Seeds of Arabidopsis thaliana were sown in a 1:1:1 mixture of vermiculite, perlite and soilrite in plastic disposable containers and kept at 4°C in dark for 3 days and then were transferred to 22–23°C to allow germination. The seedlings were grown in controlled environment growth rooms, under an 8 h light (125–150 mmol m^-2 s^-1) and 16 h dark photoperiod, with an average temperature of 22–23°C. The plants were supplied with a nutrient solution (Somerville and Ogren, 1982) or full strength Murashige and Skoog medium (salt mixture obtained from HIMEDIA), on alternate days twice a week. The plants were watered on other days.

Among the two microbial elicitors, cryptogein was prepared from Phytophthora cryptogea (Kadota et al., 2004). Harpin was prepared from Pseudomonas syringae pv. syringae and was the same as the full length harpin_Pss used by Anil and Podile (2012). Both the microbial elicitors were dissolved in milli-Q water, and stock solutions were stored at -20°C. The fluorescent probes of CM-H₂DCFDA and DAF-FM DA from Invitrogen-Molecular Probes were dissolved in DMSO. cPTIO, L-NAME, and DPI from Calbiochem were dissolved in milli-Q water.

Leaves from 5- to 6-week-old A. thaliana plants were detached and incubated in opening medium (10 mM MES-KOH, pH 6.15 and 50 mM KCl) for 3 h under light. A light intensity of 200 μmol m^-2 s^-1 was maintained with the help of a bank of tungsten lamps, and light filtered through a water jacket. The photon flux was measured with a Li-Cor quantum sensor (Li-Cor Instruments Ltd, Lincoln, NE, United States). The temperature was maintained at 25 ± 1°C. After 3 h of light incubation, the leaves were incubated in the medium containing effectors and/or modulators. After treatment for 2 h with the effectors under light, the abaxial epidermis of the leaf was stuck to the cover slip with the help of medical adhesive Telesis V (Premiere Products Inc., Pacoima, CA, United States). The remaining leaf tissue was removed and the stuck epidermis was washed immediately with water. Stomatal apertures were measured with the help of a pre-calibrated research microscope (Olympus CX21) by using NIH image for windows. Approximately 30 stomatal apertures were measured for each experiment and for each treatment.

The levels of ROS and NO were monitored by using CM-H₂DCFDA and DAF-FM DA (Excitation 488 nm, Emission 510–550 nm), respectively. The leaves of A. thaliana were incubated in an opening medium (10 mM MES-KOH, pH 6.15 and 50 mM KCl) under light for 3 h to allow the opening of the stomata. The abaxial epidermis from leaves were mounted on the cover slips with the help of silicone adhesive and were loaded separately with the 20 μM CM-H₂DCFDA or 30 μM DAF-FM DA fluorescent probes for 30 min in dark. After incubation, the epidermis of leaves was washed with the buffer to remove the excess dye and was treated with the effectors or modulators.

The fluorescence in the treated guard cells was observed by using confocal laser scanning microscope (Leica, TCS-SP-2, AOBS 4 channel UV and visible, Heidelberg, Germany) for every 5 min up to 30 min, and the images of the guard cells were captured. Average fluorescence of 30 stomata was quantified from the captured images by using NIH Image for Windows (Gonugunta et al., 2008). Averages from three different experiments on different days were presented. The fluorescence of the guard cells without the effectors was taken as control (100%) and the relative fluorescence of the guard cells for different treatment at different time points were calculated and plotted.

All the experiments were repeated at least on three different days. The presented data are averages with standard errors.

Cryptogein and harpin caused marked stomatal closure in epidermis of A. thaliana. Maximum closure already occurred at 5 μM of cryptogein, while harpin induced maximum stomatal closure at 0.5 μM (Figure 1).

The levels of ROS or NO in guard cells were monitored by using fluorescent probes of CM-H₂DCFDA and DAF-FM DA, respectively. When treated with the cryptogein, the levels of both ROS and NO increased remarkably, very similar to the effects of harpin (Figure 2). Real time monitoring of ROS or NO revealed that maximum accumulation of ROS occurred at 15 min after exposure to cryptogein and harpin. Similarly, levels of NO in the guard cells increased at 20 min after exposure to these two elicitors (Figure 3).

The role of ROS or NO during stomatal closure by microbial elicitors, was assessed by using modulators of either ROS or NO. Cryptogein and harpin induced stomatal closure was partially restricted in the presence of ROS modulators, catalase (ROS scavenger), and DPI (NADPH oxidase inhibitor) (Figure 4A). Among the modulators of NO, cPTIO (NO scavenger), could reverse the stomatal closure by cryptogein or harpin. In contrast, L-NAME (inhibitor of NOS) and sodium tungstate (NR inhibitor) could restrict the stomatal closure by cryptogein or harpin only to a partial extent (Figure 4B).

The modulators of ROS (catalase and DPI) restricted the elevation of not only ROS (Figure 5A) but also NO (Figure 6A) in the guard cells, pretreated with elicitors cryptogein and harpin. In contrast, the modulators of NO (cPTIO and L-NAME) prevented the production of NO (Figure 6B), but did not affect the rise in levels of ROS, in the guard cells (Figure 5B).

The effect of cryptogein and harpin on stomatal closure was studied in Arabidopsis mutants, deficient in NADPH oxidase (atrbohD/F) or NR (nia1 and nia2) or protein phosphatase ABI (abi1 and abi2). The results were compared with wild type, Col-0 or Ler. The stomatal closure by cryptogein and harpin was partially impaired in atrbohD/F mutant plants, compared to wild type (Figure 7A). Stomatal closure by cryptogein and harpin was partially relieved in the nia1 and nia2 mutants compared with that of their respective wild type Ler and Col-0 plants (Figure 7C). The stomatal closure by cryptogein or harpin was completely reversed in abi1 mutants (Figure 7B), compared to partial impairment in abi2 mutants.

The stomatal responses to various biotic and abiotic stress factors have been studied in different plant species including tobacco, Vicia, Pisum, Brassica napus, tomato, and Commelina. Nevertheless, A. thaliana offers as an excellent model compared to the other plant species due to the availability of a large collection of mutants, deficient in the different signaling components. Our article emphasizes the marked stomatal closure by cryptogein and harpin in A. thaliana (Figure 1). Cryptogein and harpin are well known as elicitors of microbial pathogens of tobacco. Our studies provide for the first time a detailed examination of cryptogein and harpin induced stomatal closure in epidermis of A. thaliana. Further, the kinetics and modulation of ROS/NO in guard cells by elicitors are all being reported for the first time in Arabidopsis (Figures 3, 5, 6). We suggest that A. thaliana offers a good model for comprehensive studies on stomatal signaling events, including the responses to microbial elicitors. Since several mutants of Arabidopsis are available, our article can trigger further interest to use Arabidopsis for elicitor-induced stomatal closure.

Reactive oxygen species and NO are the important signaling components during stomatal closure by a variety of signals: ABA, MeJA, elicitors like chitosan or even bicarbonate (Kolla and Raghavendra, 2007; Kolla et al., 2007; Gonugunta et al., 2009; Srivastava et al., 2009). The increase in the levels of ROS and NO in the guard cells treated with the cryptogein and harpin (Figure 2), confirms their signaling role during closure. There have been reports on increase in ROS as well as NO by cryptogein in epidermal peels of tobacco (Allan and Fluhr, 1997; Foissner et al., 2000) and tobacco BY-2 cells (Kadota et al., 2004; Leborgne-Castel et al., 2008; Stanislas et al., 2009) while inducing cell death and plant defense responses. But the increase in ROS or NO levels in guard cells by cryptogein has so far not been reported.

Harpins from different sources are known to induce production of ROS and NO in Arabidopsis suspension cells (Desikan et al., 1996; Krause and Durner, 2004) and in guard cells of tobacco (Zhang et al., 2009, 2012). Again our article is the first attempt of a comprehensive study on the production of both ROS and NO in guard cells during stomatal closure by harpin in Arabidopsis (Figure 2).

Real-time monitoring of fluorescence in guard cells indicated that on exposure to cryptogein and harpin, the levels of ROS rise and reach a peak before that of NO (Figure 3). Although the levels of ROS or NO in the guard cells were observed earlier in response to elicitors, such as chitosan, flg22, harpin, boehmerin (Lee et al., 1999; Melotto et al., 2006; Zhang et al., 2009, 2012), studies on kinetics of ROS/NO production are very few. While endorsing the suggestion that ROS production is essential for NO rise in the guard cells during closure by ABA or elicitors (Bright et al., 2006; Srivastava et al., 2009), we conclude that the ROS acts upstream of NO during cryptogein and harpin triggered stomatal closure.

In guard cells ROS, NO, cytosolic pH, and intracellular calcium are the major points of action, leading to the loss of ions/turgor and subsequent stomatal closure (Agurla and Raghavendra, 2016). Besides their direct effects, there seems to be a strong network of interactions among them. Studies with mutants suggested the upstream action of the ROS to MAP kinases, ABI2 and intracellular Ca²⁺ (Murata et al., 2001; Jammes et al., 2009; Wang et al., 2013). Among the components downstream of ROS are ABI2, NO production, K⁺_in/SLAC channels (Murata et al., 2001; Zhang et al., 2001; Vahisalu et al., 2008; Srivastava et al., 2009). A rise in NO is considered as the early signaling event in guard cell signaling toward ABA, MJ, bicarbonate (Gayatri et al., 2013). The marked interactions of NO with ROS, phospholipase D and G-protein in guard cells during stomatal closure are known (Gonugunta et al., 2008; Li et al., 2009; Distéfano et al., 2012; Zhang et al., 2012). Further studies on these interactions using elicitors as signals would be extremely interesting.

Another point of interest is the role of mitochondrial AOX. The levels of ROS and NO in mitochondria are minimized by an active AOX (Cvetkovska and Vanlerberghe, 2012; Vanlerberghe, 2013). Unlike cytochrome c oxidase (CytOX), NO resistant AOX plays a major role in preventing excessive NO generation in guard cells, thus regulating the stomatal closure (Cvetkovska et al., 2014; Gupta et al., 2014). Further experiments are warranted to understand the role of AOX in relation to other sources of NO production during stomatal closure by elicitors.

Cryptogein, a microbial elicitor triggered stomatal closure, and elevated ROS/NO levels in guard cells of A. thaliana (Figures 1, 2). Besides guard cells, cryptogein has been known to be a potent microbial elicitor in inducing several defense responses, but mostly in tobacco (Montillet et al., 2005; Sawai et al., 2010; Kurusu et al., 2013; Rosnoblet et al., 2017). Similarly, harpin has also been extensively documented for its defense responses again mostly in tobacco (Chen et al., 2008; Boureau et al., 2011; Chang et al., 2016). The marked stomatal closure associated with the rise in ROS/NO of guard cells along with the suitability of A. thaliana for these studies opens up an excellent scope for further use of cryptogein and harpin as tools to study stomatal function. The ability of cryptogein and harpin, two elicitors from microbial pathogens of tobacco, to close stomata markedly in Arabidopsis reaffirms the role of stomatal closure as a typical component of innate immunity responses of plants.