12-Hydroxyjasmonic acid glucoside causes leaf-folding of Samanea saman through ROS accumulation

Foliar nyctinasty, a circadian rhythmic movement in plants, is common among leguminous plants and has been widely studied. Biological studies on nyctinasty have been conducted using Samanea saman as a model plant. It has been shown that the circadian rhythmic potassium flux from/into motor cells triggers cell shrinking/swelling to cause nyctinastic leaf-folding/opening movement in S. saman. Recently, 12-hydroxyjasmonic acid glucoside (JAG) was identified as an endogenous chemical factor causing leaf-folding of S. saman. Additionally, SPORK2 was identified as an outward-rectifying potassium channel that causes leaf-movement in the same plant. However, the molecular mechanism linking JAG and SPORK2 remains elusive. Here, we report that JAG induces leaf-folding through accumulation of reactive oxygen species in the extensor motor cells of S. saman, and this occurs independently of plant hormone signaling. Furthermore, we show that SPORK2 is indispensable for the JAG-triggered shrinkage of the motor cell. This is the first report on JAG, which is believed to be an inactivated/storage derivative of JA, acting as a bioactive metabolite in plant.

www.nature.com/scientificreports/ mechanistic basis for the induction of K + release via activation of the potassium channel by JAG remains elusive. Here, we report that JAG induces the accumulation of reactive oxygen species (ROS) in the adaxial motor cells of S. saman to induce cell shrinking. JAG-induced shrinking of adaxial cells occurs independently of the plant hormones jasmonic acid (JA) and abscisic acid (ABA), which are also known to induce shrinking of guard cells. This finding will pave the way to a complete understanding of the molecular mechanism of JAG-triggered nyctinastic leaf-closing in S. saman.

JAG-induced accumulation of second messengers in motor cells.
The shrinking of stomatal guard cells during stomatal closure is well known in the field of cell-shrinking in plants, and a plethora of molecules involved in ABA-induced stomatal cell shrinking have been identified 30,31 . In particular, ROS and calcium ions are known to play an important role as second messengers 32,33 . ROS are effective signaling molecules that can induce guard cell shrinkage in both methyl jasmonate (MeJA)-and ABA-induced stomatal closure 34 . We compared the effect of JAG with that of plant hormones ABA and JA both in Samanea extensor motor cells and Arabidopsis guard cells. It is also possible that 12-OH-JA, the hydrolyzed product and a biosynthetic precursor of JAG, might function as the bioactive form of JAG. However, in our previous report, we confirmed that 12-OH-JA did not induce shrinkage of Samanea motor cells 26 ; hence, 12-OH-JA was excluded from the experiments in this study. Protoplasts of Arabidopsis guard cells and Samanea motor cells were prepared as previously reported 15 . Intracellular ROS accumulation in these protoplasts was monitored using the fluorescent dye 2′, 7′-dichlorofluorescin diacetate (H 2 DCF-DA), which is widely used as a ROS indicator in guard cells 35,36 . Significant ROS accumulation (p < 0.05 by SNK post-hoc test) was triggered in the guard cells of A. thaliana by 10 µM ABA treatment (Fig. 2a), which is consistent with previous reports 35,36 . No ROS accumulation was observed in guard cells of A. thaliana treated with 100 µM JAG ( Fig. 2a and c). In contrast, significant ROS accumulation (p < 0.05 by SNK post-hoc test) was triggered in protoplasts isolated from Samanea extensor motor cells by 100 µM JAG, whereas 100 µM ABA had no distinct effect on ROS generation in motor cells ( Fig. 2b and d). In addition, JAG-induced ROS accumulation was not observed in Samanea flexor motor cells, wherein JAG cannot induce cell shrinking ( Fig. 3b and f) 26 . It was concluded that JAG triggered ROS accumulation in extensor motor cell protoplasts, whereas ABA triggered ROS accumulation in guard cells, and that the responses to JAG and ABA in the Arabidopsis and  MeJA is widely used to trigger JA signaling in plants 37 . Like ABA, MeJA also induces stomatal closure 34,38 . However, JA cannot induce shrinkage of extensor motor cells 26 . In our study, treatment with 10 µM MeJA triggered ROS accumulation in Arabidopsis guard cells ( Fig. 2a and c), a result consistent with previous reports 35,36 . In extensor motor cell protoplasts, JAG treatment triggered ROS accumulation, whereas treatment with 100 µM MeJA did not affect the ROS accumulation within 15 min ( Fig. 2b and d). These results are consistent with the previous finding that JAG is an inactivated derivative of JA and cannot induce JA signaling 39 . These results suggested that JAG-triggered ROS accumulation is independent of JA signaling. Thus, we further focused on the JAG-induced accumulation of ROS in the Samanea motor cells.

JAG-induced ROS accumulation in the cytosol triggers shrinkage of extensor motor cell protoplasts.
Recent studies have shown that ROS accumulates in different subcellular components by different mechanisms 40 . In general, chloroplasts are the largest ROS producers in plants, especially in periods of light. In contrast, mitochondria produce most ROS in the dark and in non-green tissues 40 . JAG treatment triggered ROS www.nature.com/scientificreports/ accumulation in the extensor motor cell protoplasts of S. saman. Understanding the subcellular distribution of the accumulated ROS will provide insight into its mechanisms of action. We analyzed the subcellular distribution of JAG-induced ROS in extensor motor cell protoplasts using double staining experiments with H 2 DCF-DA and MitoTracker Red CMXRos. Chloroplasts were identified by autofluorescence. The background DCF signal in the Mock treatment strongly colocalized with chloroplasts and mitochondria (Fig. S1). The JAG treatment caused a remarkable increase in the DCF signal in the cytosol (Fig. S1), demonstrating that JAG-triggered ROS accumulates in the cytosol.
Next, we examined whether JAG simultaneously induced ROS accumulation and cell shrinkage in Samanea motor cell protoplasts isolated from Samanea extensor/flexor motor cells prepared from the adaxial/abaxial side of the pulvinus, respectively (Fig. 3). Although JAG induced ROS accumulation was observed in the cytosol of extensor motor cell protoplasts, there was no observable ROS accumulation in the cytosol of flexor motor cell protoplasts (Fig. 3a, b and c, e). Significant cell shrinkage (p < 0.05 by t-test) was observed when extensor motor cell protoplasts but not flexor motor cell protoplasts were treated with JAG ( Fig. 3d and f), consistent with a previous result 26 . The time-dependent change in JAG-triggered cytosolic ROS accumulation was consistent with that of JAG-triggered shrinkage of extensor motor cell protoplasts ( Fig. 3c and d). The extensor motor cell protoplasts started to shrink within several minutes and reached a plateau within 15 min of JAG treatment, and the JAG-triggered cytosolic ROS accumulation followed the same time-course. In contrast, JAG treatment had no effect on either ROS accumulation or cell shrinkage of flexor motor cell protoplasts ( Fig. 3e and f). Together, these results suggest that JAG-triggered ROS accumulation may be involved in JAG-induced extensor motor cell shrinkage.
To further assess JAG-induced ROS accumulation, a series of solutions of varying H 2 O 2 concentration was applied to the protoplasts instead of JAG. Within 20 min, both 100 µM and 1000 µM H 2 O 2 significantly increased ROS accumulation (p < 0.01 by t-test) in the cytosol of extensor motor cell protoplasts by up to 40 and 400%, respectively, whereas no increase in ROS accumulation was observed in protoplasts treated with 10 µM H 2 O 2 (Fig. S2). The effect of 100 µM H 2 O 2 on ROS accumulation in the cytosol of extensor motor cell protoplasts was similar to that of JAG (Figs. 3c and S2). Therefore, 100 µM H 2 O 2 was used in the subsequent cell volume change experiments. The effect of JAG on the cytosolic ROS accumulation in extensor motor cell protoplasts could be replicated using 100 µM H 2 O 2 , which could also induce the shrinkage of extensor motor cell protoplasts (Fig. S3). This result suggests that JAG induces extensor motor cell shrinkage through ROS accumulation.
To further assess the role of JAG-induced ROS accumulation in JAG-induced cell shrinkage, the effects of exogenous diphenyleneiodonium chloride (DPI), a widely used inhibitor of ROS production by flavoproteins including RbOH 34,41,42 , and catalase, an H 2 O 2 scavenger 43-45 , were investigated. Cytosolic ROS accumulation and JAG-induced cell shrinkage were both restricted, even after JAG treatment, in the presence of 12.5 µM DPI (Fig. 4). Furthermore, the exogenous application of 100 units mL -1 catalase prior to JAG treatment strongly repressed cytosolic ROS accumulation and eliminated JAG-induced cell shrinkage (Fig. S4). These results emphasize the importance of ROS accumulation in JAG-triggered extensor motor cell shrinkage.
Expression of outward-rectifying K + channels is indispensable for JAG-induced shrinkage of extensor motor cell protoplasts. The involvement of K + efflux systems in the JAG-induced shrinkage of Samanea extensor motor cell protoplasts was previously reported 16,17 . Recently, we revealed that SPORK2, a gene encoding the outward-rectifying K + channel of S. saman, was responsible for leaf-opening movement 28 . Accordingly, we further examined the role of SPORK2 in JAG-triggered cell shrinkage. Unfortunately, we could not observe direct activation of SPORK2 by the addition of JAG (data not shown). However, we did observe the ion transport activity of SPORK2 in whole-cell patch clamp experiment using isolated Samanea motor cells, as previously reported 28 . In these experiments, signal transduction from NADPH oxidase to SPORK2 did not occur because the cytosolic contents of Samanea motor cells were replaced by the pipette solution. However, when treated with H 2 O 2 , SPORK2 expressed in Xenopus oocyte was activated (Fig. S5).
Next, we examined the effect of JAG on extensor motor cells when the expression of SPORK2 was the lowest. To obtain extensor motor cells with the lowest expression level of SPORK2, we checked the expression of SPORK2 in a quarter period of diurnal rhythm containing leaf-folding movement (Fig. 1). SPORK2 expression was highest at Zeitgeber time (ZT) 8 and decreased as the leaves gradually folded, and the SPORK2 expression was not fluctuant in the flexor during this quarter. In the extensors, expression of SPORK2 reached a nadir at ZT 14. In addition, immunostaining using anti-SPORK2 antibody demonstrated that SPORK2 in tertiary pulvinus decreased as SPORK2 gene expression decreased (Fig. S6). We also examined the effect of JAG on the extensor motor cell protoplasts after ZT 14. Interestingly, significant ROS accumulationwas observed in extensor motor cell protoplasts under JAG treatment, whereas no cell shrinkage was observed. Thus, JAG cannot cause shrinkage of extensor motor cell protoplasts with the lowest expression level of SPORK2 gene. Meanwhile, in flexor motor cells, there was no specific ZT that caused JAG-induced ROS accumulation as well as cell shrinkage (Fig. S7).

Discussion
Samanea saman is a model plant used in legume nyctinastic leaf movement studies. Recently, JAG was identified as the bioactive metabolite that mediates this leaf-folding movement in S. saman 25,46 . JAG is perceived by the extensor motor cells in the Samanea plant body 27 , and it selectively induces extensor motor cell shrinkage to cause leaf closure 26 . This is the first report on the molecular mechanism of JAG, which is believed to be a deactivated/ storage derivative of JA 39,47 , as a bioactive metabolite in plant.
This study examined the involvement of ROS accumulation in the JAG-induced motor cell shrinkage of S. saman. The fluorescent dye H 2 DCF-DA was used to detect ROS accumulation. ABA and MeJA triggered ROS production in the guard cells of A. thaliana, as reported previously 34 Finally, the mRNA expression of the main outward-rectifying K + channel (SPORK2) was detected, as well as its effect on JAG-triggered ROS accumulation and JAG-induced cell shrinkage. The results from these experiments strongly suggest that JAG induces motor cell shrinkage through ROS accumulation and that the gene expression of SPORK2 is indispensable in JAG-induced cell shrinkage. In addition, JAG induces shrinkage of Samanea motor cells independent of ABA/JA signaling. Evidence suggests that the regulation of turgor changes in motor cells is similar to that in stomatal guard cells 49 . ABA triggers H 2 O 2 accumulation in guard cells of A. thaliana through activation of the respiratory burst oxidases multigene family (RbOHs; NADPH oxidases) located on the plasma membrane 50,51 . ABA-regulated stomatal closure is impaired in the A. thaliana Rboh D/F (AtrbohD/F) mutant 52 . All of these studies indicated that ROS functions as a second messenger in ABA-induced guard cell shrinkage 35,36,42,52 . ROS as the second messenger have been shown to be involved in various intra-and intercellular signaling events. JA signaling is believed to be involved in stomatal closure 38 . The JA signaling elicitor MeJA triggers ROS accumulation in guard cells of A. thaliana (Fig. 2a and c) 35,36 . MeJA-activated ROS production was previously shown to be mediated by the COI1-JAZ signaling module 38 . In our study, JAG did not trigger ROS accumulation in guard cells (Fig. 2b and  d), which is consistent with the non-participation of JAG in JA signaling 39 . In contrast, JAG treatment triggered ROS accumulation in extensor motor cell protoplasts, whereas MeJA treatment had no effect on ROS accumulation ( Fig. 2b and d). These results suggested that JAG-triggered ROS accumulation is independent of the COI1-JAZ signaling module, consistent with a previous report 26 . DPI chloride (Fig. 4) or catalase (Fig. S3) repressed JAG-induced ROS accumulation and resulting cell shrinkage in extensor motor cell protoplasts. Considering that catalase would be effective on extracellular ROS, the results suggest that the extracellular O 2produced by NADPH oxidases dismutate to H 2 O 2 , which is transported into the cytosol possibly via aquaporins.
Recently, SPORK2 was shown to regulate leaf-movement. We found that the effect of JAG on extensor cells required SPORK2 and was time-dependent. Notably, JAG could not shrink protoplasts prepared from folded leaves between ZT 14-18 when the expression of SPORK2 gene was at a minimum (Fig. 5), but it did induce ROS accumulation within the same time range (Fig. 5c). These results suggest that the processes of ROS accumulation Given that JAG-induced motor cell shrinkage was impeded by the co-addition of TEA (a blocker of K + channels) 26 , our current result suggests that SPORK2 is indispensable for JAG-triggered cell shrinkage. It has been reported that ROS accumulation and the subsequent calcium release activate outward-rectifying plant potassium channel, the GUARD CELL OUTWARD-RECTIFYING K + , by a calcium-dependent kinase through phosphorylation 53 . Although we did not perform calcium imaging experiments in S. saman, a similar phenomenon is presumed to be triggered by JAG in S. saman. It is also possible that ROS directly affects SPORK2, which is expressed according to circadian rhythms, to regulate its K + transport activity in extensor cells, because we confirmed that SPORK2 expressed in Xenopus oocyte was directly activated by ROS (Fig. S4). It was reported that post-translational modification of plant K + channels by ROS plays a role in the regulation of K + transport. A heterologously expressed Arabidopsis K + channel, STELAR K + OUTWARD RECTIFIER, directly induces voltage-dependent activation by ROS 54 . In transmembrane 3 within the voltage sensing complex of STELAR K + OUTWARD RECTIFIER, Cys-168 was responsible for its activation by ROS. Cys-168 is also present in transmembrane 3 of SPORK2, suggesting a similar activation mechanism for SPORK2 in extensor cells. Further studies will reveal the mechanism of JAG-mediated activation of SPORK2. Based on the current finding that ROS is involved in JAG-mediated leaf-folding, the JAG-triggered shrinking of the extensor motor cell and outward-rectifying K + -channel SPORK2 can be linked. To elucidate the mode of action on JAG-induced cell shrinkage, the upstream components of ROS accumulation in JAG-induced cell shrinkage should be investigated in future. The target protein of JAG is the key to unmask these signaling components.

Conclusion
In conclusion, our data demonstrate that JAG induces shrinkage of extensor motor cell protoplasts through ROS accumulation, independently of ABA/JA signaling. JAG has no effect on ROS accumulation in flexor motor cell protoplasts, but JAG can trigger ROS accumulation in the cytosol of extensor motor cell protoplasts to mediate cell shrinkage, which may trigger the folding of Samanea leaves. All of these results lead to the significant and novel finding that ROS is involved in JAG-induced nyctinastic leaf-folding movement. Measurement of ROS for motor cell protoplasts of S. saman using CLSM. The prepared protoplasts (10,000 cells/mL) in 130 µL wash solution were sealed in a glass-bottom Petri dish (φ 35 mm × 12 mm), coated with 200 µL of H 2 O, and incubated overnight at 24 ± 1 °C in dark. Then, the protoplasts were added to 5 µM H 2 DCF-DA (Sigma-Aldrich Co., Ltd., MO, USA) and incubated for 45 min to stabilize their initial fluorescence intensity. Thereafter, the protoplasts were imaged by CLSM (LSM 700, Carl Zeiss, Oberkochen, Germany) at 2-min intervals for 20 min after treatment with 100 µM JAG 26 dissolved in 0.1% DMSO, H 2 O 2 (FUJIFILM Wako Pure Chemical Industries Co., Osaka, Japan) at the indicated concentration, or mock solution (0.01% ethanol or DMSO for DPI); untreated protoplasts acted as the blank control. When used, 12.5 µM DPI (Sigma-Aldrich Co., Ltd., MO, USA) dissolved in DMSO or 100 U/mL catalase (Sigma Co., Ltd.) was added 30 min before treatment with the above compounds. Intercellular fluorescence was excited using 488 nm light emitted by a solid-state diode laser at 0.5% with a Plan-Apochromat 40 × /1.3 oil immersion objective and other settings as follows: emission 495-628 nm, master gain 500-650, pinhole 0.9 µm, 8-bit, frame 1024 × 1024 pixel, zoom 1.0, pixel dwell time 1.58 µs/pixel and line average of 4. Autofluorescence was negligible in this emission range using these settings. Zen 2012 Black Edition software (Carl Zeiss, Oberkochen, Germany) was used for image analysis. Photobleaching and dye leakage from the intercellular to adjacent areas was too low to detect under these conditions. Dye leakage from the cytosol to the vacuole was assessed by comparing the levels of vacuole fluorescence at the beginning and end of each experiment. The round protoplasts were selected in which vacuole fluorescence intensity was less than twice as strong as that of background solution. However, the protoplast was discarded if dye leakage from the cytosol to the vacuole had increased the intensity of vacuole fluorescence up to 200%. ROS accumulation was calculated based on the fluorescence intensity of H 2 DCF-DA. The ROS accumulation of H 2 DCF-loaded protoplasts induced by blue light and the dark conditions was recorded with untreated protoplasts as blank. ROS accumulation was estimated with the following equation: (Fc n = the fluorescence intensity of a protoplast treated with chemicals at nth minute. n = 0, 2, 4, … 20. Fu ave.of n = average of the fluorescence intensity of untreated protoplasts at nth minute. Fc 0 = the fluorescence intensity of a protoplast treated with chemicals at 0 min. Fu ave.of 0 = average of the fluorescence intensity of untreated protoplasts at 0 min). Pixel intensities of fluorescence at each given time were collected as the average intensity of three points that were away from the chloroplasts and vacuoles in each cell. Data were collected from two experiments in parallel on the same day. www.nature.com/scientificreports/ Measurement of cell shrinkage of motor cell protoplasts of S. saman using CLSM. The protoplasts selected for measuring the cytosol fluorescence intensity were used for measuring the cell shrinkage. First, the intensity of the ROS signal was adjusted into similar-level contrast in the same protoplasts at the denoted times. Then, a red circle was made to fit the edge (critical surface of ROS signal) of the round part of the protoplast. The area of the red circle was calculated based on the average area of two independent fitting processes, and the changes in normalized protoplast volume was calculated.

Quantitative RT-PCR analysis of SPORK2. To analyze time-course gene expression profiles of SPORK2
in Samanea tertiary pulvini, excised extensor and flexor motor cells were sampled every 2 h from ZT 8 to ZT 14.
The statistical analysis. All data are presented as means ± SE except denotation. The values followed by different letters are statistically different according to analysis of variance followed by SNK post hoc test. Besides, the significance of differences between data sets was assessed by Student's t-test. Differences were considered significant for P value < 0.05.

Data availability
The datasets generated and/or analyzed during the current study are reported in the references cited or available from the corresponding author, Minoru Ueda, upon request.