced 127
using RNAi, which not only led to a reduction in dark period CO2 fixation, but also 128
perturbed the operation of the central circadian clock (Boxall et al., 2017). However, 129
even the strongest PPCK1 RNAi line was still able to achieve ~33% of the dark 130
period CO2 fixation observed in the wild type (Boxall et al., 2017). These findings led 131
us here to develop transgenic K. laxiflora lines in which the CAM-associated isogene 132
of PPC itself (isogene PPC1) was downregulated using RNAi. The most strongly 133
silenced line, rPPC1-B, lacked PPC1 transcripts and activity, and this resulted in the 134
complete loss of dark CO2 fixation associated with CAM, and arrhythmia of the CAM 135
CO2 fixation rhythm under constant light and temperature (LL) free-running 136
conditions. Growth of rPPC1-B plants was reduced relative to wild type under both 137
well-watered and drought-stressed conditions. The plants reverted to fixing CO2 in 138
the light, especially in their youngest leaf pairs. Although the circadian rhythm of CO2 139
6
fixation dampened rapidly towards arrhythmia in the rPPC1-B line, the distinct 140
circadian clock output of delayed fluorescence, and the oscillations of the transcript 141
abundances of a subset of core circadian clock genes, were enhanced. Furthermore, 142
the temporal phasing of a wide range of GC specific signaling genes involved in 143
opening and closing was perturbed relative to the wild type in rPPC1-B. These 144
findings shed light on important regulators underpinning the inverse stomatal control 145
associated with CAM. 146
147 RESULTS 148
149
Initial Screening and Characterisation of PPC1 RNAi Lines of K. laxiflora 150
151
As K. laxiflora is relatively slow-growing, with flowering and seed set taking 152
approximately 9-months seed-to-seed (Hartwell et al., 2016), data are presented for 153
independent primary transformants that were propagated clonally via leaf margin 154
plantlets and/or stem cuttings. We initially screened primary transformants using 155
high-throughput leaf disc tests for starch and acidity at dawn and dusk (Cushman et 156
al., 2008; Dever et al., 2015). We screened independent transgenic lines that 157
acidified less during the dark period than the wild type for the steady-state 158
abundance of PPC1 transcripts using RT-qPCR (Figure 1). Line rPPC1-B displayed 159
a complete loss of PPC1 transcripts, whereas rPPC1-A had an intermediate level of 160
PPC1 transcripts (Figure 1A). The other plant-type PPC genes (PPC2, PPC3 and 161
PPC4) were up-regulated relative to the wild type, with their peak phased to dawn 162
(Figure 1B to 1D). PPC2 was up-regulated in line rPPC1-B at 6 and 10 h into the 12 163
h dark period (Figure 1B). 164
In line rPPC1-B, PPCK1, encoding the protein kinase that phosphorylates the 165
CAM-associated protein PPC1, was down-regulated during its dark period phased 166
peak and appeared to be slightly up-regulated at 2 h after dawn (Figure 1E). The 167
other two detectable PPCK genes, PPCK2 and PPCK3, were up-regulated in rPPC1-168
B, with PPCK2 induced 5-fold when it reached its 24 h peak in the middle of the dark 169
period, 4 h after the wild-type peak (Figure 1F). PPCK3 peaked in the middle of the 170
light period, when it reached a level almost 8-fold greater than the wild type (Figure 171
1G). 172
173
7
Loss of PPC1 Transcripts lead to Loss of PPC Protein and Activity 174
175
Immunoblotting using an antibody raised against purified CAM-specific PPC protein 176
from K. fedtschenkoi leaves (Nimmo et al., 1986) (Figure 2) revealed reduced levels 177
of PPC protein in rPPC1-A and no detectable PPC in rPPC1-B (Figure 2A). 178
We also measured the level of phospho-PPC using immunoblotting (Figure 179
2B). Phospho-PPC levels were lower in rPPC1-A than the wild type, whilst rPPC1-B 180
lacked detectable phospho-PPC (Figure 2B), which was consistent with the level of 181
PPC (Figure 2A). Although PPC2 was up-regulated in rPPC1-B (Figure 1B), it was 182
not detected at the protein level by the PPC antibody (Figure 2A). Furthermore, 183
despite PPCK2 and PPCK3 being up-regulated by 5- to 8-fold in rPPC1-B (Figure 1F 184
and 1G), any protein produced from these transcripts did not phosphorylate any 185
PPC2 protein that may have resulted from the induced PPC2 transcripts (Figure 2B), 186
at least not within the limits of detection with this immunoblotting technique. We used 187
rapidly desalted leaf extracts to measure the apparent Ki of PPC for L-malate. The Ki 188
was higher in the dark than the light for wild type and rPPC1-A, but no change in the 189
Ki was detected for rPPC1-B (Supplemental Figure 1). Furthermore, PPC activity 190
was not detected in rapidly desalted extracts from rPPC1-B leaves, whereas rPPC1-191
A displayed reduced but detectable PPC activity, at 43% of the wild-type level 192
(Figure 2C and Supplemental Table 1). 193
Pyruvate orthophosphate dikinase (PPDK), which functions in concert with 194
NAD-ME during the light-period as part of the CAM malate decarboxylation pathway 195
(Dever et al., 2015), was measured on immunoblots using specific antibodies against 196
PPDK and phospho-PPDK (Chastain et al., 2000; Chastain et al., 2002). The blots 197
showed a similar amount of PPDK protein over the diel cycle in wild type, rPPC1-A 198
and rPPC1-B (Figure 2D), confirming the even protein loading of the blots, which 199
was further supported by the images of Coomassie stained gels (Supplemental 200
Figure 1). In Kalanchoë, PPDK is inactivated in the dark by phosphorylation by 201
PPDK-regulatory protein (PPDK-RP), which also activates PPDK in the light through 202
dephosphorylation (Dever et al., 2015). In the wild type, immunoblotting of phospho-203
PPDK revealed that PPDK was dephosphorylated, and therefore likely to be fully 204
active, between 02:00 and 06:00 in the light when it is required for the conversion of 205
pyruvate, from malate decarboxylation, to PEP, thereby facilitating the recycling of 206
carbon through gluconeogenesis to starch (Figure 2E). Line rPPC1-A showed the 207
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same pattern of PPDK phosphorylation/de-phosphorylation as the wild type (Figure 208
2E). However, PPDK was phosphorylated throughout the 24 h cycle in rPPC1-B 209
(Figure 2E), and was therefore likely to be inactive. Consistent with this prediction, 210
loss of the light period dephosphorylation of PPDK in rPPC1-B correlated with a 211
significant decrease in PPDK activity in the light, whereas the wild type and line 212
rPPC1-A showed strongly light-induced levels of PPDK activity that correlated with 213
the detected level of PPDK dephosphorylation in the light (Figure 2F and 214
Supplemental Table 2). 215
216
Malate, Starch, and Soluble Sugar Levels 217
218
During CAM in Kalanchoë, primary nocturnal fixation of atmospheric CO2 (as HCO3-) 219
results in vacuolar malic acid accumulation throughout the dark period. Starch 220
accumulates during the light period and is broken down during the dark period to 221
provide PEP as the substrate for carboxylation by PPC. Starch is also broken down 222
in a rapid burst at dawn to form soluble sugars (Wild et al., 2010; Boxall et al., 2017). 223
As the lack of CAM-associated PPC1 was predicted to prevent primary nocturnal 224
carboxylation, we measured metabolites including malate, starch and soluble sugars 225
every 4 h over the 24 h cycle (Figure 3). 226
Wild-type plants accumulated 130 µmols gFW-1 malate by dawn, whereas 227
rPPC1-A and rPPC1-B accumulated 75 µmol gFW-1 and 19.5 µmols gFW-1, 228
respectively (Figure 3A). The '-malate values for wild type, rPPC1-A and rPPC1-B 229
were 124.0, 64.3 and 16.2 µmol gFW-1, respectively (Figure 3A). During the diel 230
cycle, rPPC1-A and rPPC1-B synthesized 100% and 41% of the amount of starch 231
accumulated by the wild type, respectively (Figure 3B). The '-starch values for wild 232
type, rPPC1-A and rPPC1-B were 8.5, 8.5 and 3.5 mg starch gFW-1 (Figure 3B). 233
Lines rPPC1-A and rPPC1-B accumulated 51% and 15% of the amount of sucrose 234
accumulated by the wild type (Figure 3C), and 83% and 69% of the level of glucose, 235
respectively (Figure 3D). Glucose accumulated 4 h after the sucrose peak in the wild 236
type, whereas glucose levels peaked at the same time as sucrose in lines rPPC1-A 237
and rPPC1-B (Figure 3C and 3D). Finally, lines rPPC1-A and rPPC1-B accumulated, 238
respectively, 113% and 61% of the amount of fructose compared with the wild type 239
(Figure 3E). In rPPC1-B, the daily maxima for fructose and glucose coincided with 240
9
that of sucrose at 2 h after dawn (Figure 3C to 3E). Whilst no direct evidence was 241
obtained to explain these shifts in the timing of the sucrose, glucose and fructose 242
daily maxima, the most straightforward explanation is that the observed changes in 243
the timing of photosynthetic CO2 fixation and starch accumulation and turnover in the 244
rPPC1-B transgenic line resulted in the observed changes in the timing of the soluble 245
sugar peaks. 246
247
Growth Analysis in Well-Watered versus Drought-Stressed Conditions 248
249
CAM is widely regarded as an adaptation to drought, and so it was important to 250
compare the growth performance of wild type (full CAM) with that of rPPC1-A (small 251
reduction in CAM) and rPPC1-B plants (no CAM) under both well-watered (WW) and 252
drought-stressed (D-S) conditions (Figure 3F, Supplemental Figure 2 and 253
Supplemental Data Set 1). rPPC1-B plants were significantly smaller than wild type 254
in both WW and D-S conditions (Figure 3F and Supplemental Figure 2). In WW 255
conditions, the shoot dry weight of rPPC1-B was 21% less than that of the wild type 256
(Figure 3F), but the shoot dry weight of rPPC1-A was not significantly different from 257
that of the wild type. Under D-S, the shoot dry weight of line rPPC1-B was 12% lower 258
than that of the wild type (Figure 3F). A visual inspection of representative 4-month-259
old plants demonstrated that rPPC1-B was smaller than wild type and rPPC1-A 260
(Figure 3G), which is consistent with the shoot dry weight data (Figure 3F). Shoot 261
fresh weight was also significantly reduced in WW and D-S rPPC1-B, whereas the 262
only significant difference in below-ground root tissues was for rPPC1-B under 263
drought-stress, which displayed an increase in root fresh weight that was only just 264
significant (p = 0.0483; Supplemental Figure 2). Furthermore, rPPC1-B displayed a 265
reduced degree of leaf in-rolling in response to drought relative to the wild type 266
(Supplemental Figure 3), even though it lost just as much water during drought as 267
the wild type (Supplemental Figure 2). 268
269
Gas Exchange Characteristics under Light/Dark Cycles 270
271
We measured gas exchange in mature CAM leaves (leaf pair 6, LP6) of each line 272
over a 12-h-light, 25˚C, 60% humidity/ 12-h-dark, 15˚C, 70% humidity cycle using an 273
infra-red gas analyser (IRGA; LI-COR LI-6400XT) (Figure 4; 4A to 4C). Over the 24 274
10
h diel cycle, wild type fixed 297 µmol CO2 m-2, rPPC1-A fixed 320 µmol CO2 m-2, and 275
rPPC1-B fixed 230 µmol CO2 m-2 (Figure 4A). Net CO2 fixation was 23% less than 276
the wild type in line rPPC1-B, whereas it was 15% greater in line rPPC1-A (Figure 277
4A). 278
The four phases of CAM (Osmond, 1978) are indicated on the CO2 exchange 279
graph in Figure 4A. Phase I corresponds to the period of nocturnal atmospheric CO2 280
fixation to vacuolar malic acid in the wild type, and phases II, III and IV define the 281
peaks and trough of CO2 fixation and stomatal conductance across the 12-h-light 282
period (Figure 4A and 4B). Specifically, phase II refers to the sharp peak of CO2 283
fixation in the 1 to 2 hours after dawn, phase III defines the period of refixation CO2 284
from malate decarboxylation by RuBisCO behind closed stomata that spans the 285
middle of the light period, and phase IV occurs in younger leaves of well-watered 286
plants and corresponds to the period in the late afternoon when stomata re-open and 287
atmospheric CO2 is fixed directly by RuBisCO (Osmond, 1978). Note that the data in 288
blue for rPPC1-A provide the best example of this four phases framework for CAM 289
CO2 fixation and stomatal physiology (Figure 4A). By contrast, LP6 of the wild type 290
perform full CAM defined by phase I in the dark and phase III in the light (Figure 4A). 291
By calculating the total area under or over the CO2 exchange curves in Figure 4A, 292
and calculating these areas separately for the 12-h-light and the 12-h-dark periods, it 293
was possible to quantify the amounts of CO2 fixed by each line during either the light 294
period or the dark period. In the light period (phases II through IV), wild type leaves 295
fixed negligible amounts of atmospheric CO2, whereas rPPC1-B fixed a total of 265 296
µmol m-2, and rPPC1-A fixed 89 µmol m-2 (Figure 4A). In the dark period (phase I), 297
wild type fixed 297 µmol CO2 m-2 and rPPC1-A fixed 226 µmol CO2 m-2, but rPPC1-B 298
respired 35 µmol CO2 m-2 (Figure 4A). The loss of nocturnal CO2 fixation in rPPC1-B 299
(Figure 4A) corresponded with the lack of the CAM-associated PPC1 (Figure 1 and 300
2). LP6 of rPPC1-A fixed 24% less nocturnal CO2 than wild type, but, in contrast to 301
wild type, continued some light period CO2 capture (Figure 4A). 302
Apart from opening briefly for phase II, just after lights-on, the attached LP6 of 303
the wild type closed its stomata in the light and opened them throughout the dark, 304
when stomatal conductance (gs) tracked CO2 uptake (Figure 4B). In rPPC1-B, 305
stomata stayed open in the light, closed briefly at dusk, and opened slightly 306
throughout the dark, with a small peak prior to dawn (Figure 4B). The dark 307
conductance of rPPC1-B corresponded with the release of respiratory CO2 (Figure 308
11
4A). The calculated internal partial pressure of CO2 inside the leaf (Ci) was highest 309
for the wild type in the light period, when stomata were closed, which is consistent 310
with the expected high level of CO2 inside the leaf during malate decarboxylation 311
(Figure 4C). By contrast, in rPPC1-B, Ci peaked during the dark period, when 312
stomata were slightly open, but the leaves failed to fix atmospheric CO2, and 313
respiratory CO2 escaped (Figure 4C). 314
315
Impact of Drought on CO2 uptake in Plants lacking PPC1 316
To test the importance of PPC1 for carbon assimilation during drought, we measured 317
CO2 uptake continuously in whole, young plants (9-leaf-pairs; 2-months-old) over 22-318
days of drought, followed by re-watering (Figure 4D). The soil was watered to its 319
holding capacity prior to placing the plants in the gas exchange cuvettes at the start 320
of the experiment, and then no further water was added until re-watering occurred on 321
day 22. CAM develops with leaf age in K. laxiflora; the leaves gradually reduce light 322
period CO2 fixation and increase nocturnal CO2 fixation as they mature 323
(Supplemental Figure 4). A 2-month-old wild-type plant with 9-leaf-pairs thus 324
includes young leaves fixing CO2 mainly in the light via the C3 pathway, and older 325
leaves fixing the majority of their atmospheric CO2 in the dark via PPC and CAM. 326
When WW on day 1, wild type, rPPC1-A and rPPC1-B fixed, respectively, 7%, 327
6% and -26% of their CO2 during the dark period and 93%, 94% and 126% during 328
the light period (Table 1). This indicated that, in well-watered conditions, the young 329
leaves of these young plants performed the majority of the 24 h CO2 uptake (Table 330
1). On day 1, wild type, rPPC1-A and rPPC1–B fixed a total of 1093, 1008 and 1078 331
µmol of atmospheric CO2 m-2 over 24 h, respectively (Table 1). After 7-days without 332
watering, 24 h CO2 fixation was 1345, 1367 and 1506 µmol m-2, respectively. It 333
should be noted that total leaf area was measured at the end of the experiment, so 334
leaf growth and expansion during the experiment could not be accounted for. 335
After 7-days without water, there was a substantial increase in nocturnal CO2 336
uptake in wild type and rPPC1-A relative to day 1 (52% and 40% of CO2 fixation 337
occurred in the dark, respectively; Table 1). rPPC1-B respired less (-14%) after 7-338
days of drought relative to the -26% dark respired CO2 level on day 1 (Table 1). After 339
13-days of drought, wild type, rPPC1-A and rPPC1-B fixed, respectively, 795, 802 340
and 145 µmol CO2 m-2 over the 24 h cycle (Table 1). Thus, plants performing CAM 341
(wild type and rPPC1-A) were able to fix over 5-fold more CO2 after 13-days of 342
12
drought compared to rPPC1-B. Furthermore, after 22-days of drought, wild type, 343
rPPC1-A and rPPC1-B fixed 76, 130 and -30 µmol m-2 during the 24 h light/dark 344
cycle, respectively. 345
On day 22 without water, the drought-stressed plants were re-watered (see 346
photos in Supplemental Figure 3). After re-watering, CO2 fixation increased rapidly 347
for all plants (Figure 4D). In addition, wild type and rPPC1-A displayed pronounced 348
phase III of CAM after re-watering (Figure 4D). Following soil rehydration, the wild 349
type fixed 931 µmol m-2 CO2 in the dark, compared to 768 µmol m-2 for rPPC1-A, 350
whereas prior to drought, rPPC1-A fixed more atmospheric CO2 (Table 1). rPPC1-B 351
fixed CO2 throughout the light period following re-watering, and it also resumed 352
respiratory CO2 loss throughout the dark period (Figure 4D). 353
As there was an increase in C3 photosynthesis in rPPC1-B, we measured 354
chlorophyll a and b levels in LP2 through LP7 (Figure 4E and 4F and Supplemental 355
Table 3). Line rPPC1-A contained significantly more chlorophyll a and b than wild 356
type in LP2 (Figure 4E and 4F). rPPC1-B contained significantly more chlorophyll a 357
than the wild type in LP2, but significantly less chlorophyll a in LP3 through LP7, and 358
significantly less chlorophyll b in LP3 to LP6 (Figure 4E and 4F). This decreased 359
level of chlorophyll a and b in LP3 to LP7 or LP6 in rPPC1-B was a surprising 360
finding. In general, CAM species contain reduced levels of gene transcripts and 361
encoded proteins associated with C3 photosynthesis, such as RuBisCO and light 362
harvesting complex components, when the level of CAM is induced by stress. Thus, 363
reducing the level of CAM in leaf pair 3 through 7 due to the silencing of PPC1 was 364
predicted to result in the induction of core C3 photosynthesis genes and chlorophyll 365
content in parallel with the measured increase in light period atmospheric CO2 366
fixation via the C3 pathway. Further elucidation of the reasons for this unexpected 367
chlorophyll response may come from transcriptome-wide analysis of the differentially 368
abundant transcripts in rPPC1-B compared to the wild type. 369
370
Characterisation of CAM Gene Transcript Abundance in rPPC1 Lines 371
372
Having established that rPPC1-B lacked nocturnal CO2 fixation (Figure 4A), it was 373
important to investigate the temporal regulation of other CAM-associated genes in 374
the rPPC1 RNAi lines. We investigated the transcript abundance of CAM genes in 375
CAM leaves (LP6) using samples collected every 4 h over a 12-h-light/12-h-dark 376
13
cycle (Figure 5). PPDK transcript levels were unchanged relative to the wild type in 377
both rPPC1 lines (Figure 5A), but its regulator gene PPDK-RP was up-regulated in 378
line rPPC1-B (Figure 5B), which is consistent with the continuous phosphorylation 379
and inactivation of PPDK (Figure 2E and 2F). E-NAD-ME transcript levels appeared 380
to be only slightly different from the wild type in the rPPC1 lines but appeared to be 381
lower in rPPC1-B at 22:00 (Figure 5C). 382
In light of the marked reduction in starch to half the wild-type level in rPPC1-B 383
(Figure 3B), we also measured transcripts associated with starch turnover. In rPPC1-384
B, dusk-phased starch breakdown-associated genes (GWD, AMY3a and 3b, PHS1; 385
Figure 5D to 5F and 5J) and sugar transporter genes (MEX1, pGlcT; Figure 5L and 386
5M) were up-regulated, whereas dawn-phased starch breakdown genes (BAM1, 387
BAM3, BAM9; Figure 5G to 5I) and sugar transporter genes (GPT2; Figure 5K) were 388
down-regulated compared to the wild type. 389
Finally, a CHLOROPHYLL A/B BINDING PROTEIN (CAB1) gene was up-390
regulated in rPPC1-B at dawn (Figure 5N), whereas a gene encoding a potential 391
sucrose sensor connecting growth and development to metabolic status, 392
TREHALOSE 6-PHOSPHATE SYNTHASE7 (TPS7) (Schluepmann et al., 2003), 393
was down-regulated relative to the wild type at 6 and 10 h into the 12-h-dark period 394
(Figure 5O). 395
396
Characterisation of Diel Regulation of Circadian Clock Genes in rPPC1 Lines 397
398
Recent studies using PPCK1 RNAi lines in Kalanchoë reported that a reduced 399
temporal peak of sucrose content phased to 2 h after dawn correlated with 400
perturbation of the central circadian clock (Boxall et al., 2017). Of the two 401
CIRCADIAN CLOCK ASSOCIATED1 (CCA1) genes in K. laxiflora, each of which is 402
represented by two homeologous copies in the tetraploid genome, only the two 403
homeologs of CCA1-2 were down-regulated in rPPC1-B (Figure 5P and 5Q), 404
whereas both homeologs of both TIMING OF CHLOROPHYLL A/B BINDING 405
PROTEIN1 (TOC1) genes were up-regulated in both rPPC1-A and rPPC1-B (Figure 406
5R and 5S). Two other pseudo-response regulators (PRRs) related to TOC1, namely 407
PRR7 and PRR3/7, were up-regulated in both rPPC1-A and rPPC1-B (Figure 5T and 408
14
5U), and PRR9 was induced almost 5-fold in the middle of the light period, 409
specifically in line rPPC1-B (Figure 5V). 410
Other clock associated genes, including JUMONJI DOMAIN 411
CONTAINING30/5 (JMJ30/ JMJD5), EARLY FLOWERING3 (ELF3) and CYCLING 412
DOF-FACTOR2 (CDF2) were also up-regulated in line rPPC1-B (Figure 5W to 5Y) 413
vs. the wild type, whereas the single MYB-repeat transcription factor genes 414
REVEILLE1-like (RVE1-like) and EARLY-PHYTOCHROME-RESPONSIVE1 (EPR1), 415
and LIGHT NIGHT-INDUCIBLE AND CLOCK-REGULATED3-like (LNK3-like) were 416
down-regulated (Figure 5Z to 5BB). Finally, CCA1-1, FLAVIN-BINDING KELCH-417
REPEAT F-BOX PROTEIN1 (FKF1) and GIGANTEA (GI) transcript levels were 418
consistent among all three lines (Figure 5P, 5CC and 5DD). 419
420
Gas Exchange Characteristics of rPPC1 Lines under Circadian Free Running 421
Conditions 422
423
Under constant light and temperature (LL) free-running conditions, detached K. 424
laxiflora wild type CAM leaves (LP6) with their petioles in water displayed a robust 425
circadian rhythm of CO2 uptake with a period of approximately 20 h (Figure 6; 6A). 426
This rhythm was entirely consistent with CAM rhythms reported previously for 427
detached LP6 of closely related Kalanchoë species with obligate CAM (Lüttge and 428
Ball, 1978; Anderson and Wilkins, 1989). The rhythm dampened rapidly in line 429
rPPC1-B, whereas rPPC1-A maintained a rhythm that was very similar to that of the 430
wild type (Figure 6A). 431
When LL CO2 uptake was measured for well-watered whole plants (2 months 432
old; 9-leaf pairs), rPPC1-B fixed more CO2 than the wild type and line rPPC1-A, with 433
levels of 11,269, 7414, 8383 µmol CO2 m-2, respectively (Figure 6B). Wild type 434
maintained robust oscillations of CO2 exchange under LL conditions, whereas 435
rPPC1-A dampened to arrhythmia after 3-days, and rPPC1-B was arrhythmic (Figure 436
6B). It should be noted that the LL CO2 exchange data for intact young plants with 437
their roots in soil (Figure 6B) differs from the data for detached LP6 (Figure 6A) 438
because the whole plants measured in Figure 6B included leaf pairs from very young 439
LP1, which perform C3, through to LP6 to LP9 that perform full CAM (phase I in the 440
dark and III in the light). The detached LP6 measured in Figure 6A were full CAM 441
leaves only, at least for the wild type (Supplemental Figure 4). Thus, the more rapid 442
15
and substantial dampening of the free-running CO2 fixation rhythm towards 443
arrhythmia in lines rPPC1-A and rPPC-1B relative to the wild type (Figure 6B), 444
suggests that the younger leaves (LP1 to LP5) made a dominant contribution to the 445
robust circadian rhythm in wild-type whole plants. 446
447
Delayed Fluorescence Rhythms are Enhanced in rPPC1-B Despite the Loss of 448
CAM-Associated CO2 Uptake Rhythms 449
450
Delayed fluorescence (DF) displays a robust circadian rhythm in Kalanchoë and 451
provides a measure of a chloroplast-derived clock-output that can be used for 452
statistical analysis of circadian period, robustness and accuracy (Gould et al., 2009; 453
Boxall et al., 2017). We therefore measured and analysed DF under LL (Figure 6C) 454
to calculate rhythm statistics using Biodare (Moore et al., 2014; Zielinski et al., 2014). 455
Wild type DF oscillations were very similar to those reported previously for K. 456
fedtschenkoi (Figure 6C; Gould et al., 2009; Boxall et al., 2017). rPPC1-A and 457
rPPC1-B had more robust oscillations than the wild type (Figure 6C). The rhythm 458
amplitude increased slightly with time in rPPC1-A and rPPC1-B but remained 459
relatively constant in the wild type. The relative amplitude error (RAE) plot showed a 460
wider spread of period for the wild type than for the rPPC1 lines (Figure 6D). Mean 461
periods were between 21.5 h and 22.1 h when calculated using spectral resampling 462
or fast Fourier transform (nonlinear least squares) methods, respectively (Figure 6E). 463
Average periods were similar between wild type and the rPPC1 lines. A lower mean 464
RAE was calculated for rPPC1-A and rPPC1-B compared to the wild type (Figure 465
6F), supporting statistically the visibly robust and high-amplitude DF rhythm in the 466
rPPC1 lines (Figure 6C). 467
468
Rhythm Characteristics of Core Circadian Clock and Clock-Controlled Genes 469
470
Having established that circadian control of CO2 fixation was dampened under LL in 471
plants lacking PPC1, whereas the circadian control of DF was enhanced under LL 472
(Figure 6A to 6F), it was important to investigate the regulation of both core clock 473
genes and circadian clock-controlled genes in the rPPC1 lines under LL (Figure 7). 474
Wild type displayed a rhythm in the transcript abundance of PPC1 that was 475
absent in rPPC1-B (Figure 7A). PPC2 was rhythmic in wild type and rPPC1-B, but its 476
16
abundance was lower in rPPC1-B (Figure 7B). The PPCK1 rhythm was of greater 477
amplitude in rPPC1-B, and the daily transcript peaks occurred 4 to 8 h later than the 478
wild type after the first 24 h of LL (Figure 7C). PPCK2 and PPCK3 were induced in 479
line rPPC1-B and oscillated with higher amplitude (Figure 7D and 7E). PPCK2 480
peaked after the wild type on the second and third 24 h cycles under LL (Figure 7D 481
and 7E), which is consistent with the induction detected under light/dark cycles 482
(Figure 1F and 1G). GPT2, which is involved in the transport of G6P across the 483
chloroplast membrane, was down-regulated and had lower amplitude in rPPC1-B 484
than the wild type (Figure 7F), whereas CAB1 was induced and oscillated robustly 485
(Figure 7G). 486
In rPPC1-B, the core clock gene CCA1-1 was down-regulated, phase 487
delayed, and had lower amplitude than the wild type (Figure 7H), whereas CCA1-2, 488
TOC1-1 and TOC1-2 were all up-regulated and phase delayed relative to the wild 489
type for the latter two peaks of LL (Figure 7I, 7J and 7K). PRR7 was up-regulated 490
and more robustly rhythmic in rPPC1-B (Figure 7L), whereas the rhythms of PRR3/7 491
and PRR9 dampened in rPPC1-B (Figure 7M and 7N). Finally, JMJ30/JMJD5, ELF3, 492
CDF2, RVE1-like, EPR1, FKF1 and GI were up-regulated in rPPC1-B, displaying 493
higher amplitude, phase delays and/or period lengthening during the LL time course 494
(Figure 7P to 7V), whereas LNK3-like was down-regulated but remained rhythmic, 495
with a lengthening period compared to the wild type (Figure 7O). 496
497
Diel Regulation of GC-Signalling and Ion Channel Genes in GC-Enriched 498
Epidermal Peels of rPPC1-B 499
500
As CO2 fixation and associated stomatal opening were shifted to the light period in 501
rPPC1-B, it was important to investigate the temporal regulation of known guard cell 502
signalling genes in wild type and the rPPC1-B line (Figure 8). Since epidermal peels 503
of Kalanchoë leaves are enriched for intact guard cells, we isolated RNA from 504
epidermal peels from the upper and lower leaf surfaces of LP6 every 4 h over the 12-505
h-light/12-h-dark cycle and used them for RT-qPCR. 506
PHOTOTROPIN1 (PHOT1) encodes a protein kinase that acts as a blue light 507
(BL) photoreceptor in the signal-transduction pathway leading to BL-induced 508
stomatal movements (Kinoshita et al., 2001). In rPPC1-B, PHOT1 transcripts were 509
up-regulated relative to the wild type and peaked 8 h into the 12-h-light period 510
17
(Figure 8A). CRYPTOCHROME2 (CRY2) encodes a photoreceptor that regulates BL 511
responses, including the entrainment of endogenous circadian rhythms (Somers et 512
al., 1998), and stomatal conductance via an indirect effect on ABA levels 513
(Boccalandro et al., 2012). In rPPC1-B epidermal peels, CRY2 transcript levels were 514
up-regulated and peaked at dusk, whereas CRY2 peaked 8 h into the dark in the 515
wild type (Figure 8B). 516
In Arabidopsis, GC-localised E-CARBONIC ANHYDRASE1 (CA1) and CA4 517
are involved in CO2 sensing in GCs, with the ca1 ca4 mutant displaying impaired 518
stomatal control in response to CO2 (Hu et al., 2010). In rPPC1-B, a E-carbonic 519
anhydrase gene was much more strongly induced at dawn relative to the wild type 520
(Figure 8C). PATROL1 controls the tethering of the proton ATPase AHA1 to the 521
plasma membrane and is essential for stomatal opening in response to low CO2 and 522
light levels (Hashimoto-Sugimoto et al., 2013). The cycle of PATROL1 in epidermal 523
peels was slightly different between rPPC1-B and wild type, with rPPC1-B peaking 4 524
h into the dark, but the wild type peak at 4 h into the light period (Figure 8D). 525
CONVERGENCE OF BLUE LIGHT AND CO2 1/2 (CBC1/2) stimulate stomatal 526
opening by inhibiting S-type anion channels in response to both BL and low CO2 and 527
connect BL signals perceived by PHOT1 with the protein kinase HIGH LEAF 528
TEMPERATURE1 (HT1) (Hiyama et al., 2017). Both proteins interact with and are 529
phosphorylated by HT1. In rPPC1-B, CBC1/2 was up-regulated compared to the wild 530
type both in the second half of the dark period and at dawn (Figure 8E). HT1 acts as 531
a negative regulator of high CO2 induced stomatal closure (Hashimoto et al., 2006). 532
HT1 transcript levels were reduced throughout the light/dark cycle relative to the wild 533
type in rPPC1-B (Figure 8F). OPEN STOMATA1 (OST1), encoding a protein kinase 534
that acts downstream of HT1 (Imes et al., 2013), was up-regulated in rPPC1-B both 535
at 8h into the dark period and at dawn, the time of the daily trough in the wild type 536
(Figure 8G). 537
Genes involved in ion transport across the plasma membrane and tonoplast 538
play a crucial role in the changes in cell turgor pressure that drive stomatal 539
movements (Jezek and Blatt, 2017). SLOW ACTIVATED ANION CHANNEL1 540
(SLAC1) is a key player in the closure of stomata in response to high CO2 541
concentrations (Hedrich and Geiger, 2017). SLAC1 is regulated by ABA signalling, 542
which requires de-phosphorylation steps catalysed by protein phosphatase type 2C 543
18
(PP2C). In rPPC1-B, SLAC1 was up-regulated and peaked during the light (Figure 544
8H), and PP2C was up-regulated relative to the wild type during the dark period 545
(Figure 8I). ABA is recognized and bound by the REGULATORY COMPONENT OF 546
ABA RECEPTORS (RCARs)/PYRABACTIN RESISTANCE1 (PYR1/PYL), which 547
interact with PP2C to stimulate ABA signalling (Ma et al., 2009; Park et al., 2009; 548
Santiago et al., 2009). In rPPC1-B epidermal peels, RCAR3 transcripts were induced 549
spanning dusk and the first half of the dark period, and peaked at least 4 h earlier 550
than the wild type (Figure 8J). 551
In rPPC1-B, the transcript level of GUARD CELL OUTWARD RECTIFYING 552
K+ CHANNEL (GORK), which is involved in regulating stomatal movement according 553
to water status (Ache et al., 2000), peaked 4 h earlier than wild type (Figure 8K). 554
STELAR K+ OUTSIDE RECTIFIER (SKOR) encodes a selective outward-rectifying 555
potassium channel (Gaymard et al., 1998). SKOR transcript levels peaked at dawn 556
in the wild type, whereas in rPPC1-B, SKOR transcript levels reached their daily 557
minimum at dawn (Figure 8L). The transcript level of plasma-membrane localised 558
ALUMINIUM ACTIVATED MALATE TRANSPORTER12 (ALMT12), which is involved 559
in dark-, CO2-, ABA- and water deficit-induced stomatal closure, was elevated 560
relative to the wild type at dawn in rPPC1-B (Figure 8M). The E3 UBIQUITIN-561
PROTEIN LIGASE RMA1 promotes the ubiquitination and proteasomal degradation 562
of the aquaporin PIP2:1, which plays a role in GC regulation (Grondin et al., 2015). 563
RMA1 was induced 3-fold at dawn in rPPC1-B compared to the wild type (Figure 564
8N). EMBRYO SAC DEVELOPMENT ARREST39 (EDA39) is a calmodulin binding 565
protein that promotes stomatal opening (Zhou et al., 2012). In rPPC1-B, EDA39 was 566
up-regulated relative to the wild type and peaked at dusk (Figure 8O). 567
SALT OVERLY SENSITIVE2 (SOS2) is a Calcineurin B-Like (CBL)-interacting 568
protein kinase involved in the regulatory pathway for intracellular Na+ and K+ 569
homeostasis and salt tolerance (Liu et al., 2000). SOS2 interacts with and activates 570
the vacuolar H+/Ca2+ antiporter CAX1, thereby functioning in cellular Ca2+ 571
homeostasis, an important function during stomatal opening and closing (Cheng et 572
al., 2004). SOS2 transcript levels were elevated relative to the wild type at all time 573
points, and, in particular, it was induced ~5-fold at its peak 4 h into the dark period in 574
epidermal peels of rPPC1-B relative to the wild type (Figure 8P). Ca2+-ATPase2 575
(ACA2) and ENDOMEMBRANE-TYPE Ca2+-ATPASE4 (ECA4) catalyze the 576
hydrolysis of ATP coupled with the translocation of calcium from the cytosol into the 577
19
endoplasmic reticulum and/or an endomembrane compartment (Jezek and Blatt, 578
2017). ACA2 and ECA4 were induced by ~4-fold to ~8-fold in rPPC1-B epidermal 579
peels relative to the wild type, particularly when they reached peak levels at 4 h into 580
the 12-h-dark period (Figure 8Q and 8R). 581
Finally, the transcription factor MYB60 is involved in stomatal opening in 582
response to light and promotes GC deflation in response to water deficit (Cominelli et 583
al., 2005). In rPPC1-B, MYB60 was induced ~5-fold relative to the wild type at dawn, 584
although it also maintained a dusk-phased peak of transcript abundance like the wild 585
type (Figure 8S). MYB61 functions as a transcriptional regulator of stomatal closure 586
(Liang et al., 2005). MYB61 was down-regulated in rPPC1-B relative to wild type and 587
peaked 8 h later in rPPC1-B, 4 h into the dark period, whereas in the wild type, 588
MYB61 peaked in the light 4 h before dusk (Figure 8T). 589
590
Diel Regulation of GC-Metabolism Genes in GC-Enriched Epidermal Peels of 591
rPPC1-B 592
PPC1 and PPC2 transcript levels were low in epidermal peels (Supplemental Figure 593
5A and 5B) compared to whole leaves (Figure 1A). PPC3 and PPC4 transcripts were 594
more abundant in wild type epidermal peels (Supplemental Figure 5C and 5D) 595
relative to whole leaves (Figure 1C and 1D). Furthermore, PPC3 and PPC4 were up-596
regulated in epidermal peels of rPPC1-B compared to the wild type, and their 597
transcript abundance peaked 4 h into the dark period, whereas in the wild type, the 598
abundance of both transcripts peaked at the end of the dark period or dawn 599
(Supplemental Figures 5A to 5D). 600
CAM-specific PPCK1 was up-regulated in rPPC1-B epidermal peels 601
compared to wild type, whereas the expression of PPCK2, which does not currently 602
have a proposed role in the regulation of CAM-specific PPC1 in the mesophyll, was 603
unchanged (Supplemental Figure 5E and 5F). Both PPCK2 and PPCK3 transcripts 604
were more abundant than PPCK1 in epidermal peels, suggesting their encoded 605
proteins might regulate the activity of the GC PPC(s) (Supplemental Figure 5F and 606
5G). Most strikingly, PPCK3 was up-regulated ~5-fold at 4 h after lights-on in rPPC1-607
B epidermal peels relative to the wild type (Supplemental Figure 5G). 608
In C3 plants, starch is degraded before dawn to fuel stomatal opening (Blatt, 609
2016). In Arabidopsis GCs, BAM1 encodes the major starch-degrading enzyme 610
(Valerio et al., 2010; Prasch et al., 2015; Horrer et al., 2016; Santelia and Lunn, 611
20
2017) that functions with AMY3 to mobilize starch at dawn, releasing maltose in the 612
chloroplasts (Horrer et al., 2016). In rPPC1-B epidermal peels, GWD transcript levels 613
were halved relative to wild type at its peak, i.e., 8 h into the 12 h light period 614
(Supplemental Figure 5H). AMY3a and AMY3b were up-regulated at dusk and in the 615
second half of the light period, respectively (Supplemental Figure 5I and 5J). BAM1 616
was up-regulated at dawn compared to wild type, and peaked with a very similar 617
transcript abundance to wild type at 20:00 h, 8 h into the dark period (Supplemental 618
Figure 5K), whereas BAM3 transcript levels were ~3-fold lower in rPPC1-B relative 619
to the wild type at the time of its nocturnal peak, which also occurred 8 h into the 620
dark (Supplemental Figure 5L). BAM9 expression in rPPC1-B rose later than the wild 621
type, lagging behind the wild type at dusk and 4 h into the dark, and it stayed ~5-fold 622
higher at dawn and 4 h into the light period, when the wild type reached its daily 623
trough (Supplemental Figure 5M). BAM9 in Arabidopsis is predicted to be 624
catalytically inactive and has no known function to date (Monroe and Storm, 2018). 625
The peak of PHS1 was delayed by 4 h, peaking at the light/dark transition 626
(Supplemental Figure 5N), and MEX1 was down-regulated in epidermal peels of 627
rPPC1-B, peaking at 08:00 in the light, 4 h after the wild type peak (Supplemental 628
Figure 5O). 629
630
Diel Regulation of Core Circadian Clock genes in Epidermal Peels of rPPC1-B 631
632
The circadian clock genes PRR3/7 and PRR7 were up-regulated in epidermal 633
peels of rPPC1-B relative to wild type (Supplemental Figure 5P and 5Q), with a 634
similar diel pattern to that measured in whole CAM leaves (Figure 5S and 5T). 635
PRR3/7 peaked 4 h earlier in rPPC1-B, whereas PRR7 transcript levels were greater 636
throughout the light period in rPPC1-B (Supplemental Figure 5P and 5Q). LUX is a 637
component of the morning transcriptional feedback circuit within the clock, encoding 638
a transcription factor that directly regulates the expression of PRR9 by binding to 639
specific sites in its promoter (Helfer et al., 2011). In epidermal peels, LUX was 640
downregulated >7-fold in rPPC1-B compared to the wild type at its peak, 4 h into the 641
light period, and also peaked 4 h later than the wild type (Supplemental Figure 5R). 642 643 Diel Regulation of Additional GC-Signalling Genes in GC-Enriched Epidermal 644
Peels of rPPC1-B 645
21
GCs perceive CO2 and regulate stomatal aperture via ABA and reactive oxygen 646
species signaling, with low CO2 levels mediating stomatal opening and high CO2 647
levels causing closure (Chater et al., 2015). NADP OXIDOREDUCTASE encodes an 648
NADP-binding Rossman-fold super-family protein that we only detected in epidermal 649
peels in Kalanchoë. The enzyme encoded by this gene might generate the H2O2 650
burst induced by ABA as part of the stomatal closure signalling pathway 651
(Daszkowska-Golec and Szarejko, 2013). This transcript was up-regulated ~3-fold in 652
rPPC1-B at dawn (Supplemental Figure 5S). 653
In Arabidopsis, the transcription factors MYB94 and MYB96 function together 654
in the activation of cuticular wax biosynthesis under drought stress (Lee et al., 2016). 655
MYB96 may also be involved in the response to drought stress in Arabidopsis 656
through ABA signalling that mediates stomatal closure via the RD22 pathway (Seo et 657
al., 2011). Both MYB94 and MYB96 were induced by as much as 3-fold in rPPC1-B 658
compared to wild type (Supplemental Figure 5T and 5U). Specifically, MYB94 659
transcript levels rose 8 h earlier than they did in the wild type and were already close 660
to peak levels by the light-to-dark transition, whereas in the wild type, MYB94 levels 661
peaked sharply 8 h into the dark period (Supplemental Figure 5T). MYB96 662
transcription was induced relative to the wild type at both dawn and dusk, but the 663
peak of MYB96 transcription at dawn represented the largest fold-change relative to 664
the wild type (Supplemental Figure 5U). 665
666
667
DISCUSSION 668
669
CAM Provides a Selective Advantage in the Face of Drought 670
671
The data presented here for CO2 fixation during progressive drought demonstrate 672
very clearly the importance of a fully functional CAM system for continued 673
atmospheric CO2 fixation throughout a period of drought lasting just over 3-weeks 674
(Figure 4D). Although the intermediate line rPPC1-A continued to perform 675
atmospheric CO2 fixation and malate accumulation in the dark and stomatal closure 676
in the light period, it was less able to adapt to drought by inducing CAM compared to 677
the wild type. Thus, by comparing the impacts of the different levels of PPC1 678
silencing in these transgenic lines of Kalanchoë, it is possible to conclude that the 679
22
wild type level of PPC1 transcript enables the plant to adapt to drought more rapidly 680
by increasing nocturnal CO2 fixation and malate accumulation and by decreasing the 681
magnitude and duration of phases II and IV of CAM in the light. These swift 682
adaptations allow the wild type to curtail excessive water-loss more rapidly than 683
either of the transgenic lines with reduced PPC1 transcript levels, suggesting that the 684
high level of PPC1 transcript in the wild type provides a genuine adaptive advantage 685
in terms of allowing the plant to prevent excessive water loss during gradual soil 686
drying. 687
Upon re-watering on day 23, both wild type and rPPC1-A plants returned to 688
performing strong, four-phase CAM, including pronounced phase II and IV at the 689
start and end of the light period, respectively (Figure 4D). Line rPPC1-B also 690
bounced back to its normal, pre-drought physiology after re-watering, which is 691
consistent with the re-watering response reported previously for a range of 692
facultative, weak-CAM species, including M. crystallinum, purslane (Portulaca 693
oleracea), P. umbraticola, T. triangulare, and various Calandrinia species (Winter 694
and Holtum, 2014; Holtum et al., 2017; Winter, 2019). Overall, the response of 695
rPPC1-B to progressive drought stress and subsequent re-watering is reminiscent of 696
the physiological response of facultative CAM species such as T. triangulare and M. 697
crystallinum, although both of these true facultative CAM species did achieve net 698
atmospheric CO2 fixation in the dark period following approximately 12 to 13 days of 699
drought (Winter and Holtum, 2014). 700
Although the rPPC1-B plants fixed all of their CO2 in the light, especially 701
during a pronounced and extended phase II in the hours after dawn, the CO2 uptake 702
pattern was not constant over the light period (Figure 4A). For example, in LP6 of a 703
well-watered plant, CO2 fixation dropped from ~8 µmol m-2 s-1 during phase II after 704
dawn to ~2 to 3 µmol m-2 s-1 around 4 h after lights on (Figure 4A), and stomatal 705
conductance decreased to a similar extent (Figure 4B). The observed stomatal 706
closure could not have been due to a high malate concentration and associated 707
internal release of CO2 in the mesophyll (Figure 3A). These data are consistent with 708
the current hypothesis that a signal from the core circadian clock drives the closure 709
of CAM stomata in the light and the associated decline in atmospheric CO2 fixation, 710
even when a CAM leaf has not produced malate during the preceding dark period 711
(Von Caemmerer and Griffiths, 2009). 712
23
Overall, with respect to CAM physiology, the data presented here provide 713
strong support for the long-held view that the ability to use CAM, and to use it 714
increasingly in response to drought stress, provides a genuine adaptive advantage in 715
terms of prolonging net atmospheric CO2 fixation during drought progression (Figure 716
4D) (Kluge and Fischer, 1967; Osmond, 1978). The wild type and line rPPC1-A 717
achieved, respectively, net atmospheric CO2 fixation of 18,568 and 19,071 µmoles 718
m-2 over the 22-day drought progression, whereas line rPPC1-B only achieved 719
13,983 µmoles m-2 net atmospheric CO2 fixation (Supplemental Table 4). Thus, the 720
wild type, with its fully functional CAM system, was able to fix 33% more CO2 over 721
the entire drought treatment period than the mutant. 722
723
Insights into the Evolutionary Trajectory from C3 to CAM 724
725
The discovery that the very low level of PPC activity in the rPPC1-B transgenic line 726
was associated with nocturnal refixation of respiratory CO2 revealed that even a 727
small increase in PPC activity in the dark period may be sufficient to support a 728
minimal, but functional, CAM cycle. Thus, during CAM evolution from ancestral C3 729
species, small increases in nocturnal PPC activity in photosynthetic mesophyll cells 730
behind closed stomata may have been sufficient to support the refixation of 731
respiratory CO2 to stored vacuolar malic acid, allowing its subsequent 732
decarboxylation in the light, leading to partial stomatal closure, enhanced WUE, and 733
improved RuBisCO carboxylation efficiency relative to the oxygenase (Edwards, 734
2019). However, despite our data for Kalanchoë supporting this evolutionary 735
perspective on the progression to CAM, initial attempts to increase the expression of 736
core CAM pathway enzymes in the C3 model species Arabidopsis thaliana have not 737
reciprocated the phenotype of even the most strongly silenced rPPC1-B line reported 738
here. Constitutive over-expression of the CAM-specific PPC isogene from M. 739
crystallinum in A. thaliana led to increased stomatal conductance and titratable 740
acidity (Lim et al., 2019). However, the increased stomatal conductance was 741
measured in the middle of the light period, and the timing of the titratable acidity 742
measurements relative to the light/dark cycle was not mentioned (Lim et al., 2019). 743
Therefore, it is difficult to compare these results with the results presented here 744
documenting alterations in nocturnal stomatal opening and atmospheric CO2 fixation 745
and dawn-phased malate accumulation in the rPPC1 transgenic lines of Kalanchoë. 746
24
It should be noted, however, that the K. laxiflora lines with reduced CAM-specific 747
PPC1 transcript levels still possessed succulent leaves with large mesophyll cells, 748
plus wild type levels of key CAM transcripts and enzymes such as PPDK. Thus, the 749
rPPC1 plants were by no means directly comparable to the transgenic A. thaliana 750
lines reported by Lim et al. (2019). 751
752
Impact of PPC1 Silencing on the CAM Circadian Rhythm of CO2 Fixation 753
754
The circadian rhythm of CO2 fixation observed for CAM species of Kalanchoë under 755
constant free-running conditions is a classic example of a plant clock output rhythm 756
driven by the underlying multi-gene-loop, autoregulatory core oscillator mechanism 757
in the nucleus that sets the time (Hartwell, 2006). Under continuous light and 758
temperature (LL) conditions, the CAM-associated circadian rhythm of CO2 fixation 759
dampened towards almost complete arrhythmia in detached leaves and whole, 760
young plants of rPPC1-B, and CO2 was fixed continuously (Figure 6A and 6B). The 761
collapse of the CO2 circadian rhythm suggests that CO2 fixation by RuBisCO in 762
rPPC1-B was not subject to robust and high amplitude circadian control, certainly not 763
in comparison to the rhythm of atmospheric CO2 fixation during CAM in the wild type 764
(Figure 6B). In K. daigremontiana, RuBisCO is thought to make a large contribution 765
to the observed rhythm of CO2 assimilation under LL conditions, as the level of 766
malate does not oscillate (Wyka and Lüttge, 2003). By contrast, online carbon 767
isotope discrimination measurements demonstrated that the LL CO2 rhythm of CAM 768
leaves of M. crystallinum had a RuBisCO carbon isotope discrimination early in each 769
subjective dark period, which then transitioned to a PPC carbon isotope ratio later in 770
the subjective dark (Davies and Griffiths, 2012). In rPPC1-B (with no detectable 771
PPC), the remaining RuBisCO-mediated rhythm displayed, at best, only a very weak 772
rhythm of CO2 assimilation (Figure 6A). This suggests that C3 carboxylation via 773
RuBisCO only makes a small contribution to the LL rhythm in K. laxiflora CAM 774
leaves, or perhaps the robust, high amplitude CO2 rhythm of the wild type depends 775
on interplay between high PPC activity and RuBisCO, which ceases in the absence 776
of PPC1. Overall, the weakly rhythmic to arrhythmic pattern of CO2 assimilation in 777
rPPC1-B under LL was very similar to that of C3 M. crystallinum (Davies and 778
Griffiths, 2012). 779
780
25
Regulation of Core Clock Genes in rPPC1-B 781
782
Previous reports for other CAM species have emphasised that most core circadian 783
clock genes share their pattern and phasing of diel transcript regulation between 784
Arabidopsis and, for example, Agave, pineapple (Ananas comosus), and white 785
stonecrop (Sedum album) (Sharma et al., 2017; Yin et al., 2018; Wai et al., 2019). 786
These reports therefore emphasise that the timing of core clock genes is conserved 787
between widely divergent species, regardless of the underlying mechanism of 788
photosynthetic CO2 fixation, which is consistent with the ancestral role of the core 789
circadian clock in the temporal control and optimisation of many biological processes 790
in plants. However, individual studies have identified specific core clock genes, or 791
genes closely associated with the core clock, that were differentially expressed 792
and/or temporally phased in certain CAM species relative to Arabidopsis. For 793
example, RNA-seq based transcriptome analysis in A. americana identified a gene 794
related to the clock-associated output pathway gene RVE1 (Rawat et al., 2009), 795
which was rescheduled to the dark period in Agave, but peaked phased to dawn in 796
Arabidopsis (Yin et al., 2018). The K. laxiflora RVE1-like gene measured here 797
reached its 24 h transcript peak phased to dawn in LD cycles (Figure 5) and was 798
repressed in rPPC1-B relative to wild type. Under LL, RVE1-like was induced in 799
rPPC1-B relative to wild type, and its transcript abundance oscillated robustly, with a 800
higher amplitude oscillation than the wild type (Figure 7). These findings suggest that 801
the RVE1-like gene measured here does not likely share a role in the circadian 802
coordination of the daily CAM cycle, as has been hypothesised for Agave RVE1. In 803
fact, based on our current data for the rPPC1 lines, it is possible that RVE1-like 804
might function in coupling the core clock to the robust rhythm of DF observed in the 805
rPPC1 transgenic lines (Figure 6C). 806
In pineapple, the core clock genes PRR5 and PRR9 are rhythmic specifically 807
in the green leaf tip tissue that performs CAM (Sharma et al., 2017). By contrast, the 808
PRR7, PRR3/7 and PRR9 transcripts measured in the current study were rhythmic 809
over the LD cycle in wild type, rPPC1-A and rPPC1-B, although the levels of all three 810
PRR transcripts were higher in rPPC1-B relative to the wild type (Figure 5). 811
However, the abundance and rhythmicity of PRR3/7 and PRR9 collapsed in rPPC1-812
B under LL conditions, whereas PRR7 was induced and rhythmic (Figure 7). In S. 813
album, a member of the Crassulaceae along with Kalanchoë, the induction of weak 814
26
CAM-cycling in response to drought stress led to the repression of transcripts related 815
to GI and FKF1 and the induction of transcripts related to CCA1 and TOC1 (Wai et 816
al., 2019). However, the timing of peak transcript abundance for core clock gene 817
orthologs was largely consistent between C3 and CAM-cycling leaf samples of S. 818
album, with phase delays of 2 to 4 h at most for PRR1_A, and PRR5_A, PRR5_B 819
and PRR5_D (Wai et al., 2019). In rPPC1-B under LD cycles, only changes in the 820
transcript peaks of CCA1-2 (down), TOC1-1 (up) and TOC1-2 (up) were detected 821
(Figure 5). The timing of the peaks and troughs of these transcripts remained the 822
same between wild type and both rPPC1 lines. GI and FKF1 transcript oscillations 823
and peak abundance were remarkably similar between wild type and the two rPPC1 824
lines under LD cycles (Figure 5). However, under LL conditions, CCA1-2, TOC1-1, 825
TOC1-2, GI and FKF1 were all induced relative to the wild type and oscillated more 826
robustly, with lengthening periods as the LL time course progressed (Figure 7). 827
Overall, there were no clear and consistent correlations between the transcript 828
abundance cycles of core clock gene transcripts reported here for K. laxiflora wild 829
type and RNAi lines with reduced PPC1 levels and the published RNA-seq data for 830
the LD cycles of transcript abundance of the orthologous genes in Agave, pineapple 831
and S. album. Furthermore, it was not possible to detect any temporal patterns of 832
regulation of core clock gene transcripts between different species or between C3 833
and CAM tissues in individual species, which would suggest that conserved 834
alterations in the core clock have accompanied the evolution of CAM in these 835
diverse CAM lineages. 836
837
Contrasting Core Clock Responses Between rPPC1 and rPPCK1 RNAi Lines 838
839
Similar to the LL CO2 fixation phenotype reported here for rPPC1-B, transgenic K. 840
fedtschenkoi lines lacking PPCK1 also lost the CO2 fixation rhythm, and the 841
transcript oscillations of many core clock genes were altered (Boxall et al., 2017). 842
However, different core clock genes were perturbed in rPPC1-B compared to those 843
whose rhythmic regulation changed in the rPPCK1 lines of K. fedtschenkoi (Boxall et 844
al., 2017 cf. Figure 7). In rPPC1-B, CCA1-1 transcript oscillations dampened, and 845
both TOC1-1 and TOC1-2 transcripts were up-regulated and oscillated with a 846
delayed peak phase relative to the wild type (Figure 7H, 7J and 7K). By contrast, in 847
line rPPCK1-3, the transcription of clock genes CCA1-1, CCA1-2 and TOC1-2 848
27
dampened rapidly towards arrhythmia under LL free-running conditions, whereas 849
TOC1-1 was up-regulated and rhythmic (Boxall et al., 2017). Furthermore, whilst 850
PRR7 was up-regulated and rhythmic in rPPC1-B (Figure 7), the same gene 851
dampened to arrhythmia in rPPCK1-3 after an initial induced peak during the first 6 h 852
of LL (Boxall et al., 2017). The evening phased genes GI and FKF1 were induced 853
and rhythmic under LL conditions in both rPPC1-B and rPPCK1-3, but the level of 854
transcript induction and the rhythm amplitude were much greater in rPPC1-B (Figure 855
7U and 7V), suggesting that these two clock genes are more important for robust 856
oscillations in C3 leaves. 857
Overall, these differences between the rhythms of core clock genes in rPPC1-858
B and rPPCK1-3 revealed that the clock responded very differently to the silencing of 859
two interconnected genes that lie at the heart of the circadian control of nocturnal 860
CO2 fixation. Elucidating the mechanistic basis for these differences should be a 861
fruitful avenue for further investigation, especially in light of the proposed cross-talk 862
between CAM-associated metabolites and regulation within the core clock (Boxall et 863
al., 2017), which was further supported by the clock gene phenotypes reported here 864
(Figure 7). 865
866
Interactions Between Sugars Linked to CAM and the Core Circadian Clock 867
868
In Arabidopsis, sugars associated with primary photosynthetic metabolism play a 869
role in the entrainment of the core circadian oscillator in the nucleus (Haydon et al., 870
2013). In particular, PRR7 is thought to be required for sensing metabolic status and 871
coordinating the clock with photosynthesis (Haydon et al., 2013). In rPPC1-B, the 872
low sucrose levels at 2 h after dawn (Figure 3C) may be sensed via a mechanism 873
involving PRR7 (Boxall et al., 2017). However, in Kalanchoë, a second PRR7-related 874
gene, PRR3/7, produced the more abundant transcript and displayed a transcript 875
peak 2 h before dusk under LD (Figure 5T). In the wild type, PRR3/7 may function as 876
part of a signal transduction pathway that senses metabolic status at dusk when 877
primary CO2 fixation begins (Haydon et al., 2013; Boxall et al., 2017). 878
879
rPPC1 and rPPCK1 Mutants Display Contrasting Levels of Delayed 880
Fluorescence Circadian Rhythms 881
882
28
Delayed fluorescence (DF) circadian rhythms were more robust than the wild type in 883
rPPC1-A and rPPC1-B (Figure 6C). This result represents one of the most striking 884
contrasts to the previous finding of arrhythmic DF in the K. fedtschenkoi rPPCK1-3 885
line (Boxall et al., 2017). This fundamental difference provides important insights into 886
the regulatory signal transduction network underpinning the DF rhythm, which 887
originates from inside the chloroplasts due to the changing redox state of the 888
plastoquinone pool (Gould et al., 2009). In terms of core clock gene regulation under 889
both LD and LL, CCA1-2, TOC1-2, PRR7 and the clock-controlled gene CDF2 were 890
all induced and rhythmic in rPPC1-B. Thus, CCA1-2, TOC1-2, PRR7 and CDF2 are 891
the most likely candidates for driving the induction and robust LL rhythmicity of DF in 892
rPPC1-B. Conversely, the decline in the amount and rhythmicity of these transcripts 893
in rPPCK1-3 may play a role in the dampening of the DF rhythms in the absence of 894
nocturnal phosphorylation of PPC1 (Boxall et al., 2017). 895
These results also allow us to propose that robust rhythmicity of the CAM-896
associated CO2 fixation rhythm in wild type Kalanchoë is most likely driven by an 897
underlying oscillator consisting of CCA1-1, PRR3/7, PRR9, and LNK3-like. This 898
hypothesis is supported by the finding that the rhythmicity of these core clock 899
components responded in the same way in both rPPC1-B and the previously 900
reported rPPCK1-3 transgenic lines of Kalanchoë (Boxall et al., 2017). However, 901
only a limited set of core clock and clock-associated genes has been profiled under 902
LL in rPPC1-B and rPPCK1-3, making it likely that other clock-associated genes are 903
also involved in driving robust CAM CO2 fixation rhythms and robust DF rhythms. 904
905
Perturbation of diel rhythms of gene transcript oscillations in GCs 906
907
A key gap in the current understanding of the molecular genetics underpinning the 908
physiology associated with CAM centres on the cell signalling mechanisms that 909
mediate the inverse pattern, relative to C3, of stomatal opening and closing (Borland 910
et al., 2014; Males and Griffiths, 2017). The GCs of CAM leaves and stems are 911
thought to respond directly to the internal supply of CO2. This theory has recently led 912
several groups to use whole leaf RNA-seq datasets to investigate alterations, relative 913
to C3 leaves, in the temporal phasing of known GC regulatory genes and membrane 914
transporters (Abraham et al., 2016; Wai and VanBuren, 2018; Yin et al., 2018; 915
Heyduk et al., 2019; Moseley et al., 2019). However, these studies did not use 916
29
enriched GCs as the source of RNA, and therefore the data that were mined 917
represented all leaf cell types, including palisade and spongy mesophyll, GCs, 918
subsidiary cells, phloem, phloem companion cells, xylem, bundle sheath, and water 919
storage parenchyma in Agave and pineapple. Thus, the re-scheduling of the 920
temporal patterns of candidate GC genes in these datasets may have been 921
complicated by transcripts from the same genes that were functional in other leaf cell 922
types. 923
Separated epidermal peels from Kalanchoë CAM leaves are enriched for 924
intact GCs. We leveraged this feature in order to compare the temporal pattern of 925
transcript regulation between GC-enriched epidermal peels of the wild type and 926
rPPC1-B (Figure 8). Relative to the wild type, a wide range of genes known to be 927
involved in stomatal opening and closing displayed alterations in transcript 928
abundance and/or the timing of the daily transcript peak (Figure 8A to 8U). These 929
results support the theory that the measured changes in transcript abundance and 930
temporal patterns for a range of guard cell regulatory genes were connected to the 931
measured changes in stomatal opening and closing (Figure 4B). 932
Thus, the up-regulation of HT1, SKOR and MYB61 in the wild type relative to 933
rPPC1-B, and the down-regulation of a wide range of genes in the wild type, 934
including SLAC1, PP2C, SOS2, ACA2, ECA4, and MYB60, are likely important 935
regulatory changes that facilitate nocturnal stomatal opening and light period closure 936
(Figure 4 and 8). As MYB60 is required for stomatal opening in response to light in 937
C3 Arabidopsis (Cominelli et al., 2005), it is particularly noteworthy that its transcript 938
abundance in wild type K. laxiflora GC-enriched epidermal peels peaked at dusk, 939
when stomata open in the wild type (Figure 8LL). Furthermore, in rPPC1-B, MYB60 940
also had a dramatic 3- to 4-fold induction at dawn relative to the wild type, 941
suggesting that high MYB60 transcript levels at the start of the light period may play 942
a key role in the observed light period stomatal opening of the rPPC1-B line (Figure 943
4B). It was also notable that rPPC1-B continued to have a peak of MYB60 transcripts 944
phased to dusk, as observed for the wild type, which is in agreement with the 945
observation that rPPC1-B did open its stomata slightly throughout the dark period 946
(Figure 4B). However, this nocturnal stomatal opening was futile in terms of 947
atmospheric CO2 fixation, as rPPC1-B released respired CO2 from its leaves 948
throughout the dark period (Figure 4A). Overall, MYB60, as well as several other 949
mis-regulated guard cell signalling, ion channel, and metabolite transporter genes 950
30
(Figure 8 and Supplemental Figure 5), represent key targets for future genetic 951
manipulation experiments in transgenic Kalanchoë aimed at understanding the 952
important regulators underpinning the inverse stomatal control associated with CAM. 953
954
Informing Biodesign Strategies for Engineering CAM into C3 crops 955
956
Efforts are underway to engineer CAM and its associated increased WUE into C3 957
species as a means to develop more climate-resilient crop varieties that can 958
continue to fix CO2 and grow in the face of drought, whilst using water more wisely 959
than C3 varieties (Borland et al., 2014; Borland et al., 2015; Lim et al., 2019). The 960
data presented here for the rPPC1 loss-of-function lines of K. laxiflora confirm 961
experimentally the proposed core role of PPC1 for efficient and optimised CAM. Our 962
results also provide encouragement that the level of over-expression of a CAM-963
recruited PPC1 gene introduced into an engineered C3 species may not need to be 964
as high as the level found in extant obligate CAM species, because reducing PPC1 965
levels to only 43% of wild type activity in line rPPC1-A led to plants that were still 966
capable of full CAM and fixed more CO2 than the wild type over 3 weeks of drought 967
(Figure 4D). 968
The results presented here also emphasise that only certain sub-components 969
of the core circadian clock are essential for the temporal optimisation of CAM in 970
Kalanchoë, which in turn further simplifies the challenge of achieving correct 971
temporal control of an engineered CAM pathway introduced into a C3 species. It is 972
clear from our current RT-qPCR data that the transgenic manipulation of the 973
expression and regulation of CCA1-1, PRR3/7, PRR9 and LNK3-like in Kalanchoë 974
using RNAi, over-expression and/or CRISPR-Cas mediated gene editing, will allow 975
the further refinement of the evolving model for the subset of core clock genes that 976
form the transcription-translation feedback loop that underpins the temporal 977
optimisation of CAM. However, in addition, transcriptome-wide analysis of the light/ 978
dark regulation of all detectable transcripts in LP6 of rPPC1-B compared to the wild 979
type will allow the identification of other candidate core clock genes that were not 980
profiled in the targeted RT-qPCR analysis presented here. 981
982 METHODS 983 984
31
Plant Materials 985
986
Kalanchoë laxiflora plants were propagated clonally from adventitious plantlets in the 987
leaf margin using the same clonal stock originally obtained from the Royal Botanic 988
Gardens (RBG), Kew in 2008: accession number 1982-6028, which was kindly 989
provided by the RBG-Kew Living Collection but mis-identified under the name K. 990
fedtschenkoi. Plants were initially grown in a heated transgenic greenhouse with 991
supplementary lighting (Gavita reflector lamps HPS-R Agro 600 W S-281) providing 992
year round 16-h-light/ 8-h-dark, minimum light intensity at plant height of 300–400 993
µmoles photons m-2 s-1, and a minimum temperature of 20˚C. Prior to all experiments, 994
clonal populations of developmentally synchronised plants raised under greenhouse 995
conditions were entrained in a Snijders Microclima MC-1000 growth cabinet for 7-days 996
under 12-h-light/ 12-h-dark and/ or subsequently released into constant light and 997
temperature conditions according to Boxall et al. (2017). 998
999
Time Course Experiments 1000
1001
For both light/dark and LL time course experiments, opposite pairs of leaf pair 6 (LP6) 1002
were collected every 4 h over a 12-h-light/12-h-dark cycle (12:12 LD), starting 2 h 1003
(02:00) after the lights came on at 00:00 h. Plants were entrained for 7-days in a 1004
Snijders Microclima MC-1000 growth cabinet with fluorescent lamps (20 Phillips TL5-1005
HO and 3 Phillips TLD-90 providing a mixed spectrum from 400 – 650 nm) set to 12-h-1006
light (450 µmoles photons m-2 s-1), 25˚C, 60% humidity/12-h-dark, 15˚C, 70% 1007
humidity. For constant light, constant temperature, constant humidity (LL) free-running 1008
circadian time course experiments, plants were entrained under 12-h-light/ 12-h-dark 1009
(LD) conditions as above and switched to LL after a dark period. For LL, the constant 1010
conditions were: light 100 µmoles photons m-2 s-1, temperature 15˚C and humidity 1011
70%. For both LD and LL experiments, LP6 were sampled every 4 h from three 1012
individual (clonal) plants, starting at 02:00 (2 h after lights on). All leaf samples were 1013
immediately frozen in liquid nitrogen and stored at -80°C until use. 1014
1015
Generation of Transgenic K. laxiflora Lines 1016
1017
32
An intron-containing hairpin RNAi construct was designed to target the silencing of 1018
both copies of the CAM-associated PPC1 gene in the tetraploid K. laxiflora genome 1019
(JGI Phytozome accession numbers: Kalax.0018s0056.1 and Kalax.0021s0061.1, 1020
which are the K. laxiflora orthologues of the previously characterised K. fedtschenkoi 1021
CAM-associated PPC1, GenBank accession: KM078709). A 323 bp fragment was 1022
amplified from CAM leaf cDNA using high fidelity PCR with KOD Hot Start DNA 1023
Polymerase (Merck, Germany). The amplified fragment spanned the 3' end of the 1024
PPC1 coding sequence and extended into the 3' untranslated region to ensure 1025
specificity of the silencing to both of the aforementioned CAM-associated PPC1 1026
gene copies. Alignment of the 323 bp region with the homologous regions from the 1027
three other plant-type PPC genes, and their homeologs, in the K. laxiflora genome 1028
demonstrated that none of the other PPC genes shared any 21 nucleotide stretches 1029
that were an exact match for the 323 bp PPC1 RNAi fragment. Thus, the RNAi 1030
construct in the hairpin RNA binary vector used to generate the stable transgenic 1031
lines was predicted to silence only the two homeologous copies of the CAM-1032
associated PPC1, and to be equally specific and efficient at silencing both copies. 1033
This specificity and equality of silencing efficiency was confirmed by the RT-qPCR 1034
data in Figure 1, as the qPCR primers targeted both copies of the CAM-associated 1035
PPC1 (Supplemental Figure 1), and so the quantitative signal is representative of the 1036
averaged signal for the transcript abundance of the two gene copies. 1037
The primers used for the amplification of the PPC1 gene fragment cloned to 1038
generate the hairpin RNAi binary construct were: PPC1 RNAi F 5’ 1039
CACCAAGCTACCAAGTGCCGGTG 3’, and PPC1 RNAi R 5’ 1040
CCCTCCTGCTGCTGCTGCTGC 3’. The PCR product was cloned into the pENTR/D 1041
Gateway-compatible entry vector, as described previously (Dever et al., 2015). 1042
Following confirmation of the correct sequence and orientation of the PPC1 fragment 1043
in the ENTRY vector using Sanger sequencing, the pENTR/D PPC1 RNAi clone was 1044
recombined into intron-containing hairpin RNAi binary vector pK7GWIWG2(II) 1045
(Karimi et al., 2002) using LR Clonase II enzyme mix (Life Technologies). 1046
Agrobacterium-mediated stable transformation of K. laxiflora was achieved using a 1047
method described previously for the very closely related species K. fedtschenkoi 1048
(Dever et al., 2015), with the following changes. For K. laxiflora transformation, the 1049
initial sterile leaf explants used for dipping in Agrobacterium tumefaciens GV3101 1050
carrying the engineered binary vector were generated by germinating surface-1051
33
sterilised K. laxiflora seeds on Murashige and Skoog medium with Gamborgs B-5 1052
vitamins (Murashige and Skoog, 1962; Gamborg et al., 1968) and 3% sucrose. 4- to 1053
6-week-old seedlings grown by tissue culture were chopped into explants for 1054
transformation in a sterile laminar flow bench using a sterile scalpel. The explants 1055
were dipped in the Agrobacterium suspension carrying the PPC1 RNAi binary vector, 1056
and co-cultivated and regenerated as described previously (Dever et al., 2015). 1057
1058
High-Throughput Leaf Acidity and Starch Content Screens 1059
1060
Leaf acidity (as a proxy for leaf malate content) and leaf starch content were 1061
screened in stained leaf discs using chlorophenol red and iodine solution at both 1062
dawn and dusk as described by Cushman et al. (2008). For each transgenic line, leaf 1063
discs were sampled from LP6 in triplicate at 1 hour before dawn and 1 hour before 1064
dusk and stained in a 96 well plate format. 1065
1066
Net CO2 Exchange Measurement Using Whole Young Plants 1067
1068
Whole plant gas exchange measurements were collected over a period of 27-days 1069
using 2-month-old plants at the 9-leaf-pairs stage (results shown in Figure 4D). Four 1070
individual clones per line were potted in 150 ml beakers of compost mix, which was 1071
initially watered to field capacity, and the beakers were sealed with Parafilm around 1072
the rim of the beaker and the base of the stem. Beakers with potted plants were 1073
lowered into the lower half of each of the 12 gas exchange cuvettes. Lids were sealed 1074
over each plant using bulldog clips to create a gas tight seal due to compression of 1075
foam rubber that lined the joint between the lower and upper half of each gas 1076
exchange cuvette. During the 27-day time period, the progression into drought of 1077
young, initially well-watered, wild type plants was documented as they transitioned 1078
from displaying all four phases of CAM with a relatively low proportion of dark period 1079
CO2 fixation in phase I on day 1, through to full CAM (only phase I dark CO2 fixation 1080
and phase III CO2 refixation behind closed stomata throughout the light) from day 12, 1081
followed by recovery of all 4 phases of CAM after re-watering on day 22. 1082
Measurements were made using a custom-built, 12-channel IRGA system (PP 1083
Systems, Hitchin, UK), which allowed for individual environmental control (CO2/H2O) 1084
and measurement of the rates of CO2 uptake for each of 12 gas exchange cuvettes, 1085
34
with measurements collected every ~18 min. The system was described in full by 1086
Dever et al. (2015), but was expanded here with the addition of 6 gas exchange 1087
cuvettes, thereby doubling the scope for replication and throughput. The experiment 1088
was performed using four individual clonal young plants (2 months old, 9-leaf-pairs) 1089
per line: wild type, rPPC1-A and rPPC1-B. The four-fold replicated wild type and 1090
rPPC1 lines were compared in neighbouring gas exchange cuvettes during each 1091
experimental run, such that the data are directly comparable between each line. As 1092
the entire gas exchange system was housed in a Snijders Microclima MC-1000 1093
growth cabinet, all 12 gas exchange cuvettes were under identical conditions in terms 1094
of light intensity and temperature. 1095
1096
Measurement of Circadian Rhythms of Net CO2 Exchange Under Constant Light 1097
and Temperature Free-Running Conditions 1098
Various Kalanchoë species display a persistent and robust circadian rhythm of CO2 1099
exchange when detached CAM leaves are measured with their petiole in distilled 1100
water (Wilkins, 1959). This discovery led to the current understanding that the 1101
endogenous circadian clock underpins the optimised temporal control of CAM in 1102
Kalanchoë (Hartwell, 2006; Boxall et al., 2017). The circadian rhythm of CO2 1103
exchange was measured for wild type, rPPC1-A and rPPC1-B using both detached 1104
LP6 sampled from 4-month-old mature plants and intact young plants at the 9-leaf-1105
pairs stage (2 months old). The 12-cuvette gas-switching CIRAS-DC based gas 1106
exchange system (Dever et al., 2015) was used to measure both the detached LP6 1107
and the intact young plants with their roots in compost mix. For detached LP6, leaves 1108
were detached from each line at the base of their petiole using a scalpel, and the cut 1109
petiole was immediately immersed in distilled water by pushing the petiole through a 1110
small hole cut in Parafilm covering a 150 ml beaker of distilled water. The gas 1111
exchange cuvettes were designed to hold a 150 ml beaker in the lower half of the 1112
cuvette such that the leaves were enclosed in the clear, upside-down 1 l beaker that 1113
formed the upper section of the cuvette in which gas exchange was measured. After 1114
the upper section of each cuvette was placed over the leaves or intact young plants, 1115
the upper and lower sections of the cuvettes were sealed using bulldog clips to clamp 1116
the join, which was coated in foam-rubber sealing material to generate a gas tight 1117
seal. To measure intact young plants, individual clones of each line at the 9-leaf pairs 1118
stage were potted in compost mix in 150 ml pots that fit into the base of the gas 1119
35
exchange cuvettes, and Parafilm was used to seal around the upper rim of the beaker 1120
and the central stem of the young plant to prevent soil and root respiration from 1121
interfering with the gas exchange measured for the above ground leaf pairs (LP1 to 1122
LP9). Plants were pre-entrained to 12-h-light/ 12-h-dark cycles in a Snijders 1123
Microclima MC-1000 growth cabinet for 7-days as described above prior to release 1124
into LL free-running conditions at the end of the last dark period. For LL, the constant 1125
conditions were: light 100 µmoles photons m-2 s-1, temperature 15˚C and humidity 1126
70%. The entire gas exchange system was housed inside a Snijders Microclima MC-1127
1000 growth cabinet, so all 12 cuvettes were under identical conditions. 1128
1129
Measuring Net CO2 Exchange in Attached Leaf Pair 6 Using the LI-COR 6400XT 1130
System 1131
1132
The gas exchange of mature CAM leaves (LP6) was measured over a 12-h-light, 1133
25˚C, 60% humidity: 12-h-dark, 15˚C, 70% humidity cycle using an infra-red gas 1134
analyser (LI-6400XT, LI-COR, Inc.) attached to a large CO2 gas cylinder. Data were 1135
logged every 10 minutes using an auto-program that tracked the light and temperature 1136
regimes of the growth cabinet. 1137
1138
Measuring Leaf Malate, Starch and Sucrose Contents 1139
1140
LP6 (full CAM in wild type) from mature plants were sampled into liquid nitrogen every 1141
4 hours and at dawn and dusk and stored at -80˚C until use. Biological triplicates were 1142
sampled, where each replicate was a pair of leaves of the designated developmental 1143
age from a separate, developmentally synchronised, clonal plant of each line. The 1144
frozen leaf samples were prepared and assayed for malate and starch as described 1145
by Dever et al. (2015) using the published methods for assaying malate in an enzyme-1146
linked spectrophotometric assay (Möllering, 1985) and starch (Smith and Zeeman, 1147
2006). Sucrose, glucose, and fructose were assayed according to the manufacturer’s 1148
protocol (Megazyme Technologies). 1149
1150
Chlorophyll Assays 1151
1152
36
Chlorophyll was assayed from mature greenhouse-grown plants from leaf pairs 2-7. 1153
Biological triplicates were sampled, where each replicate was a pair of leaves of the 1154
designated developmental age from a separate, developmentally synchronised, 1155
clonal plant of each line. Chlorophyll was extracted twice from 0.5 cm diameter leaf 1156
discs in 2 ml 80% (v/v) acetone and homogenised in a bead beater (PowerLyzer 24; 1157
Mo-Bio, Inc). The tubes were centrifuged at full speed in a bench top microfuge at 1158
4˚C for 2 min, and the supernatants were combined and transferred to a new tube 1159
and protected from the light. Absorbance was read at 663 nm and 645 nm, and 1160
chlorophyll contents were calculated according to the published method (Arnon, 1161
1949). 1162
1163
Total RNA Isolation and RT-qPCR 1164
Leaf samples (LP6) for RNA isolation were collected from wild type, rPPC1-A and 1165
rPPC1-B plants every 4 h starting at 2 h after dawn for both 12-h-light/ 12-h-dark 1166
cycles, and continuous light (LL) experiments. Biological triplicates were sampled, 1167
where each replicate was a pair of leaves of the designated developmental age from 1168
a separate, developmentally synchronised, clonal plant of each line. Total RNA was 1169
isolated from 100 mg of frozen, ground leaf tissue using a Qiagen RNeasy kit 1170
(Qiagen, Germany) following the manufacturer’s protocol with the addition of 13.5 µL 1171
50 mg mL-1 PEG 20000 to the 450 µL RLC buffer used for each extraction. cDNA 1172
was synthesised from the total RNA using a Qiagen Quantitect RT kit according to 1173
the manufacturer’s instructions (Qiagen, Germany). The resulting cDNA was diluted 1174
1:4 with molecular biology grade water prior to use in RT-qPCR. Transcript levels 1175
were determined using a SensiFAST SYBR No Rox kit (Bioline) in an Agilent 1176
MX3005P qPCR System Cycler. The results for each target gene transcript of 1177
interest were normalized to the reference gene THIOESTERASE/THIOL ESTER 1178
DEHYDRASE-ISOMERASE SUPERFAMILY PROTEIN (TED; Kalax.0134s0055.1 1179
and Kalax.1110s0007.1; Arabidopsis orthologue AT2G30720.1). Gene expression in 1180
a pool of RNA generated from LP6 samples collected every 4 hours over a 12-h-1181
light/12-h-dark cycle was set to 1. Primers used for RT-qPCR analyses are listed in 1182
Supplemental Data Set 2 and were designed to target both homeologs of each gene 1183
present in the tetraploid genome. It should be noted that primers that were specific to 1184
both homeologs of CCA1-1 did not target the two CCA1-2 homeologs in the K. 1185
37
laxiflora genome. Likewise, the primers that we used to quantify transcripts from both 1186
homeologs of TOC1-1 did not target the two TOC1-2 homeologs. 1187
1188
Immunoblotting 1189
1190
Total protein extracts of K. laxiflora leaves were prepared according to Dever et al. 1191
(2015). One-dimensional SDS-PAGE and immunoblotting of leaf proteins was carried 1192
out following standard methods. Blots were developed using the ECL system (GE 1193
Healthcare, UK). Immunoblot analysis was carried out using antisera to PPC raised 1194
against purified CAM leaf PPC from K. fedtschenkoi (diluted 1:5000), kindly supplied 1195
by Prof. Hugh G. Nimmo, University of Glasgow (Nimmo et al., 1986), and the 1196
phosphorylated form of PPC (diluted 1:1000) raised against a phospho-PPC peptide 1197
from wheat, and kindly supplied by Prof. Cristina Echevarría, Universidad de Sevilla, 1198
Spain (González et al., 2002; Feria et al., 2008). The PPDK antibody (diluted 1:1000) 1199
was raised against maize (Zea mays) C4 PPDK, and the phospho-PPDK antibody 1200
(diluted 1:3000) was raised against a synthetic phospho-peptide spanning the PPDK 1201
phosphorylation site in the Z. mays C4 PPDK. Both PPDK antibodies were kindly 1202
provided by Prof. Chris J. Chastain, University of Minnesota, Moorhead, USA 1203
(Chastain et al., 2000; Chastain et al., 2002). The specificity of the antibodies used 1204
and, in the case of the phospho-peptide specific antibodies for PPC and PPDK, their 1205
target phospho-site amino acid sequences are described in the Supplemental 1206
Methods. 1207
1208
Growth Measurements 1209
1210
Mature plants of wild type and the two rPPC1 lines were grown from developmentally 1211
synchronized clonal leaf plantlets in greenhouse conditions for 4 months. At the start 1212
of the drought treatment, all the plants were watered to full capacity. Water was 1213
withheld from at least six replicate plants of each line for 28-days, and at least six 1214
plants were maintained well-watered over the same period. The plants were 1215
harvested as separated above-ground (shoot) and below-ground (root) tissues, 1216
weighed to determine fresh weight, and dried in an oven at 60˚C until they reached a 1217
constant dry weight. 1218
1219
38
PPC Assays 1220
1221
LP6 samples were taken at 6 h into the light period and frozen in liquid nitrogen. 1222
Biological triplicates were sampled, where each replicate was LP6 from a separate, 1223
developmentally synchronised, clonal plant of each line. Frozen leaf tissue was 1224
ground in liquid nitrogen with a small quantity of acid washed sand and the relevant 1225
enzyme specific extraction buffer (approximately 1 g tissue to 3 ml of extraction 1226
buffer). Extracts were prepared and PPC assays were performed according to the 1227
extraction, desalting and assay buffer conditions described previously (Dever et al., 1228
2015). 1229
1230
Determination of the Apparent Ki of PPC for L-Malate in Rapidly Desalted Leaf 1231
Extracts 1232
1233
The apparent Ki of PPC for L-malate was determined using leaf extracts that were 1234
rapidly desalted as described by Carter et al. (1991). LP6 were collected at 10:00 (2 1235
h before the end of the 12-h-light period) and 18:00 (middle of the 12-h-dark period) 1236
from three biological replicates of wild type, rPPC1-A and rPPC1-B. Each biological 1237
replicate was LP6 from a separate, developmentally synchronised, clonal plant of 1238
each line. The apparent Ki of PPC activity for feedback inhibition by L-malate was 1239
determined as described by Nimmo et al. (1984), with the modifications to the range 1240
of L-malate concentrations added to the assays as described by Boxall et al. (2017). 1241
1242
PPDK Assays 1243
1244
PPDK assays were performed using the extraction and assay buffers described 1245
previously (Kondo et al., 2000; Dever et al., 2015), with the addition of NADH and 1246
G6P (Salahas et al., 1990), and Cibercron Blue (Burnell and Hatch, 1986). Biological 1247
triplicates were sampled, where each replicate was LP6 from a separate, 1248
developmentally synchronised, clonal plant of each line. Briefly, 0.3 g of powdered 1249
leaf tissue that had previously been ground to a fine powder in liquid nitrogen was 1250
extracted in 1 ml of ice-cold extraction buffer (containing 100 mM Tris pH 8.0, 10 mM 1251
DTT, 1 mM EDTA, 1% Triton, 2.5% w/v PVPP, 2% PEG-20000, 10 mM MgCl2, 1 mM 1252
PMSF, 2 µM orthovanadate, and 10 µM Cibercron Blue) by grinding with a small 1253
39
quantity of acid washed sand in a pestle and mortar. Extracts were vortexed for 30 s 1254
and the pH was adjusted to pH 8.0. Extracts were then placed on ice for 10 min 1255
before spinning them at full speed in a benchtop microfuge at 4˚C. The supernatant 1256
(500 µl) was desalted using PD minitrap Sephadex-G25 columns (GE Healthcare). 1257
The desalting buffer contained: 100 mM Tris-HCl pH 8.0, 10 mM MgCl2, 10 mM DTT, 1258
0.1 mM EDTA, 10 µM Cibercron Blue. The desalted extracts were assayed in a plate 1259
reader at 340 nm using the following assay buffer: 100 mM Tris pH 8.0, 5 mM DTT, 1260
10 mM MgCl2, 1.25 mM Pyruvic acid, 2.5 mM NaHCO3, 2.5 mM K2HPO4, 0.25 mM 1261
NADH, 2 U Malate dehydrogenase, and 6 mM glucose 6-phosphate. The reactions 1262
were started by the addition of 0.2 U of phosphoenolpyruvate carboxylase followed 1263
by 1.25 mM ATP (pH 8.0). 1264
1265
Delayed Fluorescence (DF) Measurements 1266
1267
The imaging system for DF was identical to the luciferase and delayed fluorescence 1268
imaging system described previously (Gould et al., 2009) with the exception of the 1269
CCD camera (Retiga LUMO™ CCD Camera; Qimaging). DF was quantified using 1270
Imaris image analysis software (Bitplane) to measure mean intensity for specific 1271
regions within each image. Background intensities were calculated for each image 1272
and subtracted to calculate a final DF value for each image (Gould et al., 2009). 1273
1274
DF Rhythm Analysis 1275
1276
K. laxiflora plants were grown in greenhouse conditions for 4 months and then 1277
entrained in 12-h light (450 µmoles m-2 s-1), 25°C, 60% humidity/ 12-h dark, 15°C, 1278
70% humidity cycles in a Snijders Microclima MC-1000 (Snijders Scientific) growth 1279
cabinet for 7-days as described previously (Dever et al., 2015). At dawn on the 8th 1280
day, 1.5-cm leaf discs were punched from each LP6 for three biological replicates 1281
(i.e., 7 leaf discs from each biological replicate, totalling 21 leaf discs per line) and 1282
placed on 0.3% Phytoagar (Duchefa Biochemie) on a 10-cm square Petri dish. The 1283
Petri dish was left under 12-h light/25˚C: 12-h dark/15°C for a further 24 h to 1284
synchronise the leaf discs. At subjective dawn, the Petri dish was placed in the 1285
imaging system at 14°C in constant red-blue light (LL). DF images were collected 1286
every hour for 120 h as described previously (Gould et al., 2009; Boxall et al., 2017). 1287
40
The DF images were processed as described by Gould et al. (2009). The 1288
luminescence was normalized by subtracting the Y value of the best straight line 1289
from the raw Y value. Biodare was used to carry out fast Fourier transform (nonlinear 1290
least square) analysis and spectral resampling on each DF time-course series using 1291
the time window from 24 to 120 h in order to generate period estimates and calculate 1292
the associated relative amplitude error (RAE) (Moore et al., 2014; Zielinski et al., 1293
2014). 1294
1295
Accession Numbers 1296
1297
Sequence data associated with this article are available via the JGI Phytozome 1298
portal for the K. laxiflora genome: https://phytozome.jgi.doe.gov Kalanchoë1299
laxiflora v1.1. The specific gene IDs for each gene measured in this work are 1300
provided in Supplemental Data Set 2. 1301
1302
Supplemental Data 1303
Supplemental Figure 1. Loading control for immunoblot analyses and PPC malate 1304
sensitivity assays. 1305
Supplemental Figure 2. Impact of loss of PPC activity on vegetative yield during 1306
growth under well-watered and drought-stressed conditions.1307
Supplemental Figure 3. Photographs demonstrating the visual appearance of small 1308
plants after 22 d drought prior to re-watering. 1309
Supplemental Figure 4. Impact of leaf age on the development of the characteristic 1310
24 h light/ dark pattern of CAM-associated CO2 exchange. 1311
Supplemental Figure 5. Impact of the loss of PPC1 on the light/dark regulation of 1312
the transcript abundance of CAM-, starch-, and circadian clock-associated genes in 1313
the CAM leaf epidermis. 1314
Supplemental Table 1. Student’s t-test analysis of PPC activity. 1315
Supplemental Table 2. Student’s t-test analysis of PPDK activity.1316
Supplemental Table 3. Student’s t-tests for chlorophyll a and b. 1317
Supplemental Table 4. Calculations for the cumulative total CO2 fixation (area 1318
under the curve) by each line during the 27-day drought-stress and re-watering 1319
experiment.1320
Supplemental Methods. 1321
41
Supplemental Data Set. 1. Student's t-test results for relative growth measurements 1322
between wild type (WT) and rPPC1-B. 1323
Supplemental Data Set 2. Primers used for RT-qPCR. 1324
1325
ACKNOWLEDGEMENTS1326
We thank Prof. Hugh Nimmo (University of Glasgow, UK), Prof. Cristina Echevarria 1327
(Universidad de Sevilla, Spain) and Prof. Chris Chastain (Minnesota State University, 1328
Moorhead, USA) for providing the antibodies used in this study. This work was 1329
supported by the U.S. Department of Energy (DOE) Office of Science, Genomic 1330
Science Program under Award Number DE-SC0008834, and in part by the 1331
Biotechnology and Biological Sciences Research Council, U.K. (BBSRC grant no. 1332
BB/F009313/1 awarded to J.H.). The contents of this article are solely the 1333
responsibility of the authors and do not necessarily represent the official views of the 1334
DOE. 1335
1336
AUTHOR CONTRIBUTIONS 1337
J.H. and S.F.B. designed the research. S.F.B. performed all of the experiments except1338
some of the tissue culture work for the regeneration of the transgenic lines, which was 1339
performed by N.K., and the statistical analysis of the delayed fluorescence data, which 1340
was performed by P.J.D.G. J.K. generated the RNAi binary construct for PPC1. L.V.D. 1341
performed the PPDK assays. J.L.W. grew and maintained the plants and helped with 1342
grinding and processing of leaf samples. P.J.D.G. helped S.F.B. with the delayed 1343
fluorescence experiments and the analysis and interpretation of the associated data. 1344
S.F.B. and J.H. wrote the manuscript.1345
1346
1347
Table 1. Analysis of CO2 uptake in wild type (WT), rPPC1-A and rPPC1-B during 1348 22-d of progressive drought-stress and for 4-days following re-watering.1349
1350 Night 1
Day 1
Total diel 1
Night 7
Day 7
Total diel 7
Night 13
Day 13
Total diel 13
Night 22
Day 22
Total diel 22
Night 27
Day 27
Total diel 27
WT (% of diel CO2 uptake)
7 93 100 52 48 100 111 -11 100 133 -33 100 56 44 100
WT CO2 uptake (µmol m-2)
78 1015 1093 698 647 1345 908 -113
795 152 -76 76 931 735 1666
rPPC1-A (% of diel CO2 uptake)
6 94 100 40 60 100 112 -12 100 128 -28 100 46 54 100
42
rPPC1-A CO2 uptake (µmol m-2)
73 1081 1008 553 814 1367 922 120 802 209 -81 130 768 889 1657
rPPC1-B (% of diel CO2 uptake)
-26 126 100 -14 114 100 -6 106 100 -27 -73 100 -17 117 100
rPPC1-B CO2 uptake (µmol m-2)
-575 1653 1078 -254 1759 1506 -17 161 145 -8 -22 -30 -385 1825 1440
Table Footnote: Data are presented for selected days (1, 7, 13, 22 and 27) throughout the 1351 27-day drought-stress and re-watering experiment. Results expressed as percentages were1352 calculated separately for the 12-h-light and 12-h-dark periods by determining the percentage 1353 of the total diel/ 24 h CO2 uptake that occurred in that period. The actual CO2 uptake over 1354 the 12-h-light and 12-h-dark period is also presented for each line. 1355
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Figure 1
Figure 1. Confirmation of Target Gene Silencing in Transgenic K. laxiflora RNAi Lines rPPC1-A and rPPC1-B. Gene transcript abundance in leaf pair 6 (LP6) was measured using RT-qPCR for target genes: (A) PPC1; (B) PPC2; (C) PPC3; (D) PPC4; (E) PPCK1; (F) PPCK2; and (G) PPCK3. LP6 were sampled every 4 h across the 12-h-light/12-h-dark cycle. A THIOESTERASE/THIOL ESTER DEHYDRASE-ISOMERASE superfamily gene (TEDI) was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represents the mean of 3 technical replicates for biological triplicates and were normalized to loading control gene (TEDI); error bars represent the standard error. 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 rPPC1-A, and red data rPPC1-B.
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Figure 2
Figure 2. Loss of PPC1 Transcripts Leads to Loss of PPC Protein and Activity and Failure to Activate PPDK in the Light. Total leaf protein from leaf pair 6 (LP6) was isolated from leaves sampled at dawn and dusk plus every 4 h, starting at 2 h into the light period, across the 12-h-light/12-h-dark cycle, separated using SDS-PAGE and used for immunoblot analyses withantibodies raised to PPC (A), and phospho-PPC (B). (C) PPC activity was measuredin rapidly desalted extracts from LP6, ** = rPPC1-A p= 0.0054, *** = rPPC1-B,p=0.00016. (D) Anti-PPDK immunoblot. (E) Anti-phospho-PPDK immunoblot. (F)PPDK activity was measured in leaf extracts prepared from both dark leaves andilluminated leaves, ** = rPPC1-B, p=0.003. The activities of PPC, measured 6 h intothe light period, and PPDK, measured at both 6h into the dark, and 6h into the lightperiods, were the mean of 3 technical replicates of 3 biological replicates and theerror bars represent the standard error. The white bar below each panel representsthe 12-h-light period and the black bar below each panel represents the 12-h-darkperiod. Black data are for the wild type, blue data rPPC1-A, and red data rPPC1-B.Asterisks indicate significant difference from the wild type based on the students ttest:*. All Student‘s t-test parameters are presented in Supplemental Table 1 and 2.
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Figure 3. Impact of silencing PPC1 on malate, starch, soluble sugars and growth.(A) The contents of malate, (B) starch, (C) sucrose, (D) glucose and (E) fructose were determined from leaf pair 6 (LP6) samples collected every 4 h using plants entrained under 12-h-light/12-h-dark cycles. Methanol extracts were prepared from the leaves from wild type, rPPC1-A and rPPC1-B and used for malate and soluble sugar determination, whereas starch was measured in the insoluble pellet. (F) Dry weight of 148-day-old greenhouse-grown plants, either well-watered (w) or drought-stressed for the last 28-days (d). * denotes significant difference between wild type and rPPC1-B based on the Student’s t-test. Well-watered, rPPC1-B * p = 0.017; drought-stressed rPPC1-B * p = 0.011, n = 6 to 9, developmentally synchronised, clonal plants per line. (G), 4-month-old plants raised in a greenhouse under 16-h-light/ 8-h-dark. In all graphs, black data are for the wild type, blue data rPPC1-A, and red data rPPC1-B. Error bars represent the standard error. Asterisks indicate significant difference from the wild type:*. See Supplemental Data Set 1 for full parameters from the Student’s t-test results for the relative growth data.
Figure 4. Impact of silencing PPC1 on 24 h light/ dark gas exchange profiles under well-watered and drought-stressed conditions, and on chlorophyll content. (A), CO2 uptake profile showing the 4 phases of CAM, (B), stomatal conductance (gs) profile and (C), the calculated internal partial pressure of CO2 inside the leaf (Ci) profile for CAM leaves (leaf pair 6; LP6) using plants pre-entrained for 7-days under 12-h-light/ 12-h-dark cycles. (D), Impact of drought on K. laxiflora wild type and transgenic lines with reduced PPC1. Gas exchange profile for shoots of whole young plants (9-leaf-pair stage) measured throughout 27-days. The plants were watered to full capacity on day 0 and allowed to progress into drought until day 23 when they were re-watered to full capacity (black arrow). The means for each of the four gas exchange traces for each line are shown, with error bars showing the standard error of the mean (SEM) in the paler-coloured extensions that extend above and below each of the data points. (E)and (F) Impact of silencing PPC1 on chlorophyll a (E) and chlorophyll b (F) content in leaf pairs 2 through 7. In (E) and (F) the error bars represent the standard error of the mean calculated for the three biologicalreplicates, which were individual clones of each line. Asterisks indicate significant difference from the wildtype based on the Student’s t-test: * (E), Chlorophyll a in rPPC1-A: LP2, p = 0.000160; rPPC1-B: LP2, p =0.043676; LP3, p = 0.000003; LP4, p = 0.000001; LP5, p = 0.000094; LP6, p = 0.000012; LP7, p = 0.010883.* (F), Chlorophyll b in rPPC1-A: LP2, p = 0.000304; rPPC1-B: LP3, p = 0.000019; LP4, p = 0.000016; LP5 p =0.000653; LP6, p=0.000951. All Student‘s t-test parameters are presented in Supplemental Table 3. Blackdata are for the wild type, blue data rPPC1-A, and red data rPPC1-B.
Figure 4
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Figure 5. Impact of the Loss of PPC1 Transcripts on the Light/ Dark Regulation of the Transcript Abundance of CAM- and Central Circadian Clock-Associated Genes in RNAi Lines rPPC1-A and rPPC1-B. Gene transcript abundance was measured using RT-qPCR for target genes: (A), PPDK; (B), PPDK-RP; (C), b-NAD-ME; (D), GWD; (E), AMY3a; (F), AMY3b; (G), BAM1; (H), BAM3; (I), BAM9; (J), PHS1; (K), GPT2; (L), MEX1; (M), pGlcT; (N), CAB; (O), TPS7; (P), CCA1-1; (Q), CCA1-2; (R), TOC1-1; (S), TOC1-2; (T), PRR7; (U), PRR3/7; (V), PRR9; (W), JMJ30/ JMJD5; (X), ELF3; (Y), CDF2; (Z), RVE1-like; (AA), EPR1; (BB), LNK3; (CC), FKF1 and (DD), GI. Mature leaves (leaf pair 6; LP6) were sampled every 4 h across the 12-h-light/12-h-dark cycle. A thioesterase/thiol ester dehydrase-isomerase superfamily gene (TEDI) was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represents the mean of 3 technical replicates for biological triplicates and were normalized to loading control gene (TEDI); error bars represent the standard error. 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 rPPC1-A, and red data rPPC1-B.
Figure 5
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Figure 6
Figure 6. Effects of silencing PPC1 on CAM CO2 exchange rhythms measured under constant light and temperature (LL) conditions. (A), Gas exchange profile for detached CAM leaves (leaf pair 6; LP6) was measured using leaves entrained under a 12-h-light/12-h-dark cycle followed by release into LL constant conditions (100 µmol m-2 s-1 at 15 ˚C). (B), Gas exchange profile for well-watered whole young plants (9-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 at 15 ˚C). The data represents the mean CO2 uptake of 3 individual plants. (C), Delayed Fluorescence (DF) circadian rhythms became more robust in lines rPPC1-A and rPPC1-B. Plants were entrained under 12-h-light/12-h-dark cycles before being transferred to constant red/blue light (35 µmol m2s-1) under the CCD imaging camera system. DF was assayed with a 1-h time resolution for 120 h. The plots represent normalized averages for DF measured for 21 leaf discs sampled from 3 biological replicates of LP6 from each line. Error bars indicate SE of the mean calculated from three biological replicates. (D), Relative Amplitude Error (RAE) plot for the DF rhythms. (E), Mean period length plot, and (F), Mean RAE plot; the plotted values were calculated using the Biodare package for circadian rhythm analysis. FFT = fast Fourier transform-nonlinear least squares analysis, or SR = spectral resampling analysis. Black data are for the wild type, blue data rPPC1-A, and red data rPPC1-B. LL begins at 0h in Figures 6A-C.
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Figure 7
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Figure 7. Impact of the Loss of PPC1 Transcripts on Circadian Clock Controlled GeneTranscript Abundance during Constant Light and Temperature (LL) Free Running Conditions. Mature leaves (leaf pair 6), which performed CAM in the wild type, were sampled every 4 h under constant conditions (100 µmol m-2 s-1 at 15 ˚C) for wild type and rPPC1-B. RNA was isolated and used for real-time RT-qPCR. A thioesterase/thiol ester dehydrase-isomerase superfamily gene (TEDI) was amplified as a reference gene from the same cDNAs. Gene transcript abundance data represents the mean of 3 technical replicates for biological triplicates and were normalized to loading control gene (TEDI); error bars represent the standard error. In all cases, plants were entrained under 12-h-light/12-h-dark cycles prior to release into LL free-running conditions at 0h. Black data are for the wild type and red data rPPC1-B. The grey bars behind the data represent the subjective dark period, the time when it would have been dark under the previous entrainment conditions. Likewise, the white regions represent the subjective light period.(A), Circadian rhythm of PPC1 transcript abundance under constant LL conditions (100 µmol m-2
s-1 at 15 ˚C) for wild type and rPPC1-B. (B), PPC2; (C), PPCK1; (D), PPCK2; (E), PPCK3; (F),GPT2; (G), CAB; (H), CCA1-1 ; (I), CCA1-2; (J), TOC1-1; (K), TOC1-2; (L), PRR7; (M), PRR37;(N), PRR9; (O), LNK3-like; (P), JMJ30/JMJD5; (Q), ELF3; (R), CDF2; (S), RVE1-like; (T), EPR1;(U), FKF1 and (V), GI.
Figure 8. Impact of the Loss of PPC Activity on the Light/ Dark Regulation of the Transcript Abundance of Stomatal Control Genes in the Epidermis. Plants were entrained under 12-h-light/12-h-dark for 7 days prior to sampling. Epidermal peel samples were separated from leaf pairs 6, 7 and 8, with samples collected every 4h starting at 02:00, 2 h into the 12-h-light period. Each biological sample represents a pool of epidermal peels taken from 6 leaf pair 6, 7 and 8 leaves from 3 clonal stems of each line. Each peel was frozen in liquid nitrogen immediately after it was taken and pooled later. RNA was isolated and used in RT-qPCR. A thioesterase/thiol ester dehydrase-isomerase superfamily gene (TEDI) was amplified as a reference gene from the same cDNAs. Gene transcript abundance data represent the mean of 3 technical replicates for biological triplicates, and were normalized to the reference gene (TEDI); error bars represent the standard error. 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 and red data rPPC1-B. (A), PHOT1; (B), CRY2; (C), bCA5; (D), PATROL1; (E), CBC1/2 ; (F), HT1; (G), OST1; (H), SLAC1; (I),PP2C; (J), RCAR3; (K), GORK; (L), SKOR; (M), ALMT12 ; (N), RMA1; (O), EDA39; (P), SOS2; (Q), ACA2;(R), ECA4; (S), MYB60 and (T), MYB61.
Figure 8
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DOI 10.1105/tpc.19.00481; originally published online February 12, 2020;Plant Cell
HartwellSusanna F Boxall, Nirja Kadu, Louisa V Dever, Jana Knerova, Jade L. Waller, Peter D Gould and James
Circadian Clock and Guard Cell Signaling GenesKalanchoë PPC1 is Essential for Crassulacean Acid Metabolism and the Regulation of Core
This information is current as of September 17, 2020
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