Two calcium-dependent protein kinases enhance maize drought tolerance by activating anion channel ZmSLAC1 in guard cells.

Stomatal closure is an important process to prevent water loss in plants response to drought stress, which is finely modulated by ion channels together with their regulators in guard cells, especially the S-type anion channel AtSLAC1 in Arabidopsis. However, the functional characterization and regulation analyses of anion channels in gramineous crops, such as in maize guard cells are still limited. In this study, we identified an S-type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and involved in stomatal closure under drought stress. We found that two Ca2+ -dependent protein kinases ZmCPK35 and ZmCPK37 were expressed in maize guard cells and localized on the plasma membrane. Lesion of ZmCPK37 resulted in drought-sensitive phenotypes. Mutation of ZmSLAC1 and ZmCPK37 impaired ABA-activated S-type anion currents in maize guard cells, while the S-type anion currents were increased in the guard cells of ZmCPK35- and ZmCPK37-overexpression lines. Electrophysiological characterization in maize guard cells and Xenopus oocytes indicated that ZmCPK35 and ZmCPK37 could activate ZmSLAC1-mediated Cl- and NO3 - currents. The maize inbred and hybrid lines overexpressing ZmCPK35 and ZmCPK37 exhibited enhanced tolerance and increased yield under drought conditions. In conclusion, our results demonstrate that ZmSLAC1 plays crucial roles in stomatal closure in maize, whose activity is regulated by ZmCPK35 and ZmCPK37. Elevation of ZmCPK35 and ZmCPK37 expression levels is a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress.

Introduction

Plant stomata, formed by guard cells on leaf surface, are the major pathway for water‐ and gas‐exchange with the atmosphere. Stomatal aperture decreases when plants suffer drought stress, high concentration of CO2, pathogen invasion, etc., to modulate dynamic balance between leaf transpiration and photosynthesis for better survival (Kim et al., 2010; Melotto et al., 2006; Qi et al., 2018a; Schroeder et al., 2001; Sussmilch et al., 2017; Vavasseur and Raghavendra, 2005; Zeiger, 1983). Stomatal closure is exerted by deflation of guard cells surrounding the stomatal pore. Transmembrane transporters/channels, such as K+ channels and anion channels, are responsible for the turgor change in guard cells that controls stomatal movement (Fairley‐Grenot and Assmann, 1992, 1993; Kim et al., 2015; Pandey et al., 2007; Qi et al., 2018a).

Light could induce stomatal opening by stimulating inward‐rectifying K+ channels localized on the plasma membrane (PM) of guard cells (Gao et al., 2017; Kim et al., 2010; Qi et al., 2018a). Under drought stress, ABA (abscisic acid) is rapidly accumulated in plant leaves and guard cell apoplast (Munemasa et al., 2015), then quickly activates a complex membrane transport system in guard cells to close stomata (Raghavendra et al., 2010). This ABA‐mediated stomatal closure involves ROS and Ca2+ signalling that regulate the PM‐located ion channels by different protein kinases (Gong et al., 2020; Qi et al., 2018a; Raghavendra et al., 2010).

In Arabidopsis, the slow anion channel‐associated 1 (AtSLAC1) is specifically expressed in guard cells, and functions as the main anion channel mediating the efflux of Cl‐ and NO3 ‐ from guard cells during stomatal closing (Negi et al., 2008; Vahisalu et al., 2008). The anion channel AtSLAC1 is activated by either kinases or by interaction with other proteins. So far, many protein kinases have been identified, including open stomata 1 (AtOST1/AtSnRK2.6) (Geiger et al., 2009), Ca2+‐dependent protein kinases (AtCPK3, AtCPK6, AtCPK21, AtCPK23), calcineurin‐B like protein 5 (AtCBL5)–CBL‐interacting protein kinase 11 (AtCIPK11) and AtCBL1/9‐AtCIPK23 modules, and all these kinases could directly phosphorylate and activate AtSLAC1 (Brandt et al., 2012; Geiger et al., 2010; Maierhofer et al., 2014; Mori et al., 2006; Saito et al., 2018; Scherzer et al., 2012). Besides, a receptor‐like kinase guard cell hydrogen peroxide‐resistant 1 (AtGHR1) can also interact with and activate AtSLAC1 (Hua et al., 2012; Sierla et al., 2018).

The main knowledge about stomatal closure are derived from Arabidopsis, whose stoma is formed by a pair of kidney‐shaped guard cells. By contrast, maize (Zea mays) stomatal complex is formed by a pair of dumbbell‐shaped guard cells together with the flanking subsidiary cells. During maize stomatal opening, K+ ions are released by subsidiary cells, then flux into and inflate guard cells through inward K+ channels. Our previous studies have identified some of these inward K+ channels, including KZM1/ZmK2.1, KZM2 and KZM3 in maize guard cells (Gao et al., 2017, 2019; Philippar et al., 2003). During stomatal closure, the K+ ions efflux from maize guard cells might be mediated by the outward K+ channel ZORK (Büchsenschütz et al., 2005). Recently, an S‐type anion channel ZmSLAC1, similar to AtSLAC1, was identified as a NO3 ‐ channel that is important for maize stomatal closure (Qi et al., 2018b). In addition, ZmOST1, the homologue of AtOST1/AtSnRK2.6 in maize, is reported to be involved in ABA‐induced stomatal closure and Cl‐ channel activation in maize guard cells (Wu et al., 2019). However, the anion channels mediating Cl‐ efflux in maize guard cells, as well as their regulatory mechanisms are largely unclear.

In the present study, we identified an S‐type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and localized at the PM. We found that ZmSLAC1 mediated stomatal closure in maize response to drought stress. The PM‐localized Ca2+‐dependent protein kinases, ZmCPK35 and ZmCPK37, could interact with and activate ZmSLAC1. Overexpression of ZmCPK35 and ZmCPK37 increased S‐type anion currents in guard cells and promoted stomatal closure in responses to ABA and Ca2+, which improved maize drought tolerance.

Results

Anion channel gene ZmSLAC1 is expressed in maize guard cells and induced by drought stress

In Arabidopsis thaliana, the S‐type anion channel AtSLAC1 is necessary for stomatal closure, and mediates Cl‐ and NO3 ‐ efflux across the PM of guard cells (Negi et al., 2008; Vahisalu et al., 2008). Phylogenetic analysis was performed to identify the important anion channels that are involved in maize stomatal movement. An AtSLAC1‐like anion channel, ZmSALC1 (Zm00001d002603), was found in maize B73 inbred line (Figure S1a). It shows 61% amino acid identity and similar gene structure with AtSLAC1, both contain three exons and two introns (Figure S1b).

Tissue expression analyses revealed that ZmSLAC1 was dominantly expressed in maize leaf, and its transcript level was induced by drought stress (Figure 1a and b). GUS staining of ProZmSLAC1:GUS maize transgenic lines indicated the guard cell‐specific expression of ZmSLAC1 in leaf epidermis (Figure 1c). The GUS staining after drought treatment was stronger than that under control conditions, which consolidated the induction of ZmSLAC1 transcript level by drought stress (Figure 1b and c). Subcellular localization analyses in Nicotiana benthamiana leaves showed co‐localization of ZmSLAC1‐GFP with the PM marker CBL1n‐OFP (Figure S1c).

Figure 1

Expression and localization analyses. (a) RT‐qPCR analyses of tissue expression of ZmSLAC1 in 10‐day‐old maize seedlings. (b) RT‐qPCR analyses of drought induction of ZmSLAC1. Data are presented as means ± SE of three replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (c) GUS‐staining of ProZmSLAC1:GUS line grown in well‐watered condition or with drought treatment. Bars = 40 μm. (d) Phenotypes of atslac1‐4 and atslac1‐4/ZmSLAC1 lines in soil after drought treatment. Two‐week‐old Arabidopsis seedlings grown for another 23 days without watering were photographed. (e) Water loss of detached shoots. 20‐day‐old Arabidopsis seedlings were used. Bars = 2.5 cm. (f) Leaf temperature images of atslac1‐4 and atslac1/ZmSLAC1 lines. Two‐week‐old Arabidopsis seedlings grown for another 19 days without watering were imaged. (g) Water loss of detached leaves. 20‐day‐old Arabidopsis seedlings were used. (h) ‐ (j) Stomatal aperture analyses of atslac1‐4 and atslac1‐4/ZmSLAC1 lines. Stomatal aperture in response to extracellular ABA (h), Ca2+ (i), and Dark (j) were shown. Data are presented as means ± SE. Student’s t‐test (**, P < 0.01) was used to analyse statistical significance.

ZmSLAC1 can mediate stomatal closure in Arabidopsis and maize

Mutation of AtSLAC1 impaired stomatal closure in Arabidopsis responses to external stimuli (ABA, Ca2+, dark, etc.). To test whether ZmSLAC1 could function as an anion channel and mediate stomatal closure in Arabidopsis, ZmSLAC1 was transformed into atslac1 mutants.

RT‐PCR analysis confirmed the expression of ZmSLAC1 in the atslac1 transgenic plants (Figure S1d). ZmSLAC1 complemented the mutant phenotypes to wild‐type (Col) levels. Both soil‐grown seedlings and detached shoots of the ZmSLAC1 transgenic lines wilted much slower than that of atslac1 mutants after drought stress (Figure 1d and e; Figure S2a and b). In addition, leaf temperature was also used to indicate water loss rate visually. After drought treatment the ZmSLAC1 transgenic lines (atslac1‐4/ZmSLAC1 and atslac1‐3/ZmSLAC1) and WT (Col) showed similar leaf temperature, which were higher than that in atslac1 mutants (Figure 1f and Figure S2c). Water loss of the detached leaves of ZmSLAC1 transgenic lines were much slower than that of atslac1 mutants, which was close to wild type (Figure 1g and Figure S2d). Meanwhile, ZmSLAC1 restored stomatal closure of atslac1 mutants in responses to external ABA, Ca2+ and dark treatment (Figure 1h–j and Figure S2e–g). These results indicate that ZmSLAC1 could fully complement the phenotypes of atslac1 mutants, therefore ZmSLAC1 also functions as an S‐type anion channel in guard cells.

Furthermore, the function of ZmSLAC1 was also characterized in maize. The zmslac1‐1 maize mutant (T‐DNA inserted inbred line) exhibited remarkable wilting phenotype compared with WT (W22 background) after drought treatment, no matter the seedlings were grown separately (Figure 2a) or in one pot (Figure 2b). The detached leaves of zmslac1‐1 mutant showed more obvious wilting phenotype and faster water loss rate than WT plants (Figure 2c and d). Similar like atslac1 mutants, lesion of ZmSLAC1 also impaired maize stomatal closure in responses to external ABA, Ca2+ and dark (Figure 2e–g). To further confirm the function of ZmSLAC1, another allele zmslac1‐2 mutant was generated using CRISPR/Cas9 technique, in which 70‐nt deletion was identified in the first exon of ZmSLAC1 (Figure S3a). At jointing stage, zmslac1‐2 mutant exhibited severe wilting phenotype under drought stress, whose relative water content and leaf temperature were lower than that in WT (LH244 background) plants (Figure 2h–j). These results demonstrate that ZmSLAC1 plays crucial roles in maize drought tolerance by regulating stomatal closure.

Figure 2

Phenotype analyses of zmslac1 mutant. (a) Phenotypes of zmslac1‐1 mutant in soil after drought treatment. One‐week‐old maize seedlings grown for another 5, 7 or 8 days without watering were photographed. WW, well water; Drought, drought treatment. Bars = 8 cm. (b) Phenotypes of zmslac1‐1 mutant in soil after drought treatment. 30‐day‐old maize seedlings grown for another 15 days without watering were photographed. W22 and zmslac1‐1 plants were grown in one pot. Bar = 8 cm. (c) and (d) Photograph of detached leaves (c) and statistics of detached leaf water loss (d). 18‐day‐old maize seedlings were used. Bars = 10 cm in (c); n = 4 in (d). (e) ‐ (g) Stomatal aperture analyses of zmslac1‐1 mutant. Stomatal aperture in response to extracellular ABA (e), Ca2+ (f), and Dark (g) were shown. Data are presented as means ± SE. Student’s t‐test (**, P < 0.01) was used to analyse statistical significance. (h) Phenotypes of jointing‐stage zmslac1‐2 mutant. V3 stage maize plants grown for about 18 days without watering were photographed. WW, well water; Drought, Drought treatment. Bars = 20 cm. (i) Relative water content of drought treated maize leaves. The eighth leaves were cut from drought treated plants. Data are presented as means ± SE of three replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (j) Leaf temperature images of zmslac1‐2 mutant. V3 stage maize plants grown for about 15 days without watering were photographed. Bar = 20 cm.

The Ca2+‐dependent protein kinase genes ZmCPK35 and ZmCPK37 are expressed in maize guard cells

In Arabidopsis, many Ca2+‐dependent protein kinases, such as AtCPK6 and AtCPK23, are involved in stomatal movement (Ma and Wu, 2007; Mori et al., 2006). Our previous study has shown that AtCPK8 functions in ABA‐mediated stomatal regulation in responses to drought stress by regulating CAT3 activity (Zou et al., 2015). In this follow‐up study, we further investigated the roles of AtCPK8 analogues in maize. According to the results of phylogenetic analyses, two analogue genes named ZmCPK35 (Zm00001d021835) and ZmCPK37 (Zm00001d006621) were identified (Figure S3c).

RT‐qPCR analyses showed ubiquitous expression of ZmCPK35 and ZmCPK37 in maize root and shoot tissues (Figure 3a). GUS staining of ProZmCPK35:GUS and ProZmCPK37:GUS transgenic lines indicated that both genes were highly expressed in leaf vascular tissues and guard cells (Figure S3d and e, Figure 3b and c), suggesting their functions in vascular tissue and guard cells. Transcript levels of these two genes were significantly induced in leaf by drought stress simulated by PEG treatment (Figure 3d and e). Subcellular localization analyses revealed that ZmCPK35‐GFP and ZmCPK37‐GFP were localized at the PM of mesophyll cells when expressed in Arabidopsis protoplasts (Figure S3f).

Figure 3

Activation of ZmSLAC1 by ZmCPK35 and ZmCPK37. (a) RT‐qPCR analyses of tissue expression of ZmCPK35 and ZmCPK37 in 10‐day‐old maize seedlings. (b) and (c) GUS staining of ProZmCPK35:GUS lines (b) and ProZmCPK37:GUS lines (c). Bars = 10 μm. (d) and (e) Induction of ZmCPK35 and ZmCPK37 transcripts by PEG treatment. Soil‐grown 10‐day‐old maize seedlings were treated with 20% PEG‐6000 for the indicated times, leaves were used for expression analyses. Data are presented as means ± SE of three replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (f) Patch‐clamp whole‐cell recording of S‐type anion channel currents in guard cell protoplasts of the wild type (WT) and zmslac1‐2, zmcpk37 mutants and ZmCPK35‐overexpression or ZmCPK37‐overexpression lines with or without the addition of 50 μm ABA in the bath solution. Currents were elicited by voltages step from +35 mV to −145 mV in 30 mV decrease, holding potential was 20 mV. (g) Current density–voltage data derived from the recordings as shown in (f). Data are presented as means ±SE (n ≥ 8). (h) BiFC analyses of ZmCPK35‐YFPN and ZmCPK37‐YFPN with ZmSLAC1‐YFPC in tobacco (Nicotiana benthamiana) leaves. ZmCPK14 was negative control. Bars = 50 μm. (i) and (j) Representative current traces (h) and I‐V curves (i) of ZmSLAC1 currents activated by ZmCPK35, ZmCPK37, and AtCPK8 in Xenopus oocytes. ZmCPK14 was negative control. Currents were elicited by voltages step from +40 mV to −180 mV in 20 mV decrease, holding potential was 0 mV. Dashed lines indicate zero current level. Bath solution contains 50 mm NO3 −. n ≥ 5 in (j). (k) ZmCPK35, ZmCPK37 and AtCPK8 activate ZmSLAC1 that mediates Cl− and NO3 − currents. Bath solution contains 50 mm Cl− or NO3 −. Currents at −100 mV were shown (n ≥ 5). Data are presented as means ± SE.

ZmCPK35 and ZmCPK37 interact with and activate ZmSLAC1

Activation of anion channel AtSLAC1 by protein kinases (such as AtCPK23) is necessary for guard cell anion efflux during stomatal closure in Arabidopsis (Geiger et al., 2010). We assumed that the similar mechanism may also exist in maize. Considering ZmCPK35, ZmCPK37 as well as ZmSLAC1 were all expressed in maize guard cells, we speculated that the activity of ZmSLAC1 may be regulated by ZmCPK35 and/or ZmCPK37.

We conducted whole‐cell patch‐clamp recording of S‐type anion channel currents in guard cell protoplasts. As shown in Figure 3f–g, ABA treatment (50 μm) increased S‐type anion currents in wild‐type guard cells, but not in zmslac1‐2 mutant. ABA slightly increased the currents in zmcpk37 guard cells. In contrast, the currents of ZmCPK35 and ZmCPK37 overexpression lines increased significantly, especially in ZmCPK35 OE‐1 (Figure 3f–g). Different concentrations of Ca2+ in the pipette were used to test the role of [Ca2+]cyt in regulating S‐type anion currents in maize guard cells. The results demonstrated that the S‐type anion channel currents were weak with low [Ca2+]cyt (0.15 μm), while the currents became larger when [Ca2+]cyt was increased to 2 μm (Figure 3f–g, Figure S4). Importantly, the currents in ZmCPK35 and ZmCPK37 OE lines were increased significantly compared with wild type under high Ca2+ (2 μm) conditions (Figure S4). These results indicate that ZmCPK35 and ZmCPK37 could positively regulate ZmSLAC1‐meidate S‐type anion currents in maize guard cells in a Ca2+‐ and ABA‐dependent manner.

BiFC assays in tobacco leaves revealed the protein interaction between ZmCPK35/37 and ZmSALC1 on the PM (Figure 3h). In contrast, the negative control ZmCPK14 did not interact with ZmSLAC1 (Figure 3h and Figure S1e). Then, the ZmSLAC1 regulation by ZmCPK35 and ZmCPK37 were verified in Xenopus oocytes. ZmSLAC1 alone did not show any channel activity in oocytes (Figure 3i and j). Whereas, co‐expression of ZmCPK35 or ZmCPK37 in oocytes could activate ZmSLAC1‐mediated NO3 ‐ currents that were dependent on external NO3 ‐ concentrations (Figure 3i and j, Figure S5a and b). The negative control ZmCPK14 could not activate ZmSLAC1 (Figure 3i and j). In addition, we found that AtCPK8, the analogue of ZmCPK35 and ZmCPK37 in Arabidopsis, activated ZmSLAC1 as well (Figure 3i and j). Current component analyses indicated that activated ZmSLAC1 mainly mediated NO3 ‐ currents in oocytes, but weak Cl‐ currents (Figure 3k and Figure S5c).

On the other hand, AtCPK8 was able to interact with and activate AtSLAC1 in oocytes (Figure S6a–d); meanwhile, ZmCPK35 and ZmCPK37 could activate AtSLAC1 as well (Figure S6e and f). These results demonstrate that the activation of anion channel SLAC1 by CPKs is a universal mechanism in both maize and Arabidopsis.

ZmCPK35 and ZmCPK37 regulate stomatal closure under drought stress

Considering that ZmCPK35 and ZmCPK37 are analogues of AtCPK8, we also tested their functions in Arabidopsis. Our previous study showed that atcpk8 mutant is sensitive to drought stress (Zou et al., 2015). Here, transformation of ZmCPK35 or ZmCPK37 into atcpk8 mutant was able to complement the drought‐sensitive phenotype of atcpk8 mutant (Figure S7a–d). It is suggested that ZmCPK35 and ZmCPK37 also activate AtSLAC1 in Arabidopsis response to drought stress.

Then we investigated the functions of ZmCPK35 and ZmCPK37 in vivo by determining the drought phenotypes of maize mutants. We attempted to generate the maize mutants of these two genes by using CRISPR/Cas9 technique, however only zmcpk37 mutant was obtained (Figure S3b). Phenotype observation showed that the maize inbred line of zmcpk37 mutant exhibited drought‐sensitive phenotype compared with WT (LH244 background) (Figure 4a). The relative water content and surface temperature of zmcpk37 mutant leaves were lower than that in WT plants (Figure 4b and c), suggesting that zmcpk37 mutant loss more water than WT after drought stress.

Figure 4

Drought phenotype analyses of maize inbred plants. (a) Phenotypes of jointing‐stage zmcpk37 mutant plants. V3 stage maize plants grown for about 18 days without watering were photographed. WW, well water; Drought, drought treatment. Bars = 20 cm. (b) Relative water content of drought treated zmcpk37 mutant leaves. The eighth leaves were cut from drought treated plants grown for about 18 days without watering. Data are presented as means ± SE of three replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (c) Leaf temperature images of zmcpk37 mutant. V3 stage maize plants grown for about 15 days without watering were photographed. Bar = 20 cm. (d) and (e) Expression analyses of ZmCPK35‐overexpression maize lines (d) and ZmCPK37‐overexpression maize lines (e) (n = 4). Data are presented as means ± SE. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (f) and (g) Drought phenotypes of ZmCPK35‐overexpression maize lines (f) and ZmCPK37‐overexpression maize lines (g). V3 stage maize plants grown for about 19 days without watering were photographed. WW, well water, Drought treatment. Bars = 20 cm. (h) and (i) Relative water content of ZmCPK35‐overexpression maize lines (h) and ZmCPK37‐overexpression maize lines (i). The eighth leaf were cut from drought treated plants grown for about 19 days without watering. Data are presented as means ± SE of three replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (j) and (k) Leaf temperature images of ZmCPK35‐overexpression maize lines (j) and ZmCPK37‐overexpression maize lines (k) V3 stage maize plants grown for about 15 days without watering were photographed. Bars = 20 cm.

In addition, we also generated the maize overexpression lines of ZmCPK35 and ZmCPK37, and determined their drought phenotypes. RT‐qPCR assays confirmed the high expression levels of ZmCPK35 or ZmCPK37 in these transgenic lines (Figure 4d and e). Phenotype observation indicated that WT (LH244 background) and the overexpression inbred lines of ZmCPK35 and ZmCPK37 did not show obvious differences under well‐watered conditions (Figure 4f and g). However, after drought stress, the ZmCPK35 and ZmCPK37 overexpression lines all exhibited remarkable drought‐tolerant phenotypes (Figure 4f and g), whose leaf water content (Figure 4h and i) and leaf temperature (Figure 4j and k) were higher than WT plants. These data demonstrate that overexpression of ZmCPK35 and ZmCPK37 could retain more water in leaves and enhance maize drought tolerance.

In addition, we found that the water loss rate of detached leaves in the overexpression inbred lines of ZmCPK35 and ZmCPK37 were slower than that in WT plants (Figure 5a and b). The ABA‐ and Ca2+‐induced stomatal closure was weaker in zmcpk37 mutants, and the stomatal closure degree were much stronger in the overexpression lines than that in WT plants (Figure 5c and d), suggesting that ZmCPK35 and ZmCPK37 are involved in drought tolerance by promoting ABA‐ and Ca2+‐induced stomatal closure.

Figure 5

Water loss rate and stomatal aperture analyses of ZmCPK35‐ and ZmCPK37 relative maize lines. (a) and (b) Leaf water loss of ZmCPK35‐overexpression maize lines (a) and zmcpk37 mutant and ZmCPK37‐overexpression maize lines (b), (n = 4). Data are presented as means ± SE. Student’s t‐test (#, control; *, P < 0.05; **, P < 0.01) was used to analyse statistical significance, black * for OE‐1, blue * or ** for OE‐2. (c) and (d) Stomatal aperture analyses of ZmCPK35‐ and ZmCPk37 relative maize lines. Stomatal aperture in response to extracellular ABA (c) and Ca2+ (d) were shown (n ≥ 30). Data are presented as means ± SE. Student’s t‐test (#, control; *, P < 0.05; **, P < 0.01) was used to analyse statistical significance.

Overexpression of ZmCPK35 and ZmCPK37 enhance maize yield in fields under drought stress

Since overexpression of ZmCPK35 and ZmCPK37 could enhance maize drought tolerance in inbred lines, we wondered if these two genes can be used to improve maize drought tolerance and increase yield in fields. As we know, maize hybrids are widely used in agricultural production, therefore the hybrids of ZmCPK35 and ZmCPK37 overexpression lines were generated. Here, the overexpression inbred lines (LH244 background) were used as male parents, while the T13 inbred line was used as female parent. Similar to the inbred lines, the F1 hybrids also exhibited markedly drought‐tolerant phenotypes compared with control plants when grown in pots (Figure 6).

Figure 6

Phenotype analyses of ZmCPK35‐ and ZmCPK37‐overexpression hybrid maize plants. (a) and (b) Drought phenotypes of ZmCPK35‐overexpression hybrid maize lines (a) and ZmCPK37‐overexpression hybrid maize lines (b). V3 stage maize plants grown for about 18 days without watering were photographed. WW, well water; Drought, Drought treatment. Bars = 20 cm. (c) and (d) Relative water content of ZmCPK35‐overexpression hybrid maize lines (c) and ZmCPK37‐overexpression hybrid maize lines (d). The eighth leaf was cut from drought treated plants. Data are presented as means ± SE of three replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (e) and (f) Leaf temperature images of ZmCPK35‐overexpression hybrid maize lines (e) and ZmCPK37‐overexpression hybrid maize lines (f). V3 stage maize plants grown for about 15 days without watering were photographed. Bars = 20 cm.

Furthermore, we tested the yield trait of both inbred and hybrid lines in fields. These experiments were conducted in Urumchi (Xinjiang, China) in 2018, and different irrigation schemes (Well‐watered, Drought‐1 and Drought‐2) were used during the growth stage of maize plants. Under well‐watered conditions, the WT plants and overexpression lines did not show obvious differences in yield (Figure 7). Drought stress significantly impaired the yield of both inbred and hybrid plants (Figure 7). The yield of inbred plants was reduced under both Drought conditions, but only Drought‐2 treatment impaired the yield of hybrids (Figure 7). We found that the yield of ZmCPK35 and ZmCPK37 overexpression inbred lines were much higher than that of WT under two drought conditions (Figure 7a to c). Statistical analyses indicated that the kernel number per row of ZmCPK35‐ and ZmCPK37‐overexpression lines were increased significantly (Figure 7d and e). In addition, some hybrids overexpressing ZmCPK35 and ZmCPK37 showed higher yield than WT plants under Drought‐2 conditions (Figure 7f to h). These data indicate that ZmCPK35 and ZmCPK37 may be the candidate genes used for the improvement of maize drought tolerance in future.

Figure 7

Overexpression of ZmCPK35 and ZmCPK37 enhance maize yield under drought stress. (a) Ear phenotypes of ZmCPK35‐overexpression inbred lines and ZmCPK37‐overexpression inbred lines. Bars = 20 cm. (b) and (c) Kernel weight per plant (14% water) of ZmCPK35‐overexpression inbred lines (b) and ZmCPK37‐overexpression inbred lines (c). Data are presented as means ± SE of all the ears in the two replicates. Student’s t‐test (#, control; *, P < 0.05,**, P < 0.01) was used to analyse statistical significance. (d) and (e) Kernel number per row for the ear of ZmCPK35‐overexpression inbred lines (d) and ZmCPK37‐overexpression inbred lines (e). Data are presented as means ± SE of all the ears in the two replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (f) Ear phenotypes of ZmCPK35‐overexpression hybrids (e) and ZmCPK37‐overexpression hybrids (f). Bars = 20 cm. (g) and (h) Kernel weight per plant (14% water) of ZmCPK35‐overexpression hybrids (g) and ZmCPK37‐overexpression hybrids (h). Data are presented as means ± SE of all the ears in the two replicates. Student’s t‐test (#, control; **, P < 0.01) was used to analyse statistical significance. (i) and (j) Kernel number per row for the ear of ZmCPK35‐overexpression hybrid lines (i) and ZmCPK37‐overexpression hybrid lines (j). Data are presented as means ± SE of all the ears in the two replicates.

Discussion

In Arabidopsis, stomatal closure requires efflux of anions out of guard cells. S‐type anion channel AtSLAC1 mediated Cl‐/NO3 ‐ efflux in Arabidopsis (Negi et al., 2008; Vahisalu et al., 2008), mutation of AtSLAC1 impairs stomatal closure and results in drought‐sensitive phenotype (Figure 1d–j, Figure S2). In this study, we found that ZmSLAC1, the analogue of AtSLAC1, was preferentially expressed in maize guard cells, and was able to complement the phenotype of loss‐function mutant atslac1 in Arabidopsis (Figure 1, Figure 2 and Figure S2). Mutation of ZmSLAC1 in maize resulted in constitutive opening of stomata under drought conditions, which led to faster leaf water loss and drought‐sensitive phenotype (Figure 2). It is indicated that ZmSLAC1 is the major channel that is responsible for stomatal closure in maize. The similar gene structures, expression patterns, and physiological functions of AtSLAC1 and ZmSLAC1 suggest that the SLAC1‐type channels may be evolutionarily conserved in dicotyledons and monocotyledons representing kidney‐shaped and dumbbell‐shaped guard cells respectively. However, a different regulation was discovered in this study. AtSLAC1 is mainly regulated at post‐translational level rather than transcriptional level in Arabidopsis response to drought stress (Zhang et al., 2018). Besides post‐translational regulation, the transcript of ZmSLAC1 is significantly increased after drought stress (Figure 1b and 1c), which endows maize with an additional strategy to control stomatal aperture under drought stress. It is believed that the dual regulation may facilitate maize adaptability in variable environment, especially in short‐term and/or long‐term drought environment.

In Arabidopsis, phosphorylation of SLAC1 by CPKs has been reported as one of important mechanisms in plant response to drought stress (Brandt et al., 2012; Geiger et al., 2010). Overexpression of these AtCPKs (AtCPK6, AtCPK21, AtCPK23) also strengthens plant drought tolerance by enhancing SLAC1 activity (Geiger et al., 2010; Mori et al., 2006; Xu et al., 2010). In this study, we identified two Ca2+‐dependent protein kinases ZmCPK35 and ZmCPK37 that were able to activate ZmSLAC1 and increase plant yield under drought stress when overexpressed in maize (Figure 3i, 3j and 7). Although ZmCPK35 and ZmCPK37 may also function in other cells in the overexpression lines, involvement of ZmCPK35 and ZmCPK37 in stomatal response is one of important reasons for drought tolerance in overexpression lines.

Recent report revealed that ZmOST1, the analogue of AtOST1, was able to activate ZmSLAC1 in oocytes (Qi et al., 2018b). However, AtOST1/ZmOST1‐activated ZmSLAC1 only mediated NO3 ‐ currents in oocytes. And expression of ZmSLAC1 in Arabidopsis cannot complement the Cl‐ currents in atslac1 mutant guard cells, suggesting ZmSLAC1 might function as a NO3 ‐‐selective anion channel (Qi et al., 2018b). In this study, we found that ZmSLAC1 activated by ZmCPK35 and ZmCPK37 was able to mediate both NO3 ‐ and Cl‐ currents (Figure 3k and Figure S5c).

Our previous study has revealed that AtCPK8, the analogue of ZmCPK35 and ZmCPK37, is essential for H2O2 homeostasis and inhibition of inward K+ channel in Arabidopsis guard cells in drought response (Zou et al., 2015). In this study, we found that ZmCPK35 and ZmCPK37 could also inhibit the inward K+ channels expressed in maize guard cells (Figure S8; Gao et al., 2019). More important, AtCPK8 was able to activate both AtSLAC1 and ZmSLAC1 in oocytes (Figure S6), suggesting that AtCPK8 may also regulate AtSLAC1 and promote stomatal closure in Arabidopsis in drought stress. ZmCPK35, ZmCPK37, and AtCPK8 were all able to activate both ZmSLAC1 and AtSLAC1 in oocytes, suggesting this mechanism is conserved in monocotyledons and dicotyledons. CPKs are the important components that regulate synergistically the activities of K+ channels and anion channels, so that stomata could be rapidly closed upon drought stress. It seems that several CPK proteins simultaneously regulate SLAC1 channels in both Arabidopsis and maize. Why plants need so many CPKs in drought response is still unclear.

Drought stress is one of the most serious disasters in agricultural production, especially for maize (Campos et al., 2004; Lobell et al., 2014). Improvement of water use efficiency and drought stress tolerance are critical goals in maize breeding. In this study, we identified two important Ca2+‐dependent protein kinases ZmCPK35 and ZmCPK37 involved in maize drought response. Overexpression of these two genes could markedly enhance maize drought tolerance and increase yield under drought stress (Figures 4, 6, 7). Under well‐watered conditions, overexpression of these two genes did not affect plant growth, development and yield, the overexpression lines showed similar phenotypes to WT.

We observed that stomatal closure of zmcpk37 mutant was insensitive to Ca2+ treatment, while the ZmCPK35 and ZmCPK37 OE lines were sensitive to Ca2+ treatment (Figure 5d), suggesting that the activation of ZmCPK35 and ZmCPK37 may be through Ca2+ signalling. The patch‐clamp experiments demonstrate the S‐type anion channel currents were dependent on [Ca2+]cyt. Importantly, the currents in ZmCPK35 and ZmCPK37 OE lines were increased significantly under high [Ca2+]cyt conditions (Figure 3f‐g, Figure S4). All these results demonstrate that (i) Ca2+ is essential for stomatal closure under drought stress; (ii) Ca2+ is involved in ZmCPK35/37‐regulated S‐type anion currents in maize guard cells. Therefore, these overexpression lines could fast respond to drought stress and rapidly close stomata in order to reduce water loss effectively (Figure 5). As a result, these overexpression lines, including both inbred and hybrid plants, had higher yield under drought stress in field test (Figure 7). By modification of a single gene expression level to regulate stomatal movement in maize, our study provides a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress. This study also provides important insights into drought tolerance improvement in other gramineous crops.

Methods

Growth conditions and generation of transgenic plants

Maize seedlings were grown in growth chambers with 14 h (28 ± 2°C) /10 h (24 ± 2°C) light/dark cycles, 300 μmol/m2/sec1 irradiance, 80% relative humidity. For the jointing‐stage drought treatment, the maize pot (the volume is about 15 litres) experiments were performed in solar greenhouse (Beijing) with a 14 h (28 ± 3°C)/10 h (24 ± 3°C) light/dark cycles, 400 μmol/m2/sec irradiance, and 45% relative humidity. For drought treatment, plants were well watered at V3 stage, and no water was given until exhibited drought‐tolerance phenotype (about the jointing stage, V8), for other drought treatments, methods in detail are indicated in the figure legends.

The maize field experiments were performed in Urumqi (Xinjiang, China), where the annual rainfall was 294 mm in 2018. The well‐watered blocks (seeding,750; jointing stage, 750; trumpet stage, 750; tasseling stage, 750; pollinating stage, 750; early stage of grain filling, 750; middle stage of filling, 750; late stage of filling, 750, tons of water per hectare), drought‐1 treated blocks (seeding,750; jointing stage, 375; trumpet stage, 375; tasseling stage, 375; pollinating stage, 375; early stage of grain filling, 375; middle stage of filling, 375; late stage of filling, 375, tons of water per hectare), and drought‐2 treated blocks (seeding,750; jointing stage, 375; trumpet stage, 225; tasseling stage, 225; pollinating stage, 375; early stage of grain filling, 750; middle stage of filling, 750; late stage of filling, 750; tons of water per hectare). Two repeats were performed and 20 plants were cultured for each repeat.

The zmslac1‐1 (UFMu‐04043) mutant with a Mu transposon insertion in ZmSLAC1 (Zm00001d002603) was generated by the Maize Genetics COOP Center (http://maizecoop.cropsci.uiuc.edu/). The zmslac1‐2 mutant was generated by using CRISPR/Cas9 technique. A sgRNA pair (C1, 5’‐TCTTCCACGGGGCAACAAA‐3’; and C2, 5’‐GCGGGCCGCTCAATGTCCG‐3’) targeted for ZmSLAC1 was designed, and cloned into the pBUE411 vector. The zmcpk37 mutant was generated by using CRISPR/Cas9 technique. A sgRNA (C, 5’‐GCCAAAATCAATGGCCTTA‐3’) targeted for ZmCPK37 was designed, and cloned into the pBUE411 vector. To generate ZmCPK35 and ZmCPK37 overexpression lines, the coding sequences of ZmCPK35 and ZmCPK37 were cloned into pBCXUN vector and driven by ZmUBI promotor. For the ProZmSLAC1:GUS, ProZmCPK35:GUS, and ProZmCPK37:GUS construction, the 2784/2362/3368 bp fragments upstream of their start codon (ATG) were amplified and cloned into the modified pCAMBIA1300 vector fused with GUS reporter gene respectively. The transgenic lines were obtained by using Agrobacterium‐mediated transformation in maize inbred line. The T2 or T3 homozygous maize plants were used in this study.

Arabidopsis plants were grown in growth chambers at 22°C with 12 h /12 h light/dark cycles, 120 μmol/m2/sec irradiance, 70% relative humidity. To generate atcpk8/ZmCPK35 and atcpk8/ZmCPK37 transgenic lines, the coding sequences of ZmCPK35 and ZmCPK37 were cloned into pSUPER1300 vector, and the construct was introduced into Agrobacterium tumefaciens GV3101 and transformed into atcpk8 mutant plants by the floral dip method.

Sequences alignment and phylogenetic analysis

These gene sequences and amino acid sequences were downloaded from TAIR (https://www.arabidopsis.org), MaizeGDB (http://www.maizegdb.org), and Gramene (http://ensembl.gramene.org/Zea_mays/Info/Index) database, and aligned using DNAMAN software. The phylogenetic analysis was conducted using the ClustalW. Construction of phylogenic tree using the neighbour‐joining method and confirmation of tree topology by bootstrap analysis (1,000 replicates) was performed with MEGA software (default settings except the replicates of bootstrap value).

RT‐qPCR analysis

Maize seedlings grown in soil were used for RT‐qPCR analysis. Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s instruction. DNase‐treated total RNA was reverse transcribed into cDNA using SuperScript™ II. The cDNA amplification reactions were performed on the ABI 7500 thermocycler with Power SYBR® Green PCR Master Mix (Applied Biosystems), the fluorescence signal was detected during the annealing step. The maize ZmUbiquitin gene was used as an internal standard to normalize the expression data for the tested genes. Gene specific primers were used for RT‐qPCR analysis (Table S1). At least three biological replicates were performed in each experiment.

GUS staining

The second fully expanded leaves of the 8‐day old ProZmSLAC1:GUS, ProZmCPK35:GUS and ProZmCPK37:GUS maize seedlings were cut into 0.5 cm2 pieces, and treated with −90 kPa vacuum for 20 min in GUS staining solution, which contains 1% (v/v) N, N‐dimethyl formamide, 0.1% X‐Gluc, 0.1% Triton X‐100, 0.1 m PBS, 0.05 mm K3Fe(CN)6, and 0.05 mm K4Fe(CN)6·3H2O. These leaf samples were incubated at 37°C in darkness for 72 h, then washed using 0.1 m PBS and fixed in 75% (v/v) ethanol for microscopy observation.

Subcellular localization and BiFC assay

The coding sequence of ZmSLAC1 were cloned into p16ΔS:sXVE:GFPc:Bar for the analyses of subcellular localization. The constructs, empty vector and CBLn‐OFP, were transfected into Agrobacterium tumefaciens (GV3101) via electroporation. One day after infiltration, β‐estradiol (100 μm) or 0.1% (v/v) ethanol (mock) was brushed on tobacco (Nicotiana benthamiana) leaves to induce the expression of target genes (Schlücking et al., 2013). The BiFC assays were performed as described previously (Walter et al., 2004). In brief, the coding sequences of ZmCPK35, ZmCPK37, ZmCPK14 and AtCPK8 were cloned into vector pSPYNE173, ZmSLAC1 and AtSLAC1 were cloned into pSPYCE (M). All the construct pairs were expressed 4 days in tobacco leaves before microscopy observation. The fluorescence was imaged using a confocal laser scanning microscope (LSM710; Carl Zeiss).

The coding sequences of ZmCPK35 and ZmCPK37 were cloned into pUC‐EGFP for the analysis of subcellular localization. Protoplast isolation and transformation were performed as described previously (Zhao et al., 2013). After incubation for 16 h at 23°C, the GFP fluorescence in the transformed protoplasts were imaged using a confocal laser scanning microscope (LSM710; Carl Zeiss).

Thermal imaging and relative water content measurement

Thermal imaging of plants was performed as described previously (Hua et al., 2012). Two‐week‐old Arabidopsis plants grown under normal conditions were subjected to drought stress for about 19 days. The well‐watered V3 stage maize plants were subjected to drought stress for about 15 days. Thermal images were obtained using VarioCAM HD. Images were analysed using the public domain image‐analysis program IRWIN REPORTER version 5.31. The relative water content was calculated by the following calculation: [(fresh weight ‐ dry weight)/(turgid weight ‐ dry weight)] x 100%.

Drought treatment and detached leaf water loss assay

Two‐week‐old Arabidopsis seedlings grown for another 23 days without watering were photographed. For maize seedlings drought treatment, 10‐day‐old maize seedlings grown for another 6 or 7 days without watering were photographed. For the jointing‐stage maize drought treatment, V3 stage maize plants were well watered and grown for about 2 weeks without watering.

For water loss measurement, fully expanded rosette leaves were detached from 20‐day‐old Arabidopsis seedlings and weighed, fully expanded leaves from 18‐day‐old maize seedling were detached and weighed. Weight loss of the detached leaves were monitored at the indicated time intervals. Water loss was showed as the percentage of initial fresh weight.

Stomatal aperture assay

Fully expanded rosette leaves were detached from 20‐day‐old Arabidopsis seedlings or the first leaf of 8‐day‐old maize seedlings were floated in opening buffer that contained 10 mm KCl, 50 μm CaCl2 and 10 mm Mes/Tris, pH 5.6, and kept in darkness for 2 h, then treated with light (120 or 300 μmol/m2/sec respectively). The abaxial epidermal strips were peeled from the middle part of leaves. The stomatal apertures were observed under microscope (Olympus IX‐71) and measured using ImageJ software.

Electrophysiological assays

Maize guard cell protoplasts were isolated as described (Gao et al., 2017). Whole‐cell recording techniques were applied. The bath solution contained 30 mm CsCl, 1 mm CaCl2, 2 mm MgCl2, 10 mm MES, pH 5.6, with an osmolality of 485 mmol/kg adjusted using D‐sorbitol; the pipette solution contained 150 mm CsCl, 2 mm MgCl2, 10 mm HEPES, pH 7.1, the free Ca2+ concentration was buffered with 6.7 mm EGTA and 2.29 mm CaCl2 (0.15 μm free Ca2+) or 3.35 mm CaCl2 (0.3 μm free Ca2+) or 5.87 mm CaCl2 (2 μm free Ca2+), with an osmolality of 500 mmol/kg adjusted using D‐sorbitol. Fresh ATP (5 mm) was added to the pipette solution before use. For analysis of the ABA activation of S‐type anion channels, the guard cell protoplasts were pre‐incubated with 50 μm ABA for 20 min before patch clamping, and patch clamp experiments were performed in the presence of 50 μm ABA in both bath and pipette solution. In the whole‐cell patch‐clamp experiments, the junction potentials were calculated by using the Clampex 10.3 software. The junction potential was −0.8 mV at 25°C in the used pipette/bath solutions, and corrected by using the functions of “Pipette Offset” and “Holding Command” of Axopatch 200B. S‐type anion currents were measured 10 min after whole‐cell configurations became accessible. The membrane voltage was stepped from 35 mV to −145 mV in 30‐mV decrements, and the holding potential was 20 mV.

The coding sequences of ZmCPK35, ZmCPK37, ZmCPK14, AtCPK8, AtOST1‐YFP, ZmSLAC1 and AtSLAC1‐YFP were cloned into pGEMHE vector. TEVC (Two‐Electrode Voltage Clamp) was applied using a GeneClamp 500B amplifier (Axon Instruments) at room temperature (~22°C). Whole‐cell currents recording were performed 36 h after cRNA injection. The standard bath solution contained 50 mm NaNO3 or NaCl, 1 mm Ca‐Gluconate2, 2 mm Mg‐Gluconate2, 50 mm Na‐Gluconate, and 10 mm MES, pH 5.6 with Tris. The microelectrodes were filled with 3 m KCl. Voltage pulses were applied from a holding potential of 0 mV, typically ranging from +40 mV to −180 mV in 20 mV decrements. And steady state currents were extracted at the end of the 3 s voltage step pulses.

Accession numbers

Sequence data for the genes described in this article can be found in the Arabidopsis TAIR database (https://www.arabidopsis.org), MaizeGDB database (http://www.maizegdb.org) and Gramene (http://ensembl.gramene.org/Zea_mays/Info/Index) under the following accession numbers: AT4G33950 for AtOST1, At5g19450 for AtCPK8, AT1G12480 for AtSLAC1, Zm00001d053016 for ZmCPK14, Zm00001d021835 for ZmCPK35, Zm00001d006621 for ZmCPK37, and Zm00001d002603 for ZmSLAC1.

Conflicts of interest

The authors declare no conflicts of interest related to this work.

Author contributions

X.‐D.L., Y.‐Q.G., W.‐H.W., L.‐M.C. and Y.W. designed the research. X.‐D.L. and Y.‐Q.G. performed the research and analysed the data. X.‐D.L., Y.‐Q.G. and Y.W. wrote the article. Y.W. and W.‐H.W. revised the article.

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Figure S1 Expression and localization analyses of SLAC1.

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Figure S2 Phenotype Analyses of Arabidopsis atslac1‐3/ZmSLAC1 Lines.

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Figure S3 Expression and localization analyses of ZmCPK35 and ZmCPK37.

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Figure S4 Activation of ZmSLAC1 by ZmCPK35 and ZmCPK37. Activation of ZmSLAC1 by ZmCPK35 and ZmCPK37.

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Figure S5 Ion selectivity of ZmSLAC1 in Xenopus oocytes.

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Figure S6 Activation of AtSLAC1 by AtCPK8, ZmCPK35, and ZmCPK37.

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Figure S7 Phenotype analyses of Arabidopsis atcpk8/ZmCPK35 and atcpk8/ZmCPK37 lines.

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Figure S8 Inhibition of KZM3 and KAT1 activity by ZmCPK35 and ZmCPK37.

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Table S1 The primer sequences used in this study.

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