A calcium dependent protein kinase involves H2O2 mediated guard cell signaling in Aarabidopsis

Drought is a major threat for plant growth and productivity. Plants lose over 90% of water by transpiration through stomatal pores. The cytosolic free Ca2+ elevated in guard cells in response to stress stimuli triggers stomatal closure. The plant-specific calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components of calcium signaling. In this study the biological function of Arabidopsis calcium dependent protein kinase, CPK8, in response to ABA signaling in guard cells was characterized. The plants of TDNA insertion mutant of cpk8 were more sensitive to drought stress than wild-type plants. The GUS staining studies confirmed that CPK8 expressed in leaves, specifically in guard cells. RT-PCR analysis showed that CPK8 expression was induced in response to drought stress. Further, pre-opened cpk8 stomata failed to close in response to H2O2 and Ca2+, which is consistent with the inability of cpk8 plants to reduce water loss upon drought. The drought susceptibility and stomatal impairment in response to H2O2 and Ca2+ of the cpk8 implicated that CPK8 plays a role in cellular environment in the control of H2O2 homeostasis and also as a compulsory molecule in the transduction of H2O2 signal in guard cells in response to drought stress. Tropical Agricultural Research and Extension 16(1): 2013: page 7-14


INTRODUCTION
Water is the most limiting resource for terrestrial plant growth and development and yield formation in many part of the world. Desiccation of crops during various growth stages causes severe and often irreversible damage and hence yields losses (Boyer 1982;Ainsworth and Long 2005). It would be beneficial for crop plants to show wide stomatal opening for CO 2 intake when water is available, but to close stomata during drought periods, thereby slowing desiccation and damage. Stomatal opening is driven by plasma membrane hyper polarization proposed to drive K + uptake into guard cells passively via inward-rectifying K + (K + in ) channels (Schroeder et. al. 1987). Stomatal closing involves the influx of free cytosolic Ca 2+ that down regulate the inward K + channels and activate outward channels (Li et al. 2000). Ca 2+ oscillations are a fundamental requirement for stomatal closure.
Several classes of Ca 2+ binding sensory proteins have been identified in plants. CDPKs are the largest subfamilies of plant protein kinases *Corresponding author: disnaratnasekera@gmail.com among them. The completed Arabidopsis genome sequence has revealed 34 genes encoding CDPKs and they are highly homologous to each other (Cheng et al. 2002;Hrabak et al. 2003). Recently genome wide analysis of rice found that there are 29 genes encoding CDPKs and eight closely related kinase genes (Takayuki et al. 2005). Some other plants including soybean, tomato and maize also indicate the presence of multi-gene families , but the reason for such a large number of CDPK genes is not yet known. Recent experiments indicate that functional specialization of individual CDPKs can occur through different types of regulation. For example, plants may use a combination of various strategies to functionally specialize individual CDPKs, as evidenced by two sandalwood CDPK isoforms that differ in tissue specific distribution, sub-cellular localization, and enzyme kinetics and properties (Anil et al. 2001).
CDPKs play divers roles in various biological responses by interacting with other factors and act as key regulators of many signaling path-ways. But very little is known about the particular CDPK that acts as the calcium sensor in each case. In this study we characterize the biological function of Arabidopsis calciumdependent protein kinase CPK8 in response to ABA signaling in guard cells.
Seeds were sterilized using NaOCl solution (0.5% NaOCl and 0.01% Triton x-100) for 10-15 min. and washed 5 times with sterilized distilled water under aseptic conditions in laminar flow cabinet and kept in dark at 4℃ for 72 hours to break the dormancy.
Sterilized seeds were sown on MS plates and incubated in 20-22℃ with 120 μ/mol/m 2 /s light intensity for seedling development. Seven day old seedlings were then transferred to 1:1 soil: peat medium. After transplanting, plants were covered with polythene cover to maintain high humidity and kept in growth chambers at 22℃ with illumination at 120 μ/mol/ m 2 / s for 16-h light/8-h dark cycle. The relative humidity was approximately 70% (±5%). One week later plants were disclosed.

Vector Constructions and Generation of Transgenic Plants:
The CPK8 pro :GUS construct was generated by fusing the CPK8 promoter fragment (1.96 kb) in front of the βglucuronidase (GUS) coding sequence in pCAMBIA1381 vector. The special primers for C P K 8 p r o : G U S c o n s t r u c t w e r e 5 ' -CACTCTCCTAGGAACCGATAC-3' and 5'-TTCGAATCTGAGAAGTCCTG-3'. The GUS staining assays were carried out as described by (Xu et al. 2006).

Drought tolerance measurements:
For drought experiments, Arabidopsis thaliana [Columbia ecotype, cpk8 knockout mutant] plants were grown on MS medium under continuous light for 7 days and transferred to peat soil in a controlled environment growth chamber with a 16:8 hour light: dark cycle and irrigated for 2 weeks. Then plants were subjected to drought by complete termination of irrigation. The plants (n=12 each) at the similar developmental stages were selected for the analysis. Watered plants were analyzed as control treatment. Pots were weighed after 3, 6, 9, 12, 15 and 18 days at the same time for relative water content measurements.

Water loss Measurements:
Water loss experiments were conducted on weight basis and at the same time phenotype comparison at different time intervals were carried out. For measurement of water loss, plants were transferred from high (90%) to low humidity (50%) and then leaves were detached and incubated abaxial face up in 25℃ with 50% RH, Their fresh weight was measured at different time intervals. Water loss was expressed as the percentage of initial fresh weight. To compare the phenotype, photographs were taken just after detaching and 5 hours after desiccation. Each experiment repeated 6 times with 4 replicates.

Stomatal aperture measurements:
Plants were grown on MS medium under continuous light for 7 days and transferred to peat soil in a controlled environment growth chamber with a 12:12 hour light: dark cycle for 3 weeks and then placed in overnight dark before every treatment. To measure stomatal opening, detached leaves were floated in incubation buffer containing 50mM KCl with10mM MES/KOH and 0.1mM CaCl 2 for 3 hours under light to induce stomatal opening and measured the aperture width.
To measure the stomatal closing, detached leaves were floated in incubation buffer under light for 2 hours and then kept in dark for another 2 hours to induce stomatal closing. After 2 hours, stomatal apertures were measured. For ABA-inhibition of stomatal opening, leaves were incubated with or without ABA (10 μM) under light for 2.5 hours. To study the effect of H 2 O 2 and Ca 2+ , stomata were opened by exposing plants for light and high humidity and incubating the leaves for 2 h in stomata-opening solution containing 50 mM KCl, 0.1 mM CaCl 2 , and 10 mM MES/KOH, pH 6.15, in a growth chamber at 22 to 25 0 C under a photon flux density of 0.20 to 0.30 mmolm -2 s -1 . Stomatal apertures were measured 2 h after adding 100 μM H 2 O 2 or 5mM Ca 2+ .

Phenotype Characterization of CPK8 T-DNA Insertion Mutant and Expression
Patterns of CPK8: No obvious morphological difference was observed between the cpk8 and wild-type, columbia plants under normal growth conditions (Fig. 1A). Fourteen days after withholding water, cpk8 showed severe wilting symptoms compared to wild type and re-watering did not allow the complete recovery of cpk8 plants (Fig. 1A). The site of T-DNA insertion in cpk8 was verified by conducting reverse transcription (RT)-PCR experiments (Fig.1B). RT-PCR analysis showed that there is no transcript of CPK8 in cpk8 homozygous plants (Fig. 1C), suggesting that CPK8 expression is completely eliminated in cpk8 mutant. To investigate the expression patterns of CPK8, transgenic plants harboring a GUS reporter gene fusing with CPK8 promoter was generated. High GUS activities were detected in the leaves and abundantly in stomatal guard cells, suggesting the potential role of CPK8 in regulation of stomatal movement (Fig. 1D).

Relative water content in soil (%) Water loss(% of FW)
tant plants did not display any visible phenotypic alteration. First, plants were grown with optimum irrigation up to two weeks and subjected to water stress by complete termination of watering. Fourteen days after withholding water, CPK8 T-DNA mutant showed severe wilting symptoms compared to wild type. Rewatering did not allow the complete recovery of CPK8 T-DNA mutant compared to wild type (Fig. 1A). RT-PCR verification of CPK8 expression in cpk8 is shown in figure 1C.
Relative water content (RWC) is a good indicator of a plant water status at any given time because it closely reflects the balance between water supply and transpiration rate. RWC were determined by weighing pots and expressing the weight loss as a percentage of initial fresh weight during desiccation. Transpirational water loss, as determined by RWC measurements after 3 days from the start of the treatment, was greatly decreased in the mutant compared to the wild type lines upon drought treatment (Fig 2).

Water Loss Measurements of Detached
Leaves: Water loss from detached wild-type, and cpk8 mutant rosette leaves were measured cation cpk8 leaves displayed wilting symptoms and wild type leaves still remain turgid. By 5 hours after treatment mutant leaves completely wilted due to dehydration and wild type line still remained turgid (Fig. 3A). The fresh weight of detached leaves was measured at hourly time intervals. Throughout the duration of the desiccation treatment, mutant leaves consistently lost higher amount water than wild-type leaves (Fig.3B).

Stomatal aperature(m) Stomatal aperature(m)
Stomatal Aperture Measurements: To explore whether drought sensitivity observed for the cpk8 plants correlates with stomata performance, stomatal apertures were measured with different treatments. The cpk8 stomata closed to the same extent as the wild type in response to darkness (Fig. 4A). Similarly, the cpk8 mutations had no effect on the ability of pre-closed stomata to open in response to light (Fig. 4A).
It is well characterized that plants typically synthesize ABA in response to drought, which triggers the closing of stomata, thus reducing water loss and enhancing drought stress resistance (Schroeder et al., 2001;Luan, 2002). We tested whether the gene disruption affects the stomatal movements in the mutant treated with ABA. Leaf materials were incubated in10M ABA under light. ABA induced stomatal closure partially impaired in comparison to wild type (Fig. 4B).
The exogenous H 2 O 2 induced elevations of cytosolic calcium and stomatal closure (Pei et al. 2000). However, preopened cpk8 stomata failed to close in response to H 2 O 2 ( Figure  5A), which is consistent with the inability of cpk8 plants to reduce water loss upon drought.
Further, CDPKs have been predicted to function in response to cytoplasmic Ca 2+ elevations in many physiological processes in plants. Extracellular Ca 2+ causes stomatal closing, by initiating repetitive cytoplasmic Ca 2+ elevations in guard cells (Pei et al. 2000).
To investigate the role of CPK8 in guard cell signaling, detached leaves were incubated in 5mM CaCl 2. Addition of [Ca 2+ ] ext to preopened wild-type stomata caused closure, whereas in cpk8 stomatal closure was significantly attenuated suggesting that CDPKs function in [Ca 2+ ] cyt perception and ion channel activation (Fig. 5B). These data indicate that the cpk8 mutations do not cause a general defect in stomatal functioning but specifically disrupt H 2 O 2 and Ca 2+ signaling in guard cells.

Excess H 2 O 2 Accumulation in the cpk8 Mutant under Drought Stress:
As an important signaling molecule, H 2 O 2 had been identified to mediate ABA signal transduction in stomatal guard cells (Pei et al., 2000;Murata et al. 2001;Zhang et al. 2001b;Kwak et al. 2003;Bright et al. 2006;Miao et al. 2006;Yan et al. 2007). It is known that excess ROS (Reactive Oxygen Species) accumulation in living plant cells is toxic to cellular activities, so the cytosolic concentration of ROS must be stringently controlled (Mittler 2002;Apel and Hirt 2004). To test if H 2 O 2 accumulation would be changed in cpk8 mutants, 3, 3'diaminobenzidine (DAB) uptake method (Thordal-Christensen et al. 1997;Guan and Scandalios 2000) was applied to examine the production of H 2 O 2 in leaves of cpk8, and wild-type plants. As shown in Fig. 6 homeostasis.

DISCUSSION
CDPKs have been found to function in response to cytoplasmic Ca 2+ elevations in many physiological processes in plants (Harmon et.al. 2000). Evidences for a role of CDPKs in biotic stress signaling and environmental stress signaling were previously reported (Romeis 2001, Sheen 1996, Saij, 2000. The potential of CDPKs for engineering useful traits has also been suggested by alterations in the expression of rice OsCDPK7 that influenced cold and salt/drought tolerance in transgenic rice plants (Saijo 2000). Results of this study provided that AtCDPK8 plays a role in the transduction of an H 2 O 2 signal in guard cells that mediates stomatal regulation in response to drought stress.

A B
In addition, very strong expression of ATCDPK8 promoter GUS in transgenic plants was observed in guard cells of the leaf epidermis ( Figure 1D), implying that ATCDPK8 specifically functions in guard cells. Stomatal aperture measurements showed that stomata from wild-type and AtCDPK8 plants closed to the same extent in the dark and after exposure to light, both materials displayed same extent in the opening of stomatal pores (Fig.4A). Pei et al (2000) reports that H 2 O 2 induced elevations of cytosolic calcium and stomatal closure. However, pre-opened cpk8 stomata failed to close in response to H 2 O 2 and Ca 2+ ( Figure 5A, 5B), which is consistent with the inability of cpk8 plants to reduce water loss upon drought suggesting that loss-of-function mutation in CPK8 impairs H 2 O 2 homeostasis and signal transduction in guard cells. Hence, it is possible that CPK8 acts as a key regulator that specifically modulates H 2 O 2 homeostasis and as an essential signal transduction molecule in guard cell signal transduction pathway.
These data indicate that the cpk8 mutations do not cause a general defect in stomatal functioning but specifically disrupt H 2 O 2 signaling in guard cells. Recent reports revealed that ABA-and Ca 2+ -induced stomatal closing were partially impaired in cpk3 cpk6 mutant alleles (Mori et al. 2006). Similarly, stomatal closure in AtCDPK8 recessive plants was impaired in response to Ca 2+ and H 2 O 2 but not to ABA whereas closure elicited by darkness and opening elicited by light were unaffected suggesting an alternate signaling pathway. On the basis of the results presented here as well as those reported previously (Mori et al. 2006;Pei et al. 2000), allows to describe a divergent oxidative signal transduction pathway from sensing to the drought response in plants.

CONCLUSION
Results of this study confirmed the potential location of the AtCDPK8 by GUS staining and RT-PCR verified the presence and absence of AtCDPK8 in transgenic knockout mutants and wild type plants respectively. Phenotypic expression along with stomatal and DAB staining studies revealed that AtCDPK8 plays a role in the transduction of an H 2 O 2 signal in guard cells mediating stomatal regulation in response to drought stress via divergent oxidative signal transduction pathway.