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RESEARCH ARTICLE 12
Phosphorylation of Phosphoenolpyruvate Carboxylase is Essential for 3Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock 4Operation in the Obligate Crassulacean Acid Metabolism Species 5Kalanchoë fedtschenkoi 6
7Susanna F. Boxall, Louisa V. Dever, Jana Kneřová1, Peter D. Gould, and James 8Hartwell9Department of Functional and Comparative Genomics, Institute of Integrative Biology, Crown 10Street, University of Liverpool, Liverpool L69 7ZB, UK. 111Present address: Department of Plant Sciences, Downing Street, University of Cambridge, 12Cambridge CB2 3EA, UK 13
14Corresponding author: James Hartwell, e-mail: [email protected] 15
16Short Title: Silencing PPC phosphorylation in a CAM plant 17
18One-Sentence Summary: Silencing phosphoenolpyruvate carboxylase kinase in a CAM 19species more than halves dark period CO2 fixation, and causes arrhythmia in some 20components of the central circadian clock. 21
22The author responsible for distribution of materials integral to the findings presented in this 23article in accordance with the policy described in the Instructions for Authors 24(www.plantcell.org) is: James Hartwell ([email protected]). 25
26ABSTRACT 27
28Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 29fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated post-30translationally by a circadian clock controlled protein kinase called phosphoenolpyruvate 31carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its 32sensitivity to feedback inhibition by malate, and thus enhancing nocturnal CO2 fixation to 33stored malate. Here, we report the generation and characterization of transgenic RNAi lines of 34the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 35transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to 36a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced 37malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in 38the transcript abundance of KfPPCK1 was accompanied by a loss of oscillations in the 39transcript abundance of many core circadian clock genes, suggesting that perturbing the only 40known link between CAM and the circadian clock feeds back to perturb the central circadian 41clock itself. This work shows that clock control of KfPPCK1 prolongs the activity of PPC 42throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, 43malate accumulation, CAM productivity and core circadian clock robustness. 44
45
Plant Cell Advance Publication. Published on September 8, 2017, doi:10.1105/tpc.17.00301
KM078724; KfTOC1-2, KM078726. Other accession numbers and gene IDs are presented in 820
Supplemental Table 1. 821
822
Supplemental Data 823
Supplemental Figure 1. Impact of silencing KfPPCK1 on the apparent Ki of PPC for L-malate 824in rapidly desalted leaf extracts 825Supplemental Table 1. Primers used for reverse transcription-quantitative PCR 826
827
ACKNOWLEDGEMENTS 828
26
We thank Prof. Hugh Nimmo (University of Glasgow, UK) and Prof. Cristina Echevarria 829
(Universidad de Sevilla, Spain) for providing the antibodies used in this study. This work was 830
supported in part by the Biotechnology and Biological Sciences Research Council, UK 831
(BBSRC grant no. BB/F009313/1 awarded to J.H.), and in part by US Department of Energy 832
(DOE) Office of Science, Genomic Science Program under Award Number DE-SC0008834. 833
The contents of this article are solely the responsibility of the authors and do not necessarily 834
represent the official views of the DOE. 835
836
AUTHOR CONTRIBUTIONS 837
JH, SFB, LVD and PDG designed the research. SFB performed all experiments except the 838
and delayed fluorescence measurements. LVD performed the immunoblots, starch 840
determinations and growth yield drought experiment. JK generated the binary constructs and 841
carried out the stable transformation, regeneration and initial screening of the K. fedtschenkoi 842
rPPCK1 transgenic lines, and also collaborated with LVD on the growth yield drought 843
experiment. PDG carried out the delayed fluorescence experiments in collaboration with SFB. 844
SFB, LVD, PDG and JH analyzed the data. SFB and JH wrote the manuscript. 845
846
FIGURE LEGENDS 847
Figure 1. Confirmation of target gene silencing in transgenic K. fedtschenkoi RNAi 848
lines rPPCK1-1 and rPPCK1-3. Down-regulation of the target endogenous KfPPCK1 gene 849
was confirmed at the level of KfPPCK1 transcript abundance (A), and target protein PPC 850
phosphorylation and the apparent Ki of PPC for L-malate in rapidly desalted leaf extracts; 851
measured as L-malate (mM) required for a 50% reduction in extractable PPC activity (B). 852
Gene transcript abundance was measured using RT-qPCR for wild type, rPPCK1-1 and 853
rPPCK1-3 for target genes: A, KfPPCK1; C, KfPPCK2; and D, KfPPCK3. Mature leaves (leaf 854
pair 6) were sampled from three individual, clonal, biological replicate plants collected every 4 855
h across the 12-h-light/ 12-h-dark cycle. A thioesterase/thiol ester dehydrase-isomerase 856
superfamily gene (KfTEDI) was amplified from the same cDNAs as a reference gene. Gene 857
transcript abundance data represents the mean of 3 technical replicates of each of three 858
biological replicates, and was normalized to reference gene (KfTEDI); error bars represent the 859
standard error of the mean calculated for each biological replicate. In all cases, plants were 860
27
entrained under 12-h-light/ 12-h-dark cycles for 7 days prior to sampling. B, Protein 861
abundance (right panel) and the phosphorylation state of PPC (left panel) was determined by 862
immunoblot analyses, and the apparent Ki of PPC for L-malate was measured using rapidly 863
desalted leaf extracts (Ki values in mM below the corresponding left panel immunoblot time 864
points). Total leaf protein (leaf pair 6) was isolated from leaves sampled every 4 h across the 865
12-h-light/ 12-h-dark cycle, separated using SDS-PAGE and used for immunoblot analyses 866
with antibodies raised to a phospho-PPC peptide (left panel). Sample loading was normalized 867
according to total protein and confirmed using the immunoblot for total PPC protein, which is 868
stable over the 12-h-light/ 12-h-dark cycle (right panel). The white bar below each panel 869
represents the 12-h-light period and the black bar below each panel represents the 12-h-dark 870
period. For A, C and D: black data are for the wild type, blue rPPCK1-1 and red rPPCK1-3. 871
872
Figure 2. Impact of silencing KfPPCK1 on 24 h light/ dark gas exchange profiles, and 873
malate, starch and soluble sugar levels for wild type, rPPCK1-1 and rPPCK1-3 under 874
well-watered conditions. A, Gas exchange profile for CAM leaves (leaf pair 6) using plants 875
pre-entrained for 7 days under 12-h-light/ 12-h-dark cycles. B, Gas exchange profile for well-876
watered whole young plants (8-leaf-pairs stage) using plants entrained for 7 days under 12-h-877
light/ 12-h-dark cycles. C, Malate content was determined from leaf pair 6 samples collected 878
every 4 h using plants entrained under 12-h-light/ 12-h-dark cycles using methanol extracts of 879
leaves from wild type, rPPCK1-1 and rPPCK1-3. D, Starch content was determined from leaf 880
pair 6 samples collected at 1 h before dawn and 1 h before dusk. E, F, G, Soluble sugars 881
levels were determined separately for sucrose, glucose and fructose using three biological 882
replicates of leaf pair 6 sampled every 4 h using plants entrained under 12-h-light/ 12-h-dark 883
cycles. In C, D, E, F and G, the error bars represent the standard error of the mean calculated 884
for the three biological replicates measured at each time point. Black data traces represent 885
wild type, blue data rPPCK1-1 and red data rPPCK1-3. 886
887
Figure 3. Effects of silencing KfPPCK1 on CAM CO2 exchange rhythms measured 888
under constant light and temperature (LL) conditions. A, Gas exchange profile for CAM 889
leaves (leaf pair 6) was measured using leaves entrained under a 12-h-light/ 12-h-dark cycle 890
followed by release into constant LL conditions (100 µmol m-2 s-1 light at 15 ˚C). B, Gas 891
exchange profile for well-watered whole young plants (8-leaf-pairs stage) using plants 892
entrained under 12-h-light/ 12-h-dark cycles followed by release into constant LL conditions 893
28
(100 µmol m-2 s-1 light at 15 ˚C). Black data traces represent wild type, blue data rPPCK1-1 894
and red data rPPCK1-3. 895
896
Figure 4. Impact of loss of KfPPCK1 activity on gas exchange profiles (A) and 897
vegetative yield (B) for plants subjected to drought-stress. A, Drought-stressed whole 898
plants (8-leaf-pair stage) were entrained under 12-h-light/ 12-h-dark cycles followed by 899
release into constant LL conditions (100 µmol m-2s-1 light at 15 ˚C). The black data trace 900
represents wild type, blue rPPCK1-1 and red rPPCK1-3. B, C, D and E, Fresh (B, C) and dry 901
(D, E) weight of above-ground biomass (shoot; B, D) and below-ground tissues (roots; C, E) 902
at maturity (138 d under greenhouse conditions) for wild type (WT), rPPCK1-1 and rPPCK1-3 903
under well-watered and drought-stressed conditions. n = 7 plants; error bars represent 904
standard error of the mean. * or *** = significantly different to wild type based on Student’s t-905
test, *P < 0.05 and ***P < 0.001. 906
907
Figure 5. Impact of the loss of KfPPCK1 activity on the light/ dark regulation of the 908
transcript abundance of central circadian clock genes KfTOC1-1, KfTOC1-2, KfCCA1-1, 909
KfCCA1-2, KfPRR7 and KfPRR37. Mature leaves (leaf pair 6) were sampled from three 910
biological replicates every 4 h across the 12-h-light/ 12-h-dark cycle, RNA isolated and used 911
for real-time RT-qPCR. A, TOC1-1. B, TOC1-2. C, CCA1-1. D, CCA1-2. E, PRR7. F, PRR37. 912
A thioesterase/thiol ester dehydrase-isomerase superfamily (KfTEDI) gene was amplified 913
from the same cDNAs as a reference gene. Gene transcript abundance data represents the 914
mean of 3 technical replicates for each of three biological replicates and was normalized to 915
the reference gene (KfTEDI); error bars represent the standard error of the mean calculated 916
for the three biological replicates. In all cases, plants were entrained under 12-h-light/ 12-h-917
dark cycles for 7 days prior to sampling. Black data are for the wild type, blue data rPPCK1-1, 918
and red data rPPCK1-3. 919
920
Figure 6. Impact of the loss of KfPPCK1 activity on circadian clock controlled gene 921
transcript abundance during constant light and temperature free running conditions. A, 922
Circadian rhythm of KfPPCK1 transcript abundance under constant LL conditions (100 µmol 923
m-2 s-1 light at 15 ˚C) for wild type (black line) and rPPCK1-3 (red line). B, KfPPCK2. C, 924
KfPPCK3. D, KfCCA1-1. E, KfCCA1-2. F, KfTOC1-1. G, KfTOC1-2. H, KfPRR7. I, KfPRR37. 925
J, KfPRR9. K, KfFKF1. L, KfGIGANTEA. M, KfJMJD30. N, KfLNK3-like. O, KfCDF2. Mature 926
29
leaves (leaf pair 6) were sampled from three biological replicates every 4 h under constant 927
conditions (100 µmol m-2 s-1 light at 15˚C) for wild type and rPPCK1-3. RNA was isolated and 928
used for real-time RT-qPCR. A thioesterase/thiol ester dehydrase-isomerase superfamily 929
gene (KfTEDI) was amplified as a reference gene from the same cDNAs. Gene transcript 930
abundance data represents the mean of 3 technical replicates for each of three biological 931
replicates, and was normalized to the reference gene (KfTEDI); error bars represent the 932
standard error of the mean calculated for the three biological replicates. In all cases, plants 933
were entrained under 12-h-light/ 12-h-dark cycles prior to release into LL free-running 934
conditions. Black data are for the wild type and red data rPPCK1-3. 935
936
Figure 7. Delayed fluorescence (DF) rhythms collapsed towards arrythmia in lines 937
rPPCK1-1 and rPPCK1-3. Plants were entrained under 12-h-light/12-h-dark cycles before 938
being transferred to constant red/ blue light under the CCD imaging camera (35 µmol m-2 s-1). 939
DF was assayed with a 1-h time resolution for 108 h. The plots represent normalized 940
averages for DF measured for 6 leaf discs sampled from three biological replicates of leaf pair 941
6 for each line. A, Wild type DF rhythm under LL. B, Relative amplitude error (RAE) plot for 942
wild type DF rhythm. C, Mean period length (upper graph) and RAE plots (lower graph) for 943
wild type (black/ grey), rPPCK1-1 (blue/ pale blue) and rPPCK1-3 (red/ pale red); the plotted 944
values were calculated using using Biodare package for circadian rhythm analysis. In both 945
graphs, the dark shade of the colour represents data from Fast Fourier Transform-Non Linear 946
Least Squares (FFT-NLLS) analysis and the paler shade represents data from Spectral 947
Resampling (SR) analysis. D, rPPCK1-1 DF rhythm. E, RAE plot for rPPCK1-1. F, rPPCK1-3 948
DF rhythm. G, RAE plot for rPPCK1-3. Error bars indicate standard error of the mean 949
calculated from three biological replicates. Black data are for the wild type, blue data rPPCK1-950
1 and red data rPPCK1-3. 951
952
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1152
Figure 1. Confirmation of target gene silencing in transgenic K. fedtschenkoi RNAi lines
rPPCK1-1 and rPPCK1-3. Down-regulation of the target endogenous KfPPCK1 gene was confirmed at the level of KfPPCK1 transcript abundance (A), and target protein PPC phosphorylation and the apparent Ki of PPC for L-malate in rapidly desalted leaf extracts; measured as L-malate (mM) required for a 50 % reduction in extractable PPC activity (B). Gene transcript abundance was measured using RT-qPCR for wild type, rPPCK1-1 and rPPCK1-3 for target genes: A, KfPPCK1; C, KfPPCK2; and D, KfPPCK3. Mature leaves (leaf pair 6) were sampled from three individual, clonal, biological replicate plants collected every 4 h across the 12-h-light/ 12-h-dark cycle. A thioesterase/thiol ester dehydrase-isomerase superfamily gene (KfTEDI) was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represents the mean of 3 technical replicates of each of three biological replicates, and was normalized to reference gene (KfTEDI); error bars represent the standard error of the mean calculated for each biological replicate. In all cases, plants were entrained under 12-h-light/ 12-h-dark cycles for 7 days prior to sampling. B, Protein abundance (right panel) and the phosphorylation state of PPC (left panel) was determined by immunoblot analyses, and the apparent Ki of PPC for L-malate was measured using rapidly desalted leaf extracts (Ki values in mM below the corresponding left panel immunoblot time points). Total leaf protein (leaf pair 6) was isolated from leaves sampled every 4 h across the 12-h-light/ 12-h-dark cycle, separated using SDS-PAGE and used for immunoblot analyses with antibodies raised to a phospho-PPC peptide (left panel). Sample loading was normalized according to total protein and confirmed using the immunoblot for total PPC protein, which is stable over the 12-h-light/ 12-h-dark cycle (right panel). The white bar below each panel represents the 12-h-light period and the black bar below each panel represents the 12-h-dark period. For A, C and D: black data are for the wild type, blue rPPCK1-1 and red rPPCK1-3.
Figure 1
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Figure 2. Impact of silencing KfPPCK1 on 24 h light/ dark gas exchange profiles, and
malate, starch and soluble sugar levels for wild type, rPPCK1-1 and rPPCK1-3 under
well-watered conditions. A, Gas exchange profile for CAM leaves (leaf pair 6) using plants pre-entrained for 7 days under 12-h-light/ 12-h-dark cycles. B, Gas exchange profile for well-watered whole young plants (8-leaf-pairs stage) using plants entrained for 7 days under 12-h-light/ 12-h-dark cycles. C, Malate content was determined from leaf pair 6 samples collected every 4 h using plants entrained under 12-h-light/ 12-h-dark cycles using methanol extracts of leaves from wild type, rPPCK1-1 and rPPCK1-3. D, Starch content was determined from leaf pair 6 samples collected at 1 h before dawn and 1 h before dusk. E, F, G, Soluble sugars levels were determined separately for sucrose, glucose and fructose using three biological replicates of leaf pair 6 sampled every 4 h using plants entrained under 12-h-light/ 12-h-dark cycles. In C, D, E, F and G, the error bars represent the standard error of the mean calculated for the three biological replicates measured at each time point. Black data traces represent wild type, blue data rPPCK1-1 and red data rPPCK1-3.
B A
Figure 3. Effects of silencing KfPPCK1 on CAM CO2 exchange rhythms measured under constant light and temperature (LL) conditions. A, Gas exchange profile for CAM leaves (leaf pair 6) was measured using leaves entrained under a 12-h-light/ 12-h-dark cycle followed by release into constant LL conditions (100 µmol m-2 s-1 light at 15 ˚C). B, Gas exchange profile for well-watered whole young plants (8-leaf-pairs stage) using plants entrained under 12-h-light/ 12-h-dark cycles followed by release into constant LL conditions (100 µmol m-2 s-1 light at 15 ˚C). Black data traces represent wild type, blue data rPPCK1-1 and red data rPPCK1-3.
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Figure 3
Figure 4. Impact of loss of KfPPCK1 activity on gas exchange profiles (A) and vegetative yield (B) for plants subjected to drought-stress. A, Drought-stressed whole plants (8-leaf-pair stage) were entrained under 12-h-light/ 12-h-dark cycles followed by release into constant LL conditions (100 µmol m-2s-1 light at 15 ˚C). The black data trace represents wild type, blue rPPCK1-1 and red rPPCK1-3. B, C, D and E, Fresh (B, C) and dry (D, E) weight of above-ground biomass (shoot; B, D) and below-ground tissues (roots; C, E) at maturity (138 d under greenhouse conditions) for wild type (WT), rPPCK1-1 and rPPCK1-3 under well-watered and drought-stressed conditions. n = 7 plants; error bars represent standard error of the mean. * or *** = significantly different to wild type based on Student’s t-test, *P < 0.05 and ***P < 0.001.
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Figure 5. Impact of the loss of KfPPCK1 activity on the light/ dark regulation of the
transcript abundance of central circadian clock genes KfTOC1-1, KfTOC1-2, KfCCA1-
1, KfCCA1-2, KfPRR7 and KfPRR37. Mature leaves (leaf pair 6) were sampled from three biological replicates every 4 h across the 12-h-light/ 12-h-dark cycle, RNA isolated and used for real-time RT-qPCR. A, TOC1-1. B, TOC1-2. C, CCA1-1. D, CCA1-2. E, PRR7. F, PRR37. A thioesterase/thiol ester dehydrase-isomerase superfamily (KfTEDI) gene was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represents the mean of 3 technical replicates for each of three biological replicates and was normalized to the reference gene (KfTEDI); error bars represent the standard error of the mean calculated for the three biological replicates. In all cases, plants were entrained under 12-h-light/ 12-h-dark cycles for 7 days prior to sampling. Black data are for the wild type, blue data rPPCK1-1, and red data rPPCK1-3.
Figure 5
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Figure 6. Impact of the loss of KfPPCK1 activity on circadian clock controlled genetranscript abundance during constant light and temperature free runningconditions. A, Circadian rhythm of KfPPCK1 transcript abundance under constant LLconditions (100 µmol m-2 s-1 light at 15 ˚C) for wild type (black line) and rPPCK1-3 (redline). B, KfPPCK2. C, KfPPCK3. D, KfCCA1-1. E, KfCCA1-2. F, KfTOC1-1. G, KfTOC1-2.H, KfPRR7. I, KfPPR37. J, KfPRR9. K, KfFKF1. L, KfGIGANTEA. M, KfJMJD30. N,KfLNK3-like. O, KfCDF2. Mature leaves (leaf pair 6) were sampled from three biologicalreplicates every 4 h under constant conditions (100 µmol m-2 s-1 light at 15 ˚C) for wildtype and rPPCK1-3. RNA was isolated and used for real-time RT-qPCR. Athioesterase/thiol ester dehydrase-isomerase superfamily gene (KfTEDI) was amplifiedas a reference gene from the same cDNAs. Gene transcript abundance data representsthe mean of 3 technical replicates for each of three biological replicates, and wasnormalized to loading control gene (KfTEDI); error bars represent the standard error ofthe mean calculated for the three biological replicates. In all cases, plants were entrainedunder 12-h-light/ 12-h-dark cycles prior to release into LL free-running conditions. Blacklines represent wild type and red lines rPPCK1-3.
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Figure 7. Delayed fluorescence (DF) rhythms collapsed towards arrythmia in lines
rPPCK1-1 and rPPCK1-3. Plants were entrained under 12-h-light/12-h-dark cycles before being transferred to constant red/ blue light under the CCD imaging camera (35 µmol m-2 s-
1). DF was assayed with a 1-h time resolution for 108 h. The plots represent normalized averages for DF measured for 6 leaf discs sampled from three biological replicates of leaf pair 6 for each line. A, Wild type DF rhythm under LL. B, Relative amplitude error (RAE) plot for wild type DF rhythm. C, Mean period length (upper graph) and RAE plots (lower graph) for wild type (black/ grey), rPPCK1-1 (blue/ pale blue) and rPPCK1-3 (red/ pale red); the plotted values were calculated using using Biodare package for circadian rhythm analysis. In both graphs, the dark shade of the colour represents data from Fast Fourier Transform-Non Linear Least Squares (FFT-NLLS) analysis and the paler shade represents data from Spectral Resampling (SR) analysis. D, rPPCK1-1 DF rhythm. E, RAE plot for rPPCK1-1. F, rPPCK1-3 DF rhythm. G, RAE plot for rPPCK1-3. Error bars indicate standard error of the mean calculated from three biological replicates. Black data are for the wild type, blue data rPPCK1-1 and red data rPPCK1-3.
Figure 7
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DOI 10.1105/tpc.17.00301; originally published online September 8, 2017;Plant Cell
Susanna F Boxall, Louisa V Dever, Jana Knerova, Peter D Gould and James HartwellSpecies Kalanchoë fedtschenkoi
CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Phosphorylation of Phosphoenolpyruvate Carboxylase is Essential for Maximal and Sustained Dark