Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes

Initial Screening and Characterization of PPC1 RNAi Lines of K. laxiflora

As K. laxiflora is relatively slow-growing, with flowering and seed set taking ∼9 months seed-to-seed (Hartwell et al., 2016), data are presented for independent primary transformants that were propagated clonally via leaf margin plantlets and/or stem cuttings. We initially screened primary transformants using high-throughput leaf disc tests for starch and acidity at dawn and dusk (Cushman et al., 2008; Dever et al., 2015). We screened independent transgenic lines that acidified less during the dark period than the wild type for the steady state abundance of PPC1 transcripts using RT-qPCR (Figure 1). Line rPPC1-B displayed a complete loss of PPC1 transcripts, whereas rPPC1-A had an intermediate level of PPC1 transcripts (Figure 1A). The other plant-type PPC genes (PPC2, PPC3, and PPC4) were upregulated relative to the wild type, with their peak phased to dawn (Figures 1B to 1D). PPC2 was upregulated in line rPPC1-B at 6 and 10 h into the 12-h dark period (Figure 1B).

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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 PPC1 (A), PPC2 (B), PPC3 (C), PPC4 (D), PPCK1 (E), PPCK2 (F), and PPCK3 (G). 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 represent means of three technical replicates for biological triplicates and were normalized to the loading control gene (TEDI); error bars represent the se. In all cases, plants were entrained under 12-h-light/12-h-dark cycles for 7 d prior to sampling. Black data are for the wild type, blue data for rPPC1-A, and red data for rPPC1-B.

In line rPPC1-B, PPCK1, encoding the protein kinase that phosphorylates the CAM-associated protein PPC1, was downregulated during its dark period phased peak and appeared to be slightly upregulated at 2 h after dawn (Figure 1E). The other two detectable PPCK genes, PPCK2 and PPCK3, were upregulated in rPPC1-B, with PPCK2 induced fivefold when it reached its 24-h peak in the middle of the dark period, 4 h after the wild-type peak (Figure 1F). PPCK3 peaked in the middle of the light period, when it reached a level almost eightfold greater than the wild type (Figure 1G).

Loss of PPC1 Transcripts Leads to Loss of PPC Protein and Activity

Immunoblotting using an antibody raised against purified CAM-specific PPC protein from K. fedtschenkoi leaves (Nimmo et al., 1986; Figure 2) revealed reduced levels of PPC protein in rPPC1-A and no detectable PPC in rPPC1-B (Figure 2A).

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Figure 2.

Loss of PPC1 Transcripts Leads to Loss of PPC Protein and Activity and Failure to Activate PPDK in the Light.

(A) and (B) 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 with antibodies raised to PPC (A) and phospho-PPC (B).

(C) PPC activity was measured in 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 and illuminated leaves. **, rPPC1-B, P = 0.003.

The activities of PPC, measured 6 h into the light period, and PPDK, measured at both 6 h into the dark period and 6 h into the light period, were means of three technical replicates of three biological replicates, and error bars represent the se. The white bars below panels (A), (B), (D), and (E) represent the 12-h-light period, and the black bars represent the 12-h-dark period. In panels (C) and (F), black data are for the wild type, blue data for rPPC1-A, and red data for rPPC1-B. Asterisks indicate significant differences from the wild type based on Student’s t test. All Student’s t test parameters are presented in Supplemental Tables 1 and 2.

We also measured the level of phospho-PPC using immunoblotting (Figure 2B). Phospho-PPC levels were lower in rPPC1-A than the wild type, while rPPC1-B lacked detectable phospho-PPC (Figure 2B), which was consistent with the level of PPC (Figure 2A). Although PPC2 was upregulated in rPPC1-B (Figure 1B), it was not detected at the protein level by the PPC antibody (Figure 2A). Furthermore, despite PPCK2 and PPCK3 being upregulated by fivefold to eightfold in rPPC1-B (Figures 1F and 1G), any protein produced from these transcripts did not phosphorylate any PPC2 protein that may have resulted from the induced PPC2 transcripts (Figure 2B), at least not within the limits of detection with this immunoblotting technique. We used rapidly desalted leaf extracts to measure the apparent inhibitor constant (K_i) of PPC for l-malate. The K_i was higher in the dark than in the light for the wild type and rPPC1-A, but no change in the K_i was detected for rPPC1-B (Supplemental Figure 1). Furthermore, PPC activity was not detected in rapidly desalted extracts from rPPC1-B leaves, whereas rPPC1-A displayed reduced but detectable PPC activity, at 43% of the wild-type level (Figure 2C; Supplemental Table 1).

PPDK, which functions in concert with NAD-ME during the light period as part of the CAM malate decarboxylation pathway (Dever et al., 2015), was measured on immunoblots using specific antibodies against PPDK and phospho-PPDK (Chastain et al., 2000, 2002). The blots showed a similar amount of PPDK protein over the diel cycle in the wild type, rPPC1-A, and rPPC1-B (Figure 2D), confirming the even protein loading of the blots, which was further supported by the images of Coomassie blue-stained gels (Supplemental Figure 1). In Kalanchoë, PPDK is inactivated in the dark by phosphorylation by PPDK-regulatory protein (PPDK-RP), which also activates PPDK in the light through dephosphorylation (Dever et al., 2015). In the wild type, immunoblotting of phospho-PPDK revealed that PPDK was dephosphorylated, and therefore likely to be fully active, between 02:00 and 06:00 h in the light when it is required for the conversion of pyruvate, from malate decarboxylation, to PEP, thereby facilitating the recycling of carbon through gluconeogenesis to starch (Figure 2E). Line rPPC1-A showed the same pattern of PPDK phosphorylation/dephosphorylation as the wild type (Figure 2E). However, PPDK was phosphorylated throughout the 24-h cycle in rPPC1-B (Figure 2E) and was therefore likely to be inactive. Consistent with this prediction, loss of the light period dephosphorylation of PPDK in rPPC1-B correlated with a significant decrease in PPDK activity in the light, whereas the wild type and line rPPC1-A showed strongly light-induced levels of PPDK activity that correlated with the detected level of PPDK dephosphorylation in the light (Figure 2F; Supplemental Table 2).

Malate, Starch, and Soluble Sugar Levels

During CAM in Kalanchoë, primary nocturnal fixation of atmospheric CO₂ (as HCO₃⁻) results in vacuolar malic acid accumulation throughout the dark period. Starch accumulates during the light period and is broken down during the dark period to provide PEP as the substrate for carboxylation by PPC. Starch is also broken down in a rapid burst at dawn to form soluble sugars (Wild et al., 2010; Boxall et al., 2017). As the lack of CAM-associated PPC1 was predicted to prevent primary nocturnal carboxylation, we measured metabolites including malate, starch, and soluble sugars every 4 h over the 24-h cycle (Figure 3).

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Figure 3.

Impact of Silencing PPC1 on Malate, Starch, Soluble Sugars, and Growth.

(A) to (E) The contents of malate (A), starch (B), sucrose (C), glucose (D), and fructose (E) 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 the wild type, rPPC1-A, and rPPC1-B and used for malate and soluble sugar determination, whereas starch was measured in the insoluble pellet. FW, fresh weight.

(F) Dry weight (DW) of 148-d-old greenhouse-grown plants, either well-watered (w) or drought-stressed for the last 28 d (d). Asterisks denote significant differences between the wild type and rPPC1-B based on Student’s t test. Well-watered rPPC1-B, *, P = 0.017; drought-stressed rPPC1-B, *, P = 0.011; n = 6 to 9 developmentally synchronized, clonal plants per line. See Supplemental Data Set 1 for full parameters from Student’s t test results for the relative growth data.

(G) Four-month-old plants raised in a greenhouse under 16-h-light/8-h-dark cycles.

In all graphs, black data are for the wild type, blue data for rPPC1-A, and red data for rPPC1-B. Error bars represent the se.

Wild-type plants accumulated 130 µmol g⁻¹ fresh weight malate by dawn, whereas rPPC1-A and rPPC1-B accumulated 75 and 19.5 µmol g⁻¹ fresh weight, respectively (Figure 3A). The Δ-malate values for the wild type, rPPC1-A, and rPPC1-B were 124.0, 64.3, and 16.2 µmol g⁻¹ fresh weight, respectively (Figure 3A). During the diel cycle, rPPC1-A and rPPC1-B synthesized 100 and 41% of the amount of starch accumulated by the wild type, respectively (Figure 3B). The Δ-starch values for the wild type, rPPC1-A, and rPPC1-B were 8.5, 8.5, and 3.5 mg starch g⁻¹ fresh weight (Figure 3B). Lines rPPC1-A and rPPC1-B accumulated 51 and 15% of the amount of sucrose accumulated by the wild type (Figure 3C) and 83 and 69% of the level of glucose, respectively (Figure 3D). Glucose accumulated 4 h after the sucrose peak in the wild type, whereas glucose levels peaked at the same time as sucrose in lines rPPC1-A and rPPC1-B (Figures 3C and 3D). Finally, lines rPPC1-A and rPPC1-B accumulated 113 and 61% of the amount of fructose, respectively, compared with the wild type (Figure 3E). In rPPC1-B, the daily maxima for fructose and glucose coincided with that of sucrose at 2 h after dawn (Figures 3C to 3E). While no direct evidence was obtained to explain these shifts in the timing of the sucrose, glucose, and fructose daily maxima, the most straightforward explanation is that the observed changes in the timing of photosynthetic CO₂ fixation and starch accumulation and turnover in the rPPC1-B transgenic line resulted in the observed changes in the timing of the soluble sugar peaks.

Growth Analysis in Well-Watered Versus Drought-Stressed Conditions

CAM is widely regarded as an adaptation to drought, and so it was important to compare the growth performance of the wild type (full CAM) with that of rPPC1-A (small reduction in CAM) and rPPC1-B (no CAM) plants under both well-watered and drought-stressed conditions (Figure 3F; Supplemental Figure 2; Supplemental Data Set 1). rPPC1-B plants were significantly smaller than wild-type plants in both well-watered and drought-stressed conditions (Figure 3F; Supplemental Figure 2). In well-watered conditions, the shoot dry weight of rPPC1-B was 21% less than that of the wild type (Figure 3F), but the shoot dry weight of rPPC1-A was not significantly different from that of the wild type. Under drought-stressed conditions, the shoot dry weight of line rPPC1-B was 12% lower than that of the wild type (Figure 3F). A visual inspection of representative 4-month-old plants demonstrated that rPPC1-B was smaller than the wild type and rPPC1-A (Figure 3G), which is consistent with the shoot dry weight data (Figure 3F). Shoot fresh weight was also significantly reduced in well-watered and drought-stressed rPPC1-B, whereas the only significant difference in belowground root tissues was for rPPC1-B under drought stress, which displayed an increase in root fresh weight that was only just significant (P = 0.0483; Supplemental Figure 2). Furthermore, rPPC1-B displayed a reduced degree of leaf in-rolling in response to drought relative to the wild type (Supplemental Figure 3), even though it lost just as much water during drought as the wild type (Supplemental Figure 2).

Gas-Exchange Characteristics under Light/Dark Cycles

We measured gas exchange in mature CAM leaves (leaf pair 6 [LP6]) of each line over a 12-h-light, 25°C, 60% humidity/12-h-dark, 15°C, 70% humidity cycle using an infrared gas analyzer (LI-COR LI-6400XT; Figures 4A to 4C). Over the 24-h diel cycle, the wild type fixed 297 µmol CO₂ m⁻², rPPC1-A fixed 320 µmol CO₂ m⁻², and rPPC1-B fixed 230 µmol CO₂ m⁻² (Figure 4A). Net CO₂ fixation was 23% less than in the wild type in line rPPC1-B, whereas it was 15% greater in line rPPC1-A (Figure 4A).

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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) to (C) CO₂ uptake profiles showing the four phases of CAM (A), stomatal conductance (g_s) profile (B), and the calculated internal partial pressure of CO₂ inside the leaf (C_i) profile (C) for CAM leaves (leaf pair 6 [LP6]) using plants pre-entrained for 7 d under 12-h-light/12-h-dark cycles.

(D) Impact of drought on K. laxiflora wild-type and transgenic lines with reduced PPC1 expression. Gas-exchange profiles for shoots of whole young plants (nine-leaf-pair stage) measured throughout 27 d are shown. The plants were watered to full capacity on day 0 and allowed to progress into drought until day 23, when they were rewatered 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 se in the paler-colored 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) contents in LP2 through LP7. The error bars represent the se calculated for the three biological replicates, which were individual clones of each line. Asterisks indicate significant differences from the wild type based on Student’s t test. For (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. For (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. Black data are for the wild type, blue data for rPPC1-A, and red data for rPPC1-B.

The four phases of CAM (Osmond, 1978) are indicated on the CO₂ exchange graph in Figure 4A. Phase I corresponds to the period of nocturnal atmospheric CO₂ fixation to vacuolar malic acid in the wild type, and phases II, III, and IV define the peaks and troughs of CO₂ fixation and stomatal conductance across the 12-h-light period (Figures 4A and 4B). Specifically, phase II refers to the sharp peak of CO₂ fixation in the 1 to 2 h after dawn, phase III defines the period of refixation of CO₂ from malate decarboxylation by Rubisco behind closed stomata that spans the middle of the light period, and phase IV occurs in younger leaves of well-watered plants and corresponds to the period in the late afternoon when stomata reopen and atmospheric CO₂ is fixed directly by Rubisco (Osmond, 1978). Note that the data in blue for rPPC1-A provide the best example of this four-phase framework for CAM CO₂ fixation and stomatal physiology (Figure 4A). By contrast, LP6 of the wild type performed full CAM defined by phase I in the dark and phase III in the light (Figure 4A). By calculating the total area under or over the CO₂-exchange curves in Figure 4A, and calculating these areas separately for the 12-h-light and 12-h-dark periods, it was possible to quantify the amounts of CO₂ fixed by each line during either the light period or the dark period. In the light period (phases II through IV), wild-type leaves fixed negligible amounts of atmospheric CO₂, whereas rPPC1-B fixed a total of 265 µmol m⁻² and rPPC1-A fixed 89 µmol m⁻² (Figure 4A). In the dark period (phase I), the wild type fixed 297 µmol CO₂ m⁻² and rPPC1-A fixed 226 µmol CO₂ m⁻², but rPPC1-B respired 35 µmol CO₂ m⁻² (Figure 4A). The loss of nocturnal CO₂ fixation in rPPC1-B (Figure 4A) corresponded with the lack of the CAM-associated PPC1 (Figures 1 and 2). LP6 of rPPC1-A fixed 24% less nocturnal CO₂ than the wild type, but, in contrast to the wild type, it continued some light period CO₂ capture (Figure 4A).

Apart from opening briefly for phase II, just after lights-on, the attached LP6 of the wild type closed its stomata in the light and opened them throughout the dark, when stomatal conductance tracked CO₂ uptake (Figure 4B). In rPPC1-B, stomata stayed open in the light, closed briefly at dusk, and opened slightly throughout the dark, with a small peak prior to dawn (Figure 4B). The dark conductance of rPPC1-B corresponded with the release of respiratory CO₂ (Figure 4A). The calculated internal partial pressure of CO₂ inside the leaf was highest for the wild type in the light period, when stomata were closed, which is consistent with the expected high level of CO₂ inside the leaf during malate decarboxylation (Figure 4C). By contrast, in rPPC1-B, internal partial pressure of CO₂ inside the leaf peaked during the dark period, when stomata were slightly open, but the leaves failed to fix atmospheric CO₂ and respiratory CO₂ escaped (Figure 4C).

Impact of Drought on CO₂ Uptake in Plants Lacking PPC1

To test the importance of PPC1 for carbon assimilation during drought, we measured CO₂ uptake continuously in whole, young plants (nine leaf pairs, 2 months old) over 22 d of drought, followed by rewatering (Figure 4D). The soil was watered to its holding capacity prior to placing the plants in the gas-exchange cuvettes at the start of the experiment, and then no further water was added until rewatering occurred on day 22. CAM develops with leaf age in K. laxiflora; the leaves gradually reduce light period CO₂ fixation and increase nocturnal CO₂ fixation as they mature (Supplemental Figure 4). A 2-month-old wild-type plant with nine leaf pairs thus includes young leaves fixing CO₂ mainly in the light via the C₃ pathway and older leaves fixing the majority of their atmospheric CO₂ in the dark via PPC and CAM.

When well-watered on day 1, the wild type, rPPC1-A, and rPPC1-B fixed, respectively, 7, 6, and −26% of their CO₂ during the dark period and 93, 94, and 126% during the light period (Table 1). This indicated that, in well-watered conditions, the young leaves of these young plants performed the majority of the 24-h CO₂ uptake (Table 1). On day 1, the wild type, rPPC1-A, and rPPC1-B fixed a total of 1093, 1008, and 1078 µmol atmospheric CO₂ m⁻² over 24 h, respectively (Table 1). After 7 d without watering, 24-h CO₂ fixation was 1345, 1367, and 1506 µmol m⁻², respectively. It should be noted that total leaf area was measured at the end of the experiment, so leaf growth and expansion during the experiment could not be accounted for.

After 7 d without water, there was a substantial increase in nocturnal CO₂ uptake in the wild type and rPPC1-A relative to day 1 (52 and 40% of CO₂ fixation occurred in the dark, respectively; Table 1). rPPC1-B respired less (−14%) after 7 d of drought relative to the −26% dark-respired CO₂ level on day 1 (Table 1). After 13 d of drought, the wild type, rPPC1-A, and rPPC1-B fixed, respectively, 795, 802, and 145 µmol CO₂ m⁻² over the 24-h cycle (Table 1). Thus, plants performing CAM (the wild type and rPPC1-A) were able to fix over fivefold more CO₂ after 13 d of drought compared with rPPC1-B. Furthermore, after 22 d of drought, the wild type, rPPC1-A, and rPPC1-B fixed 76, 130, and −30 µmol m⁻² during the 24-h light/dark cycle, respectively.

On day 22 without water, the drought-stressed plants were rewatered (see photographs in Supplemental Figure 3). After rewatering, CO₂ fixation increased rapidly for all plants (Figure 4D). In addition, the wild type and rPPC1-A displayed pronounced phase III of CAM after rewatering (Figure 4D). Following soil rehydration, the wild type fixed 931 µmol m⁻² CO₂ in the dark compared with 768 µmol m⁻² for rPPC1-A, whereas prior to drought, rPPC1-A fixed more atmospheric CO₂ (Table 1). rPPC1-B fixed CO₂ throughout the light period following rewatering, and it also resumed respiratory CO₂ loss throughout the dark period (Figure 4D).

As there was an increase in C₃ photosynthesis in rPPC1-B, we measured chlorophyll a and b levels in LP2 through LP7 (Figures 4E and 4F; Supplemental Table 3). Line rPPC1-A contained significantly more chlorophyll a and b than the wild type in LP2 (Figures 4E and 4F). rPPC1-B contained significantly more chlorophyll a than the wild type in LP2, significantly less chlorophyll a in LP3 through LP7, and significantly less chlorophyll b in LP3 to LP6 (Figures 4E and 4F). These decreased levels of chlorophyll a and b in LP3 to LP7 or LP6 in rPPC1-B were a surprising finding. In general, CAM species contain reduced levels of gene transcripts and encoded proteins associated with C₃ photosynthesis, such as Rubisco and light-harvesting complex components, when the level of CAM is induced by stress. Thus, reducing the level of CAM in LP3 through LP7 due to the silencing of PPC1 was predicted to result in the induction of core C₃ photosynthesis genes and chlorophyll content in parallel with the measured increase in light period atmospheric CO₂ fixation via the C₃ pathway. Further elucidation of the reasons for this unexpected chlorophyll response may come from transcriptome-wide analysis of the differentially abundant transcripts in rPPC1-B compared with the wild type.

Characterization of CAM Gene Transcript Abundance in rPPC1 Lines

Having established that rPPC1-B lacked nocturnal CO₂ fixation (Figure 4A), it was important to investigate the temporal regulation of other CAM-associated genes in the rPPC1 RNAi lines. We investigated the transcript abundance of CAM genes in CAM leaves (LP6) using samples collected every 4 h over a 12-h-light/12-h-dark (LD) cycle (Figure 5). PPDK transcript levels were unchanged relative to the wild type in both rPPC1 lines (Figure 5A), but its regulator gene PPDK-RP was upregulated in line rPPC1-B (Figure 5B), which is consistent with the continuous phosphorylation and inactivation of PPDK (Figures 2E and 2F). β-NAD-ME transcript levels appeared to be only slightly different from the wild type in the rPPC1 lines but appeared to be lower in rPPC1-B at 22:00 h (Figure 5C).

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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 PPDK (A), PPDK-RP (B), β-NAD-ME (C), GWD (D), AMY3a (E), AMY3b (F), BAM1 (G), BAM3 (H), BAM9 (I), PHS1 (J), GPT2 (K), MEX1 (L), pGlcT (M), CAB (N), TPS7 (O), CCA1-1 (P), CCA1-2 (Q), TOC1-1 (R), TOC1-2 (S), PRR7 (T), PRR3/7 (U), PRR9 (V), JMJ30/JMJD5 (W), ELF3 (X), CDF2 (Y), RVE1-like (Z), EPR1 (AA), LNK3 (BB), FKF1 (CC), and GI (DD). Mature leaves (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 represent means of three technical replicates for biological triplicates and were normalized to the loading control gene (TEDI); error bars represent the se. In all cases, plants were entrained under 12-h-light/12-h-dark cycles for 7 d prior to sampling. Black data are for the wild type, blue data for rPPC1-A, and red data for rPPC1-B.

In light of the marked reduction in starch to half the wild-type level in rPPC1-B (Figure 3B), we also measured transcripts associated with starch turnover. In rPPC1-B, dusk-phased starch breakdown-associated genes (GWD, AMY3a and AMY3b, and PHS1; Figures 5D to 5F and 5J) and sugar transporter genes (MEX1 and pGlcT; Figures 5L and 5M) were upregulated, whereas dawn-phased starch breakdown genes (BAM1, BAM3, and BAM9; Figures 5G to 5I) and sugar transporter genes (GPT2; Figure 5K) were downregulated compared with the wild type.

Finally, a CHLOROPHYLL A/B BINDING PROTEIN (CAB1) gene was upregulated in rPPC1-B at dawn (Figure 5N), whereas a gene encoding a potential sucrose sensor connecting growth and development to metabolic status, TREHALOSE 6-PHOSPHATE SYNTHASE7 (TPS7; Schluepmann et al., 2003), was downregulated relative to the wild type at 6 and 10 h into the 12-h-dark period (Figure 5O).

Characterization of Diel Regulation of Circadian Clock Genes in rPPC1 Lines

Recent studies using PPCK1 RNAi lines in Kalanchoë reported that a reduced temporal peak of sucrose content phased to 2 h after dawn correlated with perturbation of the central circadian clock (Boxall et al., 2017). Of the two CIRCADIAN CLOCK ASSOCIATED1 (CCA1) genes in K. laxiflora, each of which is represented by two homeologous copies in the tetraploid genome, only the two homeologs of CCA1-2 were downregulated in rPPC1-B (Figures 5P and 5Q), whereas both homeologs of both TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (TOC1) genes were upregulated in both rPPC1-A and rPPC1-B (Figures 5R and 5S). Two other pseudoresponse regulators (PRRs) related to TOC1, namely PRR7 and PRR3/7, were upregulated in both rPPC1-A and rPPC1-B (Figures 5T and 5U), and PRR9 was induced almost fivefold in the middle of the light period, specifically in line rPPC1-B (Figure 5V).

Other-clock associated genes, including JUMONJI DOMAIN CONTAINING30/5 (JMJ30/JMJD5), EARLY FLOWERING3 (ELF3), and CYCLING DOF-FACTOR2 (CDF2), were also upregulated in line rPPC1-B (Figures 5W to 5Y) versus the wild type, whereas the single MYB-repeat transcription factor genes REVEILLE1-like (RVE1-like) and EARLY-PHYTOCHROME-RESPONSIVE1 (EPR1) as well as LIGHT NIGHT-INDUCIBLE AND CLOCK-REGULATED3-like (LNK3-like) were downregulated (Figure 5Z to 5BB). Finally, CCA1-1, FLAVIN BINDING KELCH-REPEAT F-BOX PROTEIN1 (FKF1), and GIGANTEA (GI) transcript levels were consistent among all three lines (Figures 5P, 5CC, and 5DD).

Gas-Exchange Characteristics of rPPC1 Lines under Circadian Free-Running Conditions

Under LL free-running conditions, detached K. laxiflora wild-type CAM leaves (LP6) with their petioles in water displayed a robust circadian rhythm of CO₂ uptake with a period of ∼20 h (Figure 6A). This rhythm was entirely consistent with CAM rhythms reported previously for detached LP6 of closely related Kalanchoë species with obligate CAM (Lüttge and Ball, 1978; Anderson and Wilkins, 1989). The rhythm dampened rapidly in line rPPC1-B, whereas rPPC1-A maintained a rhythm that was very similar to that of the wild type (Figure 6A).

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Figure 6.

Effects of Silencing PPC1 on CAM CO₂-Exchange Rhythms Measured under LL Conditions.

(A) Gas-exchange profile for detached CAM leaves (LP6) 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 at 15°C).

(B) Gas-exchange profile for well-watered whole young plants (nine-leaf-pair 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 in (A) and (B) represent mean CO₂ uptake of three individual plants.

(C) 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 m⁻² s⁻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 three biological replicates of LP6 from each line. Error bars indicate se calculated from three biological replicates.

(D) RAE plot for the DF rhythms.

(E) and (F) Mean period length plot (E) and mean RAE plot (F). The plotted values were calculated using the BioDare package for circadian rhythm analysis. FFT, fast Fourier transform-nonlinear least squares analysis; SR, spectral resampling analysis.

For all panels, black data are for the wild type, blue data for rPPC1-A, and red data for rPPC1-B. LL begins at 0 h in (A) to (C).

When LL CO₂ uptake was measured for well-watered whole plants (2 months old, nine leaf pairs), rPPC1-B fixed more CO₂ than the wild type and line rPPC1-A, with levels of 11,269, 7414, and 8383 µmol CO₂ m⁻², respectively (Figure 6B). The wild type maintained robust oscillations of CO₂ exchange under LL conditions, whereas rPPC1-A dampened to arrhythmia after 3 d and rPPC1-B was arrhythmic (Figure 6B). It should be noted that the LL CO₂-exchange data for intact young plants with their roots in soil (Figure 6B) differ from the data for detached LP6 (Figure 6A) because the whole plants measured in Figure 6B included leaf pairs from very young LP1, which perform C₃, through to LP6 to LP9, which perform full CAM (phase I in the dark and phase III in the light). The detached LP6 measured in Figure 6A were full CAM leaves only, at least for the wild type (Supplemental Figure 4). Thus, the more rapid and substantial dampening of the free-running CO₂ fixation rhythm toward arrhythmia in lines rPPC1-A and rPPC-1B relative to the wild type (Figure 6B) suggests that the younger leaves (LP1 to LP5) made a dominant contribution to the robust circadian rhythm in wild-type whole plants.

DF Rhythms Are Enhanced in rPPC1-B Despite the Loss of CAM-Associated CO₂ Uptake Rhythms

DF displays a robust circadian rhythm in Kalanchoë and provides a measure of a chloroplast-derived clock output that can be used for statistical analysis of circadian period, robustness, and accuracy (Gould et al., 2009; Boxall et al., 2017). We therefore measured and analyzed DF under LL (Figure 6C) to calculate rhythm statistics using BioDare (Moore et al., 2014; Zielinski et al., 2014).

Wild-type DF oscillations were very similar to those reported previously for K. fedtschenkoi (Figure 6C; Gould et al., 2009; Boxall et al., 2017). rPPC1-A and rPPC1-B had more robust oscillations than the wild type (Figure 6C). The rhythm amplitude increased slightly with time in rPPC1-A and rPPC1-B but remained relatively constant in the wild type. The relative amplitude error (RAE) plot showed a wider spread of period for the wild type than for the rPPC1 lines (Figure 6D). Mean periods were between 21.5 and 22.1 h when calculated using spectral resampling or fast Fourier transform (nonlinear least squares) methods, respectively (Figure 6E). Average periods were similar between the wild type and the rPPC1 lines. A lower mean RAE was calculated for rPPC1-A and rPPC1-B compared with the wild type (Figure 6F), supporting statistically the visibly robust and high-amplitude DF rhythm in the rPPC1 lines (Figure 6C).

Rhythm Characteristics of Core Circadian Clock and Clock-Controlled Genes

Having established that circadian control of CO₂ fixation was dampened under LL in plants lacking PPC1, whereas the circadian control of DF was enhanced under LL (Figures 6A to 6F), it was important to investigate the regulation of both core clock genes and circadian clock-controlled genes in the rPPC1 lines under LL (Figure 7).

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Figure 7.

Impact of the Loss of PPC1 Transcripts on Circadian Clock-Controlled Gene Transcript Abundance during LL Free-Running Conditions.

Mature leaves (LP6), which performed CAM in the wild type, were sampled every 4 h under constant LL conditions (100 μmol m⁻² s⁻¹ at 15°C) for the wild type and rPPC1-B. RNA was isolated and used for 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 means of three technical replicates for biological triplicates and were normalized to the loading control gene (TEDI); error bars represent the se. In all cases, plants were entrained under 12-h-light/12-h-dark cycles prior to release into LL free-running conditions at 0 h. Black data are for the wild type and red data for rPPC1-B. The gray 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. Panels show the circadian rhythm of transcript abundance under constant LL conditions (100 μmol m⁻² s⁻¹ at 15°C) for the wild type and rPPC1-B for PPC1 (A), PPC2 (B), PPCK1 (C), PPCK2 (D), PPCK3 (E), GPT2 (F), CAB (G), CCA1-1 (H), CCA1-2 (I), TOC1-1 (J), TOC1-2 (K), PRR7 (L), PRR37 (M), PRR9 (N), LNK3-like (O), JMJ30/JMJD5 (P), ELF3 (Q), CDF2 (R), RVE1-like (S), EPR1 (T), FKF1 (U), and GI (V).

The wild type displayed a rhythm in the transcript abundance of PPC1 that was absent in rPPC1-B (Figure 7A). PPC2 was rhythmic in the wild type and rPPC1-B, but its abundance was lower in rPPC1-B (Figure 7B). The PPCK1 rhythm was of greater amplitude in rPPC1-B, and the daily transcript peaks occurred 4 to 8 h later than in the wild type after the first 24 h of LL (Figure 7C). PPCK2 and PPCK3 were induced in line rPPC1-B and oscillated with higher amplitude (Figures 7D and 7E). PPCK2 peaked after the wild type on the second and third 24-h cycles under LL (Figures 7D and 7E), which is consistent with the induction detected under light/dark cycles (Figures 1F and 1G). GPT2, which is involved in the transport of G6P across the chloroplast membrane, was downregulated and had lower amplitude in rPPC1-B than the wild type (Figure 7F), whereas CAB1 was induced and oscillated robustly (Figure 7G).

In rPPC1-B, the core clock gene CCA1-1 was downregulated, phase delayed, and had lower amplitude than the wild type (Figure 7H), whereas CCA1-2, TOC1-1, and TOC1-2 were all upregulated and phase delayed relative to the wild type for the latter two peaks of LL (Figures 7I to 7K). PRR7 was upregulated and more robustly rhythmic in rPPC1-B (Figure 7L), whereas the rhythms of PRR3/7 and PRR9 dampened in rPPC1-B (Figures 7M and 7N). Finally, JMJ30/JMJD5, ELF3, CDF2, RVE1-like, EPR1, FKF1, and GI were upregulated in rPPC1-B, displaying higher amplitude, phase delays, and/or period lengthening during the LL time course (Figures 7P to 7V), whereas LNK3-like was downregulated but remained rhythmic, with a lengthening period compared with the wild type (Figure 7O).

Diel Regulation of GC Signaling and Ion Channel Genes in GC-Enriched Epidermal Peels of rPPC1-B

As CO₂ fixation and associated stomatal opening were shifted to the light period in rPPC1-B, it was important to investigate the temporal regulation of known GC signaling genes in the wild type and the rPPC1-B line (Figure 8). Since epidermal peels of Kalanchoë leaves are enriched for intact GCs, we isolated RNA from epidermal peels from the upper and lower leaf surfaces of LP6 every 4 h over the LD cycle and used them for RT-qPCR.

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Figure 8.

Impact of the Loss of PPC Activity on the Light/Dark Regulation of the Transcript Abundance of Stomatal Genes in the Epidermis.

Plants were entrained under 12-h-light/12-h-dark conditions for 7 d prior to sampling. Epidermal peel samples were separated from LP6, LP7, and LP8, with samples collected every 4 h starting at 02:00 h, 2 h into the 12-h-light period. Each biological sample represents a pool of epidermal peels taken from six LP6 leaves from three 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 means of three technical replicates for biological triplicates and were normalized to the reference gene (TEDI); error bars represent the se. In all cases, plants were entrained under 12-h-light/12-h-dark cycles for 7 d prior to sampling. Black data are for the wild type and red data for rPPC1-B. Panels show the light/dark regulation of transcript abundance under constant LL conditions (100 μmol m⁻² s⁻¹ at 15°C) for the wild type and rPPC1-B for PHOT1 (A), CRY2 (B), βCA5 (C), PATROL1 (D), CBC1/2 (E), HT1 (F), OST1 (G), SLAC1 (H), PP2C (I), RCAR3 (J), GORK (K), SKOR (L), ALMT12 (M), RMA1 (N), EDA39 (O), SOS2 (P), ACA2 (Q), ECA4 (R), MYB60 (S), and MYB61 (T).

PHOTOTROPIN1 (PHOT1) encodes a protein kinase that acts as a blue light (BL) photoreceptor in the signal transduction pathway leading to BL-induced stomatal movements (Kinoshita et al., 2001). In rPPC1-B, PHOT1 transcripts were upregulated relative to the wild type and peaked 8 h into the 12-h-light period (Figure 8A). CRYPTOCHROME2 (CRY2) encodes a photoreceptor that regulates BL responses, including the entrainment of endogenous circadian rhythms (Somers et al., 1998), and stomatal conductance via an indirect effect on ABA levels (Boccalandro et al., 2012). In rPPC1-B epidermal peels, CRY2 transcript levels were upregulated and peaked at dusk, whereas CRY2 peaked 8 h into the dark in the wild type (Figure 8B).

In Arabidopsis, GC-localized β-CARBONIC ANHYDRASE1 (CA1) and CA4 are involved in CO₂ sensing in GCs, with the ca1 ca4 mutant displaying impaired stomatal control in response to CO₂ (Hu et al., 2010). In rPPC1-B, a β-carbonic anhydrase gene was much more strongly induced at dawn relative to the wild type (Figure 8C). PATROL1 controls the tethering of the proton ATPase AHA1 to the plasma membrane and is essential for stomatal opening in response to low CO₂ and light levels (Hashimoto-Sugimoto et al., 2013). The cycle of PATROL1 in epidermal peels was slightly different between rPPC1-B and the wild type, with rPPC1-B peaking 4 h into the dark but the wild type peaking at 4 h into the light period (Figure 8D).

CONVERGENCE OF BLUE LIGHT AND CO₂ 1/2 (CBC1/2) stimulate stomatal opening by inhibiting S-type anion channels in response to both BL and low CO₂ and connect BL signals perceived by PHOT1 with the protein kinase HIGH LEAF TEMPERATURE1 (HT1; Hiyama et al., 2017). Both proteins interact with and are phosphorylated by HT1. In rPPC1-B, CBC1/2 was upregulated compared with the wild type both in the second half of the dark period and at dawn (Figure 8E). HT1 acts as a negative regulator of high-CO₂-induced stomatal closure (Hashimoto et al., 2006). HT1 transcript levels were reduced throughout the light/dark cycle relative to the wild type in rPPC1-B (Figure 8F). OPEN STOMATA1 (OST1), encoding a protein kinase that acts downstream of HT1 (Imes et al., 2013), was upregulated in rPPC1-B both at 8 h into the dark period and at dawn, the time of the daily trough in the wild type (Figure 8G).

Genes involved in ion transport across the plasma membrane and tonoplast play a crucial role in the changes in cell turgor pressure that drive stomatal movements (Jezek and Blatt, 2017). SLOW ACTIVATED ANION CHANNEL1 (SLAC1) is a key player in the closure of stomata in response to high CO₂ concentrations (Hedrich and Geiger, 2017). SLAC1 is regulated by ABA signaling, which requires dephosphorylation steps catalyzed by PP2C. In rPPC1-B, SLAC1 was upregulated and peaked during the light (Figure 8H) and PP2C was upregulated relative to the wild type during the dark period (Figure 8I). ABA is recognized and bound by the REGULATORY COMPONENT OF ABA RECEPTORS (RCARs)/PYRABACTIN RESISTANCE1, which interact with PP2C to stimulate ABA signaling (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009). In rPPC1-B epidermal peels, RCAR3 transcripts were induced spanning dusk and the first half of the dark period and peaked at least 4 h earlier than in the wild type (Figure 8J).

In rPPC1-B, the transcript level of GUARD CELL OUTWARD RECTIFYING K⁺ CHANNEL (GORK), which is involved in regulating stomatal movement according to water status (Ache et al., 2000), peaked 4 h earlier than in the wild type (Figure 8K). STELAR K⁺ OUTSIDE RECTIFIER (SKOR) encodes a selective outward-rectifying potassium channel (Gaymard et al., 1998). SKOR transcript levels peaked at dawn in the wild type, whereas in rPPC1-B, SKOR transcript levels reached their daily minimum at dawn (Figure 8L). The transcript level of plasma membrane-localized ALUMINUM ACTIVATED MALATE TRANSPORTER12 (ALMT12), which is involved in dark-, CO₂-, ABA-, and water deficit-induced stomatal closure, was elevated relative to the wild type at dawn in rPPC1-B (Figure 8M). The E3 UBIQUITIN-PROTEIN LIGASE RMA1 promotes the ubiquitination and proteasomal degradation of the aquaporin PIP2:1, which plays a role in GC regulation (Grondin et al., 2015). RMA1 was induced threefold at dawn in rPPC1-B compared with the wild type (Figure 8N). EMBRYO SAC DEVELOPMENT ARREST39 (EDA39) is a calmodulin binding protein that promotes stomatal opening (Zhou et al., 2012). In rPPC1-B, EDA39 was upregulated relative to the wild type and peaked at dusk (Figure 8O).

SALT OVERLY SENSITIVE2 (SOS2) is a Calcineurin B-Like-interacting protein kinase involved in the regulatory pathway for intracellular Na⁺ and K⁺ homeostasis and salt tolerance (Liu et al., 2000). SOS2 interacts with and activates the vacuolar H⁺/Ca²⁺ antiporter CAX1, thereby functioning in cellular Ca²⁺ homeostasis, an important function during stomatal opening and closing (Cheng et al., 2004). SOS2 transcript levels were elevated relative to the wild type at all time points; in particular, it was induced approximately fivefold at its peak 4 h into the dark period in epidermal peels of rPPC1-B relative to the wild type (Figure 8P). Ca²⁺-ATPase2 (ACA2) and ENDOMEMBRANE-TYPE Ca²⁺-ATPASE4 (ECA4) catalyze the hydrolysis of ATP coupled with the translocation of calcium from the cytosol into the endoplasmic reticulum and/or an endomembrane compartment (Jezek and Blatt, 2017). ACA2 and ECA4 were induced by approximately fourfold to approximately eightfold in rPPC1-B epidermal peels relative to the wild type, particularly when they reached peak levels at 4 h into the 12-h-dark period (Figures 8Q and 8R).

Finally, the transcription factor MYB60 is involved in stomatal opening in response to light and promotes GC deflation in response to water deficit (Cominelli et al., 2005). In rPPC1-B, MYB60 was induced approximately fivefold relative to the wild type at dawn, although it also maintained a dusk-phased peak of transcript abundance like the wild type (Figure 8S). MYB61 functions as a transcriptional regulator of stomatal closure (Liang et al., 2005). MYB61 was downregulated in rPPC1-B relative to the wild type and peaked 8 h later in rPPC1-B, 4 h into the dark period, whereas in the wild type, MYB61 peaked in the light 4 h before dusk (Figure 8T).

Diel Regulation of GC Metabolism Genes in GC-Enriched Epidermal Peels of rPPC1-B

PPC1 and PPC2 transcript levels were low in epidermal peels (Supplemental Figures 5A and 5B) compared with whole leaves (Figure 1A). PPC3 and PPC4 transcripts were more abundant in wild-type epidermal peels (Supplemental Figures 5C and 5D) relative to whole leaves (Figures 1C and 1D). Furthermore, PPC3 and PPC4 were upregulated in epidermal peels of rPPC1-B compared with the wild type, and their transcript abundance peaked 4 h into the dark period, whereas in the wild type, the abundance of both transcripts peaked at the end of the dark period or dawn (Supplemental Figures 5A to 5D).

CAM-specific PPCK1 was upregulated in rPPC1-B epidermal peels compared with the wild type, whereas the expression of PPCK2, which does not currently have a proposed role in the regulation of CAM-specific PPC1 in the mesophyll, was unchanged (Supplemental Figures 5E and 5F). Both PPCK2 and PPCK3 transcripts were more abundant than PPCK1 in epidermal peels, suggesting that their encoded proteins might regulate the activity of the GC PPCs (Supplemental Figures 5F and 5G). Most strikingly, PPCK3 was upregulated approximately fivefold at 4 h after lights-on in rPPC1-B epidermal peels relative to the wild type (Supplemental Figure 5G).

In C₃ plants, starch is degraded before dawn to fuel stomatal opening (Blatt, 2016). In Arabidopsis GCs, BAM1 encodes the major starch-degrading enzyme (Valerio et al., 2011; Prasch et al., 2015; Horrer et al., 2016; Santelia and Lunn, 2017) that functions with AMY3 to mobilize starch at dawn, releasing maltose in the chloroplasts (Horrer et al., 2016). In rPPC1-B epidermal peels, GWD transcript levels were halved relative to the wild type at its peak (i.e., 8 h into the 12-h light period; Supplemental Figure 5H). AMY3a and AMY3b were upregulated at dusk and in the second half of the light period, respectively (Supplemental Figures 5I and 5J). BAM1 was upregulated at dawn compared with the wild type and peaked with a very similar transcript abundance to the wild type at 20:00 h, 8 h into the dark period (Supplemental Figure 5K), whereas BAM3 transcript levels were approximately threefold lower in rPPC1-B relative to the wild type at the time of its nocturnal peak, which also occurred 8 h into the dark (Supplemental Figure 5L). BAM9 expression in rPPC1-B rose later than in the wild type, lagging behind the wild type at dusk and 4 h into the dark, and it stayed approximately fivefold higher at dawn and 4 h into the light period, when the wild type reached its daily trough (Supplemental Figure 5M). BAM9 in Arabidopsis is predicted to be catalytically inactive and has no known function to date (Monroe and Storm, 2018). The peak of PHS1 was delayed by 4 h, peaking at the light/dark transition (Supplemental Figure 5N), and MEX1 was downregulated in epidermal peels of rPPC1-B, peaking at 08:00 h in the light, 4 h after the wild-type peak (Supplemental Figure 5O).

Diel Regulation of Core Circadian Clock Genes in Epidermal Peels of rPPC1-B

The circadian clock genes PRR3/7 and PRR7 were upregulated in epidermal peels of rPPC1-B relative to the wild type (Supplemental Figures 5P and 5Q), with a similar diel pattern to that measured in whole CAM leaves (Figures 5S and 5T). PRR3/7 peaked 4 h earlier in rPPC1-B, whereas PRR7 transcript levels were greater throughout the light period in rPPC1-B (Supplemental Figures 5P and 5Q). LUX is a component of the morning transcriptional feedback circuit within the clock, encoding a transcription factor that directly regulates the expression of PRR9 by binding to specific sites in its promoter (Helfer et al., 2011). In epidermal peels, LUX was downregulated greater than sevenfold in rPPC1-B compared with the wild type at its peak, 4 h into the light period, and also peaked 4 h later than the wild type (Supplemental Figure 5R).

Diel Regulation of Additional GC Signaling Genes in GC-Enriched Epidermal Peels of rPPC1-B

GCs perceive CO₂ and regulate stomatal aperture via ABA and reactive oxygen species signaling, with low CO₂ levels mediating stomatal opening and high CO₂ levels causing closure (Chater et al., 2015). NADP OXIDOREDUCTASE encodes an NADP binding Rossman-fold superfamily protein that we only detected in epidermal peels in Kalanchoë. The enzyme encoded by this gene might generate the H₂O₂ burst induced by ABA as part of the stomatal closure signaling pathway (Daszkowska-Golec and Szarejko, 2013). This transcript was upregulated approximately threefold in rPPC1-B at dawn (Supplemental Figure 5S).

In Arabidopsis, the transcription factors MYB94 and MYB96 function together in the activation of cuticular wax biosynthesis under drought stress (Lee et al., 2016). MYB96 may also be involved in the response to drought stress in Arabidopsis through ABA signaling that mediates stomatal closure via the RD22 pathway (Seo et al., 2011). Both MYB94 and MYB96 were induced by as much as threefold in rPPC1-B compared with the wild type (Supplemental Figures 5T and 5U). Specifically, MYB94 transcript levels rose 8 h earlier than they did in the wild type and were already close to peak levels by the light-to-dark transition, whereas in the wild type, MYB94 levels peaked sharply 8 h into the dark period (Supplemental Figure 5T). MYB96 transcription was induced relative to the wild type at both dawn and dusk, but the peak of MYB96 transcription at dawn represented the largest fold change relative to the wild type (Supplemental Figure 5U).