The Calcium-Dependent Protein Kinase CPK33 Mediates Strigolactone-Induced Stomatal Closure in Arabidopsis thaliana

Strigolactones (SLs) are known to mediate plant acclimation to environmental stress. We recently reported that SLs acted as prominent regulators in promotion of stomatal closure. However, the detailed mechanism by which SLs induce stomatal closure requires further investigation. Here we studied the essential role of the calcium (Ca2+) signal mediating by the calcium-dependent protein kinase (CPK) in SL-induced stomatal closure. SL-induced stomatal closure was strongly inhibited by a Ca2+ chelator and Ca2+ channel blockers, indicating that Ca2+ functions in SL promotion of stomatal closure. Through examining a collection of cpk mutants, we identified CPK33, potentially acting as a Ca2+ transducer, which is implicated in guard cell SL signaling. SL- and Ca2+-induced stomatal closure were impaired in cpk33 mutants. CPK33 kinase activity is essential for SL induction of stomatal closure as SL-induced stomatal closure is blocked in the dead kinase mutant of CPK33. The cpk33 mutant is impaired in H2O2-induced stomatal closure, but not in SL-mediated H2O2 production. Our study thus uncovers an important player CPK33 which functions as an essential Ca2+ signals mediator in Arabidopsis guard cell SL signaling.


INTRODUCTION
Plants are sessile organisms that confront with a wide range of biotic and abiotic stress conditions during their life cycle. As a strategy to cope with environmental stress, plants utilize stomatal pores, each consisting of a pair of guard cells, that open and close to modulate gas exchange for photosynthesis, transpirational water loss, and stomatal immunity, thereby allowing plants to respond properly to diverse environmental stress (Hetherington and Woodward, 2003;Ruszala et al., 2011;Blatt et al., 2017;Melotto et al., 2017). Guard cells have developed sophisticated mechanisms which enable plants to appropriately control of stomatal apertures in mediating response of environmental stimuli such as light, drought, and external calcium ion (Ca 2+ ) (Kim et al., 2010;Murata et al., 2015). Particularly, phytohormones, including abscisic acid (ABA), ethylene, brassinosteroids, strigolactones (SLs), salicylic acid (SA), jasmonic acid (JA), and small signaling peptides have been found to play pivotal roles through their coordination with various key guard cell signaling components to modulate stomatal apertures in response to fluctuating environmental stress (Kim et al., 2010;Daszkowska-Golec and Szarejko, 2013;Munemasa et al., 2015;Murata et al., 2015;Cardinale et al., 2018;Mostofa et al., 2018;Qu et al., 2019;Zhang et al., 2019).
Other than their notable roles in shoot branching (Al-Babili and Bouwmeester, 2015), SLs have been found to be implicated in many plant developmental processes such as primary root development (Kapulnik et al., 2011;Ruyter-Spira et al., 2011), adventitious root formation (Rasmussen et al., 2012;Sun et al., 2015), secondary growth (Agusti et al., 2011), photomorphogenesis (Shen et al., 2007;Shen et al., 2012), flower development (Snowden et al., 2005;Kohlen et al., 2012;Liu et al., 2013), and hypocotyl elongation (Tsuchiya et al., 2010;Jia et al., 2014). Notably, accumulating data indicated that SLs are also involved in mediating plant responses to environmental stress, rendering plants to defend against abiotic stress as well as against specific bacterial and fungal species (Marzec, 2016;Cardinale et al., 2018;Mostofa et al., 2018). Specifically, SLdeficient and SL-signaling mutants exhibited drought hypersensitivity, whereas SLs application rescued droughtsensitive phenotypes of SL-deficient mutants and strengthened drought tolerance of wild-type (WT) plants (Bu et al., 2014;Ha et al., 2014;Liu et al., 2015;Li et al., 2017;Zhang et al., 2018). We recently revealed that SLs could induce stomatal closure through enhancing hydrogen peroxide (H 2 O 2 ) and nitric oxide production in an ABA-independent manner, possibly preventing water loss and pathogen invasion and thereby resulting in plant acclimation to environmental stress Zhang et al., 2018). However, the detail molecular mechanism, especially the intracellular events that are initiated by SLs in guard cells, remains largely unclear. To this end, the potential downstream component(s) that transduces guard cell SL signaling is thus required to be determined.
It has long been known that calcium functions as a secondary messenger in stomatal closure (Blatt, 2000;Bowler and Fluhr, 2000;Kim et al., 2010;Murata et al., 2015;Ray, 2017). For instance, through H 2 O 2 activation of Ca 2+ -permeable cation channels, ABA triggers an increment of cytosolic Ca 2+ [(Ca 2+ ) cyt ] that includes Ca 2+ influx elevation from extracellular spaces and Ca 2+ release from intracellular stores (Pei et al., 2000). In addition, JA-induced stomatal closure is mediated by cytosolic Ca 2+ since JA signaling in guard cells is inhibited by Ca 2+ channel blockers (Suhita et al., 2003;Suhita et al., 2004). Likewise, Ca 2+ signaling is found to be implicated in SA induction of stomatal closure in a mode of action similar to studies of ABA-and JA-mediated stomatal closure (Prodhan et al., 2018). Eventually, the resultant guard cell cytosolic Ca 2+ elevation promotes stomatal closure by stimulation of SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) anion channels and/or the GATED OUTWARDLY-RECTIFYING K + (GORK) channel (Kim et al., 2010;Murata et al., 2015;Roux and Leonhardt, 2018).
In this study, the importance of Ca 2+ in SL-induced stomatal closure was firstly determined by performing pharmacological studies, which indicate that Ca 2+ acts as a prominent mediator functions in SL induction of stomatal closure. Through examining a collection of cpk mutants, we identified CPK33 (and possibly CPK10) which is implicated in guard cell SL signaling. SL activation of stomatal closure, as well as Ca 2+ -induced stomatal closure, was greatly impaired in cpk33 mutants. Additionally, CPK33 kinase activity is essential for SL induction of stomatal closure. We further found that H 2 O 2induced stomatal closure was moderately impaired in cpk33 mutants whereas SL-mediated H 2 O 2 production was maintained in cpk33 mutants. Taken together, our study uncovers an important player CPK33 that functions as an important mediator in SL signaling in Arabidopsis guard cells.
To obtain the double mutant cpk10 cpk33, the cpk10 mutant and cpk33 mutants (cpk33-1 and cpk33-2) were crossed. Homozygous double mutants were determined by PCRs using a combination of a gene-specific primer and a T-DNA border primer. The primers used were listed in Table S1.
Seeds were sterilized and sown for germination in 1/2 Murashige and Skoog (MS) medium supplemented with 0.8% (w/v) agar and 1% sucrose (w/v). Seedlings were transplanted into pots and subsequently kept in a growth chamber with a 16h/ 8h (light/darkness) regime at 21°C. Fully expanded rosette leaves detached from 4-to 6-week-old healthy plants were harvested for immediate use.

Stomatal Aperture Bioassay
Stomatal apertures were measured as described previously Zhang et al., 2019). In brief, epidermal strips of fully expanded leaves were incubated in the MES-KCl buffer (10 mM MES-KOH/50 mM KCl, pH 6.15) to promote stomatal opening following treatments as described in each experiment. Finally, stomatal apertures on the abaxial epidermis were measured and presented as mean ± SE of three replicates.

H 2 O 2 Content Detection in Guard Cells
H 2 DCF-DA was utilized to determine H 2 O 2 content in stomata. The measurement of H 2 O 2 content was performed according to Lv et al. (2018) and Zhang et al. (2019). Briefly, epidermal strips with open stomata were incubated in the Tris-KCl solution containing 50 µM H 2 DCF-DA for 10 min of darkness at 25°C in the absence or presence of GR24. Epidermal strips were washed three times with Tris-KCl buffer to remove excess dye under darkness. The fluorescence in stomata was visualized using a TCS SP2 confocal laser scanning microscope (Leica Lasertechnik GmbH, Germany). The fluorescence intensity representing the endogenous H 2 O 2 content was determined using ImageJ software. Fluorescence intensities were normalized to those of controls. The data for fluorescence intensities represent the mean ± SE of three replicates.

Statistical Analyses
Statistical analyses were performed using a one-way ANOVA to discriminate significant differences followed by the least significant difference test.
It was reported that exogenous Ca 2+ could promote stomatal closure (Blatt, 2000;Ray, 2017). Since we have shown that Ca 2+ plays a crucial role in SL-induced stomatal closure, we further examined the stomatal response of SL-related mutants in response to Ca 2+ . We found that addition of Ca 2+ (CaCl 2 ) induced a similar stomatal closure in SL-related mutants as in WT plants, whereas SL-signaling mutants max2 and d14 were insensitive to GR24 as reported previously ( Figure S1; Lv et al., 2018). This result indicates that Ca 2+ might act as a signaling molecule downstream of SL-signaling and SL-biosynthetic genes.

SL-and Ca 2+ -Induced Stomatal Closure
Were Impaired in the Cpk33 Mutant Given that Ca 2+ is essential for SL-induced stomatal closure (Figure 1), we sought to identify the specific Ca 2+ sensor(s) that is involved in guard-cell SL signaling. CPKs, as Ca 2+ sensors, are important in the regulation of ABA-mediated and Ca 2+ -mediated stomatal closure (Kim et al., 2010;Boudsocq and Sheen, 2013;Schulz et al., 2013;Simeunovic et al., 2016). To determine whether any CPK is required for SL signaling in guard cells, we firstly examined SL-induced stomatal closure in the absence and presence of CPK inhibitors TFP and staurosporine (ST) (Li et al., 1998). TFP and ST significantly inhibited GR24-induced stomatal closure ( Figure S2), suggesting that CPK(s) is truly indispensable for SL-induced stomatal closure.
Furthermore, to identify CPK(s) that might be involved in SL signaling in guard cells, the effect of GR24 on stomatal aperture was examined in a collection of cpk mutants, including cpk4, cpk8, cpk10, cpk11, cpk23, cpk33-1, cpk33-2, cpk3 cpk5 cpk6 cpk11, and cpk5 cpk6 cpk11 cpk23 (Figure 2). GR24 significantly induced stomatal closure in most of examined cpk mutants. Conversely, SL-induced stomatal closure is greatly impaired in cpk33-1 and cpk33-2 mutants, and marginally impaired in cpk10 mutants ( Figure 2). To overcome the potential redundant function between CPK10 and CPK33, we generated double mutants cpk10 cpk33-1 and cpk10 cpk33-2. The double mutants cpk10 cpk33 exhibited insensitivity to SL-induced stomatal closure to a similar extent to that of the cpk33 mutants (Figure 2), suggesting that CPK10 exerted slight effect on SL-induced stomatal closure. Nevertheless, we found that CPK33 is predominantly involved in guard cell SL signaling, and we thus concentrated on CPK33 in subsequent studies.
Because SL promotion of stomatal closure depends on Ca 2+ , we hypothesized that disruption of CPK33 would impair Ca 2+ signaling transduction. To test this hypothesis, we investigated the stomatal response following exogenous Ca 2+ application in cpk33-1 and cpk33-2 mutants. Exogenous Ca 2+ stimulated stomatal closure in WT plants, but not in cpk33-1 and cpk33-2 mutants (Figure 3). Taken together, our results indicate that CPK33 acts as an important Ca 2+ sensor that is involved in guard-cell SL signaling.

CPK33 Kinase Activity Is Essential for SL-Induced Stomatal Closure
Previous studies have shown that the kinase activity of CPK33 is essential for SLAC1 channels and GORK channels activities and ABA-induced stomatal movement (Li C.L et al., 2016;Corratgé-Faillie et al., 2017). To investigate whether the in vivo kinase activity of CPK33 is also required for SL-induced stomatal closure, we performed stomatal bioassay analysis upon GR24 treatment using cpk33-1 and cpk33-2 mutants, two independent cpk33-1 complementation lines (35::CPK33 cpk33 #1 and #2), and two independent lines expressing a kinase-inactive CPK33 K102R construct in cpk33-1 (35::CPK33 K102R cpk33 #1 and #2). Two 35::CPK33 cpk33 lines exhibited GR24-sensitive phenotype similar to that of WT plants, while we found that 35:: CPK33 K102R cpk33 plants, as well as cpk33-1 and cpk33-2 mutants, failed to close stomata in response to GR24 ( Figure  4). Thus, this result suggests that CPK33 kinase activity is essential for stomatal closure induced by SLs.
FIGURE 2 | Strigolactone-stimulated stomatal closure is mediated by CPK33. Leaf epidermal peels of WT plants and a collection of cpk mutants were exposed to the MES-KCl buffer in the absence and presence of 1 mM GR24. Stomatal apertures were measured and presented as means ± SE of three independent experiments. Bars with different letters represent statistically significant differences.

Effects of the cpk33 Mutation on SL-Induced H 2 O 2 Production and H 2 O 2 -Mediated Stomatal Closure in Guard Cells
It was reported previously that SLs stimulate H 2 O 2 accumulation, and the resultant H 2 O 2 acts as an early signal component in the induction of stomatal closure triggered by SLs . To investigate the genetic relationship between H 2 O 2 and CPK33 in guard cell SL signaling, we examined the SL effect on H 2 O 2 production in cpk33 mutants using the H 2 O 2 fluorescent probe H 2 DCF-DA. In line with previous data, GR24 stimulated H 2 O 2 production in WT plants. Similarly, GR24 also stimulated H 2 O 2 production in cpk33 mutants ( Figure 5A), suggesting that CPK33 disruption did not affect SL-induced H 2 O 2 production.
To further investigate the position of H 2 O 2 production and CPK33 in guard cell SL signaling pathway, we next examined the stomatal aperture upon exogenous H 2 O 2 in cpk33 mutants. Exogenous application of H 2 O 2 significantly stimulated stomatal closure in WT plants, but H 2 O 2 -induced stomatal closure is partially impaired in cpk33 mutants in comparison with WT plants ( Figure 5B). Altogether, our results indicate that CPK33 possibly functions downstream of H 2 O 2 production in guard-cell SL signaling. It appears that, as a paradigm, H 2 O 2 activates CPK(s) that function as signal mediator(s) in guard cells. However, it remains to be explored whether and how CPK33 is activated by SL-induced H 2 O 2

DISCUSSION
It has been shown that SLs were implicated in various developmental processes of plants and mediate their responses to environmental stress (Al-Babili and Bouwmeester, 2015;Marzec, 2016;Cardinale et al., 2018;Mostofa et al., 2018). Recently we found that SLs function as common regulators to induce stomatal closure Zhang et al., 2018). We further elucidated that SLinduced stomatal closure is accomplished through enhancing production of H 2 O 2 and nitric oxide, eventually promoting plant resilience to environmental stress Zhang et al., 2018). However, no significant effect on stomatal aperture was observed when spraying GR24 onto intact plants, although SL-related mutants were genetically confirmed to display higher stomatal conductance (Kalliola et al., 2019). The discrepancy in SLinduced stomatal closure may be due to the different materials used, intact plants and epidermal strips, which possibly results in different efficacy of GR24 such as permeability problem. It has been reported that Ca 2+ and its sensors CPKs are crucial for stomatal closing (Blatt, 2000;Bowler and Fluhr, 2000;Pei et al., 2000;Kim et al., 2010;Murata et al., 2015;Ray, 2017). OST1, a Ca 2 + -independent kinase, was found to be dispensable for SLinduced stomatal closure . Based on these observations, we thus hypothesize that Ca 2+ as well as its transducers CPK(s) is implicated in SL-triggered stomatal closure.
In this study we found that Ca 2+ chelator EGTA, and Ca 2+ channel blockers LaCl 3 and AlCl 3 significantly suppressed SL-induced stomatal closure (Figure 1), suggesting the requirement of extracellular free Ca 2+ and Ca 2+ channels in the modulation of SL-induced stomatal closure. Our pharmacological and further genetic analyses suggest that SL requires cytosolic Ca 2+ signals to promote stomatal closure (Figures 1  and 3). It is possible that SLs stimulate either Ca 2+ sensitivity priming or cytosolic Ca 2+ elevation to perturb Ca 2+ signals, thereby resulting in stomatal closure. It is thus demanded to detect the alteration of Ca 2+ signals through Ca 2+ detection fluorescence dyes and/or different Ca 2+ biosensors in the future analyses.
It has long been known that CPKs, acting as Ca 2+ sensors, are important mediators of Ca 2+ -dependent stomatal closure and ion FIGURE 3 | Ca 2+ -induced stomatal closure is significantly impaired in cpk33 mutants. Leaves of WT plants, cpk33-1, and cpk33-2 mutants with open stomata were exposed to the MES-KCl buffer containing different concentrations of CaCl 2 . Stomatal apertures were subsequently measured and presented as means ± SE of three independent experiments. Bars with different letters represent statistically significant differences.
FIGURE 4 | CPK33 kinase activity is essential for strigolactone-induced stomatal closure. Leaves of WT, cpk33-1, cpk33-2, 35::CPK33 cpk33 #1, 35::CPK33 cpk33 #2, 35::CPK33 K102R cpk33 #1, and 35::CPK33 K102R cpk33 #2 plants with open stomata were exposed to the MES-KCl buffer in the absence or presence of 1 µM GR24. Stomatal apertures were measured and presented as means ± SE of three independent experiments. Bars with different letters represent statistically significant differences. channel activation (Kim et al., 2010;Boudsocq and Sheen, 2013;Schulz et al., 2013;Simeunovic et al., 2016 -induced stomatal closure, were impaired in cpk33 mutants (Figures 2 and 3). Interestingly, CPK10 disruption slightly impaired SL-induced stomatal closure ( Figure 2). The double mutant cpk10 cpk33 exhibited insensitivity to SL-induced stomatal closure to a similar extent to that of the cpk33 mutant (Figure 2), suggesting a prime role of CPK33 and a differential contribution of CPK10 and CPK33 in SL-induced stomatal closure. It was found previously that CPK10 was involved in plant responses to drought stress via modulation of ABA-and Ca 2+ -mediated stomatal closure (Zou et al., 2010). Considering the complexity of crosstalk of distinct signaling pathways, it is plausible that CPK10 might indirectly impact on the SL activation of stomatal closure. Alternatively, given that there are 34 CPKs in Arabidopsis (Boudsocq and Sheen, 2013), a yet unidentified CPK, rather than CPK33, may function redundant with CPK10 in modulation of SLinduced stomatal closure. Nevertheless, the role of CPK10 (possible with another redundant CPK) in SL promotion of stomatal closure needs to be investigated. Intriguingly, guard cell outward potassium channel GORK is specifically stimulated by CPK33 to active outward potassium ion currents, showing that, unlike its negative regulation of anion channels (Li C.L et al., 2016), CPK33 positively modulates the GORK channel activity to promote stomatal closure (Corratgé-Faillie et al., 2017). Consequently, cpk33 mutants were delayed in stomatal closure under normal conditions (Corratgé-Faillie et al., 2017). In this regard, it is possible that SLs could exploit CPK33activated GORK channels to promote stomatal closure. It thus remains to be investigated whether SLs could stimulate CPK33activated GORK channel activity. CPK33 was reported as a negative regulator in ABA-modulated stomatal closure (Li C.L et al., 2016). SL activation of stomatal closure, however, were inhibited in cpk33 mutants (Figure 2), indicating that CPK33 positively functions in SL-induced stomatal closure. Taken together, our results and previous reports suggest that the CPK-dependent Ca 2+ recognition conveying by CPK33 could be essential for SL signaling, as well as ABA signaling in guard cells. Nevertheless, the role of opposite effect of CPK33 on anion and potassium channels, as well as the discrepant role of CPK33 in guard cell ABA and SL signaling, is required to be further studied.
Integrated previous studies with our present genetic and physiological analyses, we propose that CPK33, as a Ca 2+ transducer, acts downstream of H 2 O 2 and Ca 2+ (Figure 6). In guard cells, following perception by D14 and MAX2, SLs stimulate the production of H 2 O 2 that possibly activates CPK33, which likely modulates anion and potassium channels to promote stomatal closure (Li C.L et al., 2016;Corratgé-Faillie et al., 2017; Figure 6). Indeed, it has been reported that CPK33 positively stimulates the GORK channels while negatively regulates SLAC1 channels to promote stomatal closure (Li C.L et al., 2016;Corratgé-Faillie et al., 2017). However, it remains to be examined whether and how CPK33 is activated by SL-induced H 2 O 2 . It has been reported that H 2 O 2 -stimulated cytosolic Ca 2+ elevation is crucial for stomatal closure (Pei et al., 2000). CPK33 was confirmed to be a typical Ca 2 + -dependent kinase (Corratgé-Faillie et al., 2017), indicating that CPK33 activity requires cytosolic Ca 2+ increase. Therefore, it will be intriguing to investigate whether SLs and/or SL-induced H 2 O 2 are able to promote cytosolic Ca 2+ which presumably is sensed by CPK33 in SL-mediated guard cell signaling. The finding that the stomata of cpk33 mutants remain considerable response to H 2 O 2 indicates that other factor(s) might also required for SL-triggered stomatal closure. In the meanwhile, our results emphasizes the essential role of H 2 O 2 serving as a hub in the complicated hormonemediated stomatal closure. Altogether, our study reinforces the understanding of the molecular mechanism by which SLs induce stomatal closure and provides new insights to improve stress acclimatization of plants.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

AUTHOR CONTRIBUTIONS
YZ and GW designed the experiments. XW, SL, XH, XG, XS, JK, ZL, and YZ performed the experiments. BC assisted in microscope. WZ contributed materials. YZ, CL, and GW wrote the manuscript. All authors reviewed the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019. 01630/full#supplementary-material FIGURE 6 | A working model for CPK33-mediated strigolactone (SL) signaling in guard cells. Following perception by D14 and MAX2, SLs stimulate the production of H 2 O 2 that possibly activates the Ca 2+ transducer CPK33 which likely modulates anion and potassium channels to promote stomatal closure. The question mark stands for an unknown molecule(s) that is downstream of CPK33 in SL-mediated stomatal closure. It remains to be explored whether and how SLs and/or SL-induced H 2 O 2 could promote cytosolic Ca 2+ which is presumably sensed by CPK33 in SL-mediated guard cell signaling.