Response of lettuce seeds undergoing dormancy break and early senescence to plasma irradiation

This study reports the response of lettuce seeds undergoing dormancy breaking and early senescence to DBD plasma irradiation. A heat map of germination percentages at 12 h reveals that dormancy has broken at 39 days' storage, and that one minute of plasma irradiation enhances germination in dormant seeds. Plasma irradiation does not affect those seeds where dormancy has already broken. Early senescence via storage was estimated using ESR measurements and the molecular modification of quercetin. This study reveals that lettuce seed susceptibility to plasma irradiation depends on storage duration and conditions, with dormancy state as a critical variable modulating the impact of plasma irradiation.

][3] The irradiation of plant seeds with plasma can promote growth and increase harvest yield. 4)We will demonstrate that three minutes of plasma irradiation (at a discharge power of 2.3 W) of Arabidopsis thaliana seeds results in growth enhancement at all growth stages, from germination to harvest. 5)Growth response induced via the plasma treatment of seeds has also been demonstrated in numerous other plants, such as radish, 6,7) wheat, 8) red clover, 9) Sunflower, 10) red clover, and purple coneflower. 11)urthermore, studies of the molecular mechanisms underlying these responses (reviewed recently by Mildaziene et al. 12) ) have revealed that plasma irradiation alters the balance of abscisic acid (ABA) and gibberellic acid (GA) in seeds 13,14) as well as their deoxyribonucleic acid (DNA) methylation levels. 15)Recently, a method has been designed to directly detect the reactive oxygen and nitrogen species (RONS) induced in seeds by plasma irradiation, employing mass spectrometry in order to quantitatively estimate the biological effects of plasma irradiation. 16)The majority of this research uses plasma to induce plant response.Research into plasma agriculture, however, has tended to focus on the correlation between plant phenotypes and plasma conditions such as voltage, power, and gas configuration.It has been pointed out that in order to properly comprehend the mechanism of plasma-induced plant response, an understanding of plant molecular biology is crucial.Research has focused on dormancy with respect to RONSs, given that the dormancy state of seeds is an essential physiological mechanism controlling germination. 17)Bailly et al. showed that seeds produce endogenous ROS during storage after seed harvesting, 18) and that storage conditions (such as humidity 19) ) can alter their dormancy state.However, there have to date been limited reports discussing plant responses induced by seed irradiation with plasma, where dormancy is included as a relevant parameter.In this study, we provide new insights into the effects of plasma irradiation, focusing on their dependence on changes in the seed's dormancy state.
Seeds of Lactuca sativa L. (lettuce) were purchased from Asahi Farm, Japan.Lettuce seeds were selected as a model for seeds whose dormancy state changes over a short period of time. 19)Seeds from the same batch, delivered on the same day were used in all of the experiments in this study.The physiological state of the seeds was modulated by two methods: humidification and storage time.Humidification was achieved by leaving the seeds in the dark for periods of 0, 0.5, 1, and 2 days, respectively, at a temperature of 22 °C, and a humidity of 85%Rh 19) for the purpose of a short-term study of the effects of plasma irradiation.For the estimation of storage effects, seeds were preserved in the dark at 4 °C, in sealed bags containing 20 ml of seeds, for 152 days.During the storage period, one bag was used per germination test.An experiment using the stored seeds was conducted to study the relationship between plasma irradiation and germination without humidification.A scalable dielectric barrier discharge (SDBD) electrode was employed to irradiate the seed with plasma. 4,16,20,21)Lettuce seeds (3000 mg or about 3500 seeds) were distributed equally on a quartz plate placed under the SDBD electrode in such a way that the distance between the electrode and the seed surface was 5 mm.Temperature and humidity were maintained at 23.6 ± 2.3 °C and 52.9 ± 6.1%Rh, respectively.Plasma was generated intermittently (5 s ON/55 s OFF) near the electrode at a 13 kV pp discharge voltage and at a 9.4 kHz reputation frequency.The discharge power was measured via the V-Q Lissajous method, using a capacitor inserted between the SDBD electrode and the ground.The ozone (O 3 ) concentration was measured in the same manner as in Ref. 22.The O 3 concentration was measured using O 3 detection tubes No. 18 M (Gastec, Kanagawa, Japan) connected via a hole in a glass plate vertically placed at 5 mm below the center of the SDBD electrode by means of a silicon rubber tube.The indicated O 3 concentrations were corrected by the dead volume of the rubber tube to the hole.O 3 concentration was separately obtained at 0, 10, 30, and 50 s elapsed following a 5 s burst of plasma generation.For the germination test, the samples of seeds (450 mg or about 520 seeds) were taken out from the plate after 1, 3, and 5 min of irradiation.For the test, 30 seeds were placed in a 5 × 6 array using tweezers, onto filter paper (Advantec; 00021090) soaked with 3 ml of tap water and placed onto a Petri dish (Kanto Kagaku; CSPD90-15S).The germination test was carried out in an incubation chamber under the following controlled conditions: a temperature of 22 °C, a humidity of 55%, and a photon flux of 100 μmol m −2 s −1 .The number of germinated seeds was counted every 12 h.The number of seeds for one replicate was 50, with 3 biological replicates.The water content of seeds was measured using an infrared moisture analyzer (Kett; FD-660) on 1.0 g, or about 1160 seeds (the number of experimental replications was 14).Electron spin resonance (ESR) spectroscopy (Bruker, ER072) was carried out to measure the quantity of organic radicals in the seeds 21,23,24) at a microwave power of 2.15 mW, a frequency of 100 kHz, a g-factor range from 1.84 to 2.19, a gain of 1.00 × 10 5 , a temperature 300 K, and with 1024 data points.The seed samples employed in the germination test were used for the ESR measurements.The ESR signal was divided by the weight of 50 seeds (see Table S2).Means of the various parameters between the control and treatment groups were compared using Student's t-tests for independent samples.
The characteristics of the SDBD plasma were evaluated in terms of power consumption and O 3 concentration.The discharge power of the SDBD plasma was 15.8 ± 0.96 W. Figure 1 shows the O 3 concentration.In Fig. 1, t = 0 stands for the time immediately after plasma generation for 5 s.The O 3 concentration was 97.0 ppm at 0 s, then gradually decreased.The O 3 concentration was below the limit of detection (4 ppm) at 50 s.The maximum O 3 concentration remained at 93.5-97.0ppm when 5 s of plasma generation was performed 12, 36, and 60 times, with an OFF-time interval of 55 s.The seeds thus experienced multiple pulses of O 3 exposure that decayed within approximately 50-55 s.The O 3 exposure amount was evaluated by integrating the result of Fig. 1, assuming that the slope from 30 s to 50 s was maintained until 60 s.As a result, when one cycle of 5 s ON/ 55 s OFF was performed, the level of O 3 exposure to the seeds is 950 ppm s (=1.90 mg l −1 s), calculated at a temperature of 23.6 °C, a pressure of 1013 hPa, and at a molecular weight of 48.Therefore, the total level of O 3 exposure to the seeds measured 22.8, 68.4, and 114 mg l −1 s for plasma irradiation at 1, 3, and 5 min, respectively.Consideration of other RONSs specific to plasma irradiation will be provided in future studies.
Table I shows the percentage of germination (at 12 h after imbibition) of seeds exposed to different treatments, such as humidification, and exposure to plasma irradiation, in different combinations.The average percentage of germination under all conditions was 0% at 0 h, and over 90% at 24 h (see Table S1), indicating that the maximal germination of lettuce seeds was not affected within 2 days of humidification.A progressive increase in germination percentage of seeds subjected to humidification only (without plasma irradiation) was observed at 12 h after imbibition, and increased with the duration of humidification (the percentage of germinated seeds was enhanced by 85 and 128% after 1 and 2 days of humidification, respectively).Plasma irradiation for 1 min duration did not impact germination at 12 h, whereas 3 min, or prolonged treatment, stimulated germination at 12 h (by 39%) in seeds humidified for 1 day.However, this effect was not statistically significant due to the large scatter of results.Negative effects (−31%) of plasma irradiation for periods of 3 and 5 min respectively, was observed in seeds humidified for 2 days.These results indicate that the effects of plasma irradiation are dependent on the humidification period.On the other hand, the dependence of germination characteristics on the period of humidification and that of plasma irradiation also suggests that an optimum condition for treatment duration may be determined.It is important to note that the germination percentage at 24 h after imbibition was above 90%, even in seeds irradiated with plasma for 5 min (Table S1).Thus, plasma irradiation did not negatively affect the final germination percentage.A notable finding is the lack of a cancelling effect between humidification and plasma irradiation.The germination rate at 12 h was significantly (156%) increased for seeds subject to humidification for 1 day and plasma irradiation for 3 min, as compared to the control (no humidification and no plasma irradiation).The ratio of 3-min plasma irradiation to 0-min irradiation was consistently 1.4 for both 0-and 1-day humidification.Furthermore, the ratio of 1-day humidification to 0-day humidification remained at 1.8 for both 0-and 3-min plasma irradiation.A maximum germination rate of 20.0% at 12 h was not achieved when humidification and plasma irradiation were applied independently, indicating a positive additive effect between humidification and plasma irradiation.Such additive effects were also found for other conditions.We found a significant increase in germination rate at 12 h, compared to the control, for 0.5 day of humidification and 3 min of plasma irradiation (71%), 1 day of humidification and 5 min of plasma irradiation (100%), and 2 days of humidification and 1, 3, and 5 min of plasma irradiation (114%, 56%, and 56%, respectively).These results indicate that both the intrinsic effect of humidification and the extrinsic effect of plasma irradiation may be important factors in determining germination kinetics.This result is consistent with the results shown by August et al. 25) who found that the stimulation of germination by means of plasma irradiation in Arabidopsis thaliana seeds was far more effective in seeds containing 30% water, as compared to seeds with lower (3 or 10%) water content, and concluded that increasing the water content of the seed improved the plasma-triggered dormancy release.
Aiming to gain more information on such effects, we estimated the impact of humidification on the seeds' water content.Figure 2 shows the dependence of water content in seeds on humidification time.The obtained results revealed that water content monotonically increases from 4.7% to 10.8% with an increase in the duration of the humidification from 0 day to 2 days.For the first day of humidification, the water content increased linearly.The fitted equation is given by: where y and x denote the water content (%) and humidification duration (days), respectively.The water absorption rate into the seeds from air at 85%Rh was constant until the level of water in the seeds reached 9%.The water absorption rate, R abs , of early 1 day of humidification is given by: where a is the slope of water content per day, 4.0% day −1 from Eq. (1), M s is the mass of a lettuce seed, 0.8 mg seed −1 , 16) and M w is the mass of an H 2 O molecule, 18 g mol −1 .R abs is 1.8 μmol day −1 seed −1 for 85%Rh air at 22 °C.Thus, the change in sensitivity of germination to plasma irradiation due to humidification (shown in Table I) may be explained by an increase in the water content of the seeds.According to August et al, the increase in water content results in a transition of the cellular cytoplasm from a glassy to a rubbery state, followed by a better diffusion of plasma-generated RONS that releases dormancy. 25)herefore, examining the effects of plasma irradiation on seeds with changing physiological states while maintaining low moisture content is intriguing.
Besides humidification, we attempted to change the physiological state of seeds by other means, such as seed storage time, which requires long-term experiments to eliminate other effects caused by moisture absorption.Figure 3 shows the dependence of seed water content on storage time.The results below indicate that seed water content remained constant for 152 days.The estimated mean and standard deviation were 4.7% and 0.3%, respectively.These results indicate that the seeds were stored under appropriate conditions to maintain low water content during the storage period.
It is well known that seeds undergo after-ripening and dormancy breaking during longer periods of storage, due to ROS production accompanying the changes in the balance of phytohormones. 26,27)We carried out a study into the effects of plasma irradiation on lettuce seeds stored for different time durations, aiming to estimate the impact of the seed dormancy state on plasma-induced changes in the parameters of germination.The results presented in Figs.4(a) and 4(b) show how the percentage of germination at 12 and 24 h after imbibition depends on the duration of seed exposure to plasma discharge.As one can see, the differences in plasma effects on germination are more obvious at 12 h [Fig.4(a)], as Table I.The germination percentage of lettuce seeds at 12 h after imbibition.The symbol * indicates statistically significant effects of plasma treatment (i.e., the difference from 0 min plasma irradiation and 0 humidification duration).The symbol # indicates a statistically significant difference between nonhumidified (0 humidification duration) and humidified groups for the same duration of plasma treatment (the differences were assumed to be statistically significant at p ⩽ 0.05).The means of three replicates ±SD are presented here., germination at 24 h or maximal seed germination for all experimental groups was similarly high and did not fall below 90% for 152 days.The presence of optimal time for the germination percentage at 12 h indicated that the germination rate of seeds decreased after 39 days of storage, likely due to seed senescence.Thus, during a 152-day storage period, seeds undergo both dormancy break and early senescence.

Plasma irradiation duration (min
The finding that both control seeds and seeds irradiated with plasma for 1, 3, and 5 min had an optimal germination percentage after 39 days of storage [Fig.4(a)] indicates that the SDBD plasma had little effect on the timing of the dormancy break.Seed treatment with plasma for 1 min tended to improve germination characteristics, but only until dormancy break (the mean percentage of germination at 39 days was 1.14 times, as compared to the control).Lengthening the duration of seed exposure to plasma (3 min) did not help to increase positive plasma effects, and 5 min irradiation actually decreased the germination percentage at 12 h after 39 days of storage.These results indicate that the susceptibility of seeds to plasma irradiation varies depending on the physiological state of seeds.In other words, for lettuce seeds, plasma irradiation for 1 min positively affected on the germination rate (estimated by the germination percentage at 12 h) only until the point of dormancy break.
To comprehensively examine the effect of plasma irradiation on germination by the number of days elapsed, we employed a heat map method.Figure 5 shows a heat map of the average germination percentage at 12 h after seed imbibition.The heat map was obtained by spline fitting of the germination percentage data.This analysis revealed that the strongest positive effect of seed irradiation with plasma on germination percentage (a hot spot) appears on day 39 for 1 min treatment duration.Increasing the duration of plasma irradiation above 1 min does not have any apparent effect on the germination percentage.Therefore, we conclude that the dormancy state of lettuce seeds is an important parameter in terms of the effect of plasma irradiation.Since seeds become senescent due to excessive storage, 19) aging is thought to be an important additional factor when considering the decrease in germination in the storage period after dormancy break in this study.
Early senescence following dormancy break was studied using ESR analysis.Figure 6 shows typical spectra of ESR signals in the region of g = 1.84 to 2.19 after plasma irradiation for 0, 1, 3 and 5 min, respectively, for seeds stored for 82 days.In line with standard practice, ESR analysis is typically conducted after seeds have shown signs of ageing, and their final germination rate has declined. 28)Consistent with this, our study initially planned to perform ESR analysis after observing a decrease in the final germination rate.Despite the lettuce seeds maintaining a final germination rate of over 90% at 82 days of storage (and even after 152 days, finally, as shown in Fig. 4), the possibility of early senescence could not be ruled out.Therefore, we decided to initiate ESR analysis after 82 days of storage, earlier than originally planned.A peak appeared at g = 2.00, assigned as the semiquinone radical. 12,13,29)No new peak appeared after plasma irradiation.This result is in agreement with previous studies, which used radish seeds. 21)The peak at g = 2.00 became larger due to plasma irradiation.The width of negative and positive peaks, and the intersection with signal intensity at 0 revealed no difference between samples.Consequently, the signal intensity obtained by subtracting the minimum value from the maximum value indicates the number of semiquinone radicals.Figure 7 shows the signal intensity for seeds irradiated with plasma for 0-, 1-, 3-and 5-min, respectively (raw data available in Table S3).The signal intensity was 0.97 × 10 4  without plasma irradiation (0 min), increasing to 1.30 × 10 4 after 1 min of plasma irradiation, and further increasing to 1.63 × 10 4 after 3 min of plasma irradiation.However, for a plasma irradiation period of 5 min, the intensity measured 1.52 × 10 4 , which is almost the same as that for 3 min-plasma irradiation.Figure 7 also shows the intensity dependency at g = 2.00 on plasma irradiation time for quercetin.This result was obtained by irradiating quercetin powder (ChromaDex; ASB-00017030-100) with plasma.For this, 1 mg of quercetin was placed on a quartz plate yielding density of 1 mg/100 μl and followed by ESR measurement.One sample of quercetin was prepared per condition.Quercetin is one of the typical phenolic compounds found in plants such as lettuce 30) which is able to scavenge superoxide anions. 31)The signal intensities obtained for quercetin powder irradiated with plasma for 0, 1, 3, and 5 min, were 1.30 × 10 4 , 2.55 × 10 4 , 2.77 × 10 4 , and 2.77 × 10 4 , respectively.This result qualitatively resembles the behavior of lettuce seeds as shown in Fig. 7.The formation of organic radicals from phenolic compounds is responsible for this change in signal intensit, and the results show that it depends on the duration of plasma irradiation.The possible mechanism involved in the increasing intensity at g = 2.00 is that the -OH group in hydroquinone C 6 H 4 (OH) 2 in the seeds is converted to -O* by plasma irradiation, yielding the semiquinone radical C 6 H 4 OHO*. 32,33) he semiquinone radical is further oxidized to a form of quinone, C 6 H 4 (=O) 2 , that does not contribute to the signal intensity of ESR at g = 2.00. 34)owever, in Fig. 7, a significant decrease in signal intensity with increasing irradiation time is not observed.This can be explained by considering the following equilibrium reaction (3): where Q, O 2 * − , and SQ* − show levels of quinone, superoxide, and semiquinone radicals, respectively.Therefore, Fig. 7 suggests that quinone increased by plasma irradiation reacts with relatively long-lived O 2 * − , and the reverse reaction that returns to semiquinone radicals becomes noticeable especially in the range of plasma irradiation for 3 min or more.Reaction (3) also shows that oxygen molecules, which are more transport friendly than flavonoids, accept electrons from semiquinone radicals.This suggests that oxygen molecules act as carriers in the signal network for propagating the stimulation of plasma irradiation to other tissues in seeds.This may be a unique characteristic of plasma irradiation to dry seeds.Figure 8 shows the signal intensity ratio on the storage duration.The signal intensity ratio was obtained by normalization based on the signal intensity without plasma irradiation.R 1 , R 3 , and R 5 show the signal intensity ratios for plasma irradiation duration of 1-, 3-, and 5-min, respectively.After 82 days of storage, R 1 = 1.33,R 3 = 1.67, and R 5 = 1.57, all of which are maximal.After that, although the relative intensity fluctuates, it tends to decrease with storage time (correlation coefficients of R 1 , R 2 , and R 5 are −0.59,−0.67, and −0.57, respectively).This result suggests that the quantity of antioxidant molecules (e.g., glutathione 35) and   The Japan Society of Applied Physics by IOP Publishing Ltd vitamin E 36) ) able to non-enzymatically scavenge ROS by becoming semiquinone radicals decreases with storage time. 18)According to S. Yu et al., the number of antioxidant flavonoids in seeds decreases with ageing. 37)Therefore, ESR results indicate that the seeds had started to undergo senescence after their dormancy break, supporting the result of Fig. 4. At 152 days of storage, R 1 = 1.11,R 2 = 1.14, and R 5 = 1.10, i.e., not below the intensity level without plasma irradiation, but closely approaching 1.00.This finding suggests that flavonoids are greatly reduced compared to the levels for the 82nd day of seed storage.
In summary, we carried out an experimental study of the treatment of lettuce seeds with plasma, focusing on the dependence of plasma effects on germination based on the physiological state of seeds, modified by humidification and storage.The germination percentage of seeds without plasma irradiation varied depending on the duration of humidification and storage.Experiments using seeds with humidification showed that germination rate increased with the duration of plasma irradiation and humidification, before decreasing.The amount of water in the seeds increases with humidification, leading to faster activation of metabolic processes in the seeds, which leads in turn to an increase in the germination rate.The heat map result suggests that controlling the dormancy state by regulating seed storage duration may improve the reproducibility of the plasma irradiation effects.Furthermore, in the case of lettuce seeds and SDBD plasma irradiation, the effect of plasma irradiation was not observed after seed dormancy breaking.ESR measurement indicates that seeds were undergoing early senescence during this storage period.These results show that the dormancy state of seeds is an important parameter impacting the effects of seed irradiation with plasma.

Fig. 1 .
Fig. 1.The ozone concentration at 5 mm below the electrode at 0, 10, 30, 50 s elapsed after plasma generation for 5 s.Marks show the mean values, and error bars show standard deviations.The grey area shows the limit of detection at 4 ppm.

Fig. 5 .
Fig. 5.A heat map of the average germination percentage ate 12 h after imbibition, obtained by spline fitting of germination percentage data.The vertical axis shows the time of plasma irradiation (min) and the horizontal axis shows the time of storage (days).The color bar indicates the germination percentage.

Fig. 8 .
Fig. 8.The dependence of the relative signal intensity in seeds irradiated for 1, 3, and 5 min by plasma, normalized by the intensity in seeds without plasma irradiation.The relative intensities R 1 , R 3 , and R 5 show the plasma irradiation duration for 1, 3, and 5 min, respectively.

Fig. 6 . 5 ©
Fig. 6.Typical spectra of ESR signals in the region of g = 1.84 to 2.19 for lettuce seeds without plasma irradiation and with 1-, 3-and 5-min-plasma irradiation for stored for 82 days.