HE Rui-rui, XI Gang, LIU Kai, ZHAO Yan-yan

Department of Applied Physics，Institute of Science，Xi’an University of Technology，Xi’an 710048，China

Effect of Pulsed Electric Field on Drought Resistance of Maize Seedling Based on Delayed Fluorescence Induced with LED

HE Rui-rui, XI Gang*, LIU Kai, ZHAO Yan-yan

Department of Applied Physics，Institute of Science，Xi’an University of Technology，Xi’an 710048，China

Effect of pulsed electric field on the drought resistance of crops is an important topic in biological effect of electric field. The changes in the photosynthetic system of leaf cells can be sensitively reflected by the kinetics parameters of delayed fluorescence. In order to reveal the effect of pulsed electric field and its mechanism on drought resistance of crop seedling, the germinating maize seeds were treated by pulsed electric field with electric field strength 200 kV·m-1, frequency 1Hz and pulse width 80ms. Then, PEG-6000 solution with -0.1 MPa osmotic potential to was used to form physiological drought of maize seedlings, the changes of dry leaf mass and the kinetics parameters, induced by LED were studied in this paper. The result showed that the dry leaf mass gradually increased under drought stress after applied with the electric field, which was significantly higher than that without external field, the relative growth rate was 45.6% (p<0.01). Besides, during the processes, the relative growth rate was between 5.8%～18.7%, the difference was significant (p<0.05) when there was no electric field, which indicated that the pulsed electric field promoted the leaf growth of maize seedling. The analysis of delayed fluorescence kinetic about leaf of maize seedling showed that the value of delayed fluorescence kinetics parameters, initial photon numberI0, coherence timeτ, decay factorβand integral intensityI(T), under drought stress, showed fluctuation, These changes were response to drought stress made by leaf cells. The study also found that pulsed electric field increased delayed fluorescence kinetics parameters and the integrated intensity of leaf cells, which indicated that the pulsed electric field could improve the photosynthesis potential and the organize sequence of photosynthetic electron transport system in leaf cells, as the interaction between functional molecules was strengthened, the leaf photosynthetic capacity was enhanced under drought stress. The result of this study provides a reference to explain clearly the effect of the pulsed electric field on drought resistance of plants seedlings.

Delayed fluorescence； Electric field； Drought stress； Kinetics parameters； Maize leaf

Introduction

The effect of electric field to crops drought resistance has been an important project in biological effects of electric field[1-2]. People estimate the effect of electric field with two methods: the first one is to study shape index, such as seedling size, root length, fresh mass, dry mass, seedling survival rate, and so on; the second is to study the change of physiological and biochemical indexes, such as cell membrane permeability, content of MDA, activity of superoxide dismutase, chlorophyll content and gene expression, etc[2-4]. However, the first method judges the drought conditions only after the shape index changed, so it cannot be applied for early diagnosis. The basic data of the latter comes from test tube experiments, separating the relationship of cell components, which has destructive effect, also the information between components and hierarchies was lost, so there is no reflection of the dynamic changes in crop cells under drought stress and the drought resistance of cells cannot be evaluated as a whole.

Delayed fluorescence is a luminescence phenomenon which emerges in life system after the external light stops. Its intensity of radiation is lower than fluorescence and phosphorescence, however, its relaxation time is longer than that of fluorescence[5]. The biological delayed fluorescence comes from excitation and decay of electrons inside the cooperating molecules, the long attenuation process reflects the molecular level related to spatial structure sequence. Therefore, biological delayed fluorescence is related to functional state of biological system, which can be a sensitive index of physiological state of cells. For now, the analysis technique has been applied to estimate the functional state in plant system and detect the environmental change[6-8].

In the research of biological effects of electric field, pulsed electric field has drawn a lot attention recently because of the following advantages: it can produce induced current within the cell to impact cellular activity; it change the chemical binding between chemical reaction rate molecules, it change the shape and structure of protein molecules by inducing the translation and rotation motion of dipole and charge, which affect the biological system directly or indirectly, on the other hand it has a strong impact on membrane, which can change the cell membrane penetrability. In particular, by coupling resonance with membrane potential[9-10], the extremely low frequency pulse electric field can cause a strong impact on physiological state of cells easily, thus, such field is worth to study.

Owing to the technical advantages of delayed fluorescence analytical method, low frequency pulse electric field was used to treat the germination of maize seeds, and drought stress was formed by PEG-6000. The change of delayed fluorescence of maize seedling leaf cells induced by LED was studied in this paper, by dynamically analyzing the fluorescence; the effect of extremely low frequency pulse electric field to drought resistance of maize seedling was illustrated.

1 Materials and methods

1.1 Materials and training

Maize seeds of the same size and similar outlook called Yandan 8 were chosen as material whose surface were sterilized with 0.2% HgCl2for 2 min and thoroughly washed, and then put into distilled water to fully imbibe about 24 h then germinated for 48 h in 30 ℃ constant temperature box. The seeds were selected and divided into two groups one was applied by electric field, called control group another, without external field, was called treatment group, each group had 200 seeds.

1.2 Treatment of PEF

The output of system was at extremely low frequency whose amplitude can be continuously adjusted at the range of 6 to 20 kV and frequency from 0.1 to 15 Hz. Two copper plates with size 60 cm×60 cm was placed in the system sample room, Put the maize seeds between two copper plates electrodes in the sample room. According to the literature[11], field intensity was downwards. The seeds of treatment group were applied with field intensity 200 kV·m-1, frequency 1 Hz ELF-PEF and pulse width 80 ms, for 30 minutes each day, the process lasted for 5 days. At the same time, the similar seeds were chosen as control group under the same condition except that they were not applied with external field.

1.3 Drought stress

Remove the water of the test specimens after the sample treated with PEF, then add the PEG-6000 solution with the osmotic potential is -0.1 MPa, and measure the related metrics at a reasonable time.

1.4 Measurement of dry leaf mass of maize seedling

Maize seedlings were selected from control group and treatment group, their leaves were cut off. The oven was turned on before the experiment until temperature rose to 100 ℃. Snipped leaves of maize seedlings were filled into bag then the bag was put into oven for 10 min under 100 ℃, and then, the leaves were dried to a constant weight under 80 ℃. Took out the samples and cooled them down to room temperature then the dry leaves were weighed with electronic balance. Every group was repeated 3 times then average value was obtained.

1.5 The measurement method of spontaneous luminescence

BPCL ultra weak luminescence analyzer (made by Institute of Biophysics, Chinese Academy of Science) was used for the measurement of spontaneous luminescence. After turned on, the instrument was warmed up for 1 h, and the sample cup was washed with absolute ethyl alcohol to avoid the influence of bacteria. The measure time, time interval, operating voltage and current temperature in the darkroom were set as 60 and 1 s, -1 100 V, 25 ℃, respectively. At the begin of the experiment, maize seedlings were selected from control group and treatment group, and the leaves were cut off then the surface was rubbed to dry, then removed into sample room. The samples were kept in dark for 5 min, then background of sample room was measured before experiment and then the spontaneous luminescence was measured which was the difference between the measured value and background value. The measurement was repeated 3 times then the average value was calculated.

1.6 The measurement method of delayed fluorescence

The measurement system and measurement method of delayed fluorescence could be found in literature[12], after turned on, the instrument was warmed-up for 1h, as what were did when spontaneous luminescence was measured, after the sample be illuminated 30 s by LED,the delayed fluorescence was measured . The measuring time, time interval, operating voltage and current temperature in the darkroom were set as 60 s, 1 s, -1 100 V, 25 ℃, respectively. Also, background value was subtracted. The measurement was repeated 3 times then the average value was calculated.

1.7 The dynamical analysis of delayed fluorescence

According to literature[12], the delayed fluorescence was fitted follow the below formula

(1)

In the formula,tis the measure time,I0，τandβare the fitting parameter, called initial photon number (counts·s-1), coherence time (s), decay factor (dimensionless parameter),ISLis the spontaneous luminescence of per unit time.

Delayed fluorescence integral intensityI(T) is calculated by the below formula

(2)

In the formula,Tis the measurement cycle.

1.8 Data processing

Origin9.0 was used to date processing after experiment, with SPSS software to judge the significance of variance,p<0.05 meant significantly, andp<0.01 meant extremely significant.

2 Results and analysis

2.1 Effect of PEF on dry leaf mass of maize seedlings under osmotic stress

Leaf cells are a place for plant to carry out photosynthesis. The dry leaf mass can reflect the process of material synthesis, distribution and transport in leaves to some extent. Figure 1 showed the change of dry leaf mass in both group. From the figure 1, the dry leaf mass of maize seedling which was treated with PEF was higher than that in control group at the beginning of experiment, and the relative growth rate reached to 45.6%, the difference between groups was extremely significant (p<0.01). After that, with drought stress, the dry leaf mass of maize seedling increased a little at second day and third day in both group as time being. The dry mass in treated group was always higher than control group during the whole test, and the relative growth rate was between 5.8% and 18.7%, differences between two groups was significant (p<0.05). According to what have been discussed, growth of leaf was not stopped by PEG-6000 solution with the osmotic potential is -0.1 MPa. So PEF promoted the growth of leaf and increased the accumulation of dry matter.

Fig.1 Effect of PEF on dry leaf mass of maize seedlings under drought stress

Fig.2 Changes of the delayed fluorescence of maize seedling leavesunder drought stress

2.2 Effect of ELF-PEF on spontaneous luminescence of maize seedling leaves under drought stress

The dynamic changes of leaf cells and the effect of PEF in maize seedling leave under drought stress can be obtained by analysis the delayed fluorescence. Figure 2 was the decay curves of the delayed fluorescence at first day, second day, third day, fourth day and fifth day. As shown here, photon emission of maize seedling leaf followed a trend of decline after the external excitation light stopped，and the process of decline was about a few seconds. Thus, delayed fluorescence was different from general!fluorescence. To explain the characteristics and differences of delayed fluorescence quantitatively, the curves in figure 2 was fitted according to the formula (1), and the result showed in table 1 and table 2. According to table, the goodness (R2) was greater than 0.99, which indicated that the parametersI0，τandβshowed in table could describe the characteristic of dynamic parameters of delayed fluorescence accurately.

Table 1 The kinetic parameters of delayed fluorescence from leaf cells of maize seedlings about control group

Osmoticstresstime/dI0/(counts·s-1)τ/sβR205348.051.0931.6330.99814287.211.4871.6620.99924636.172.2391.6070.99934774.011.4011.4310.99944255.551.6721.5270.99855010.241.8611.4580.999

Table 2 The kinetic parameters of delayed fluorescence from leaf cells of maize seedlings about treated group

Osmoticstresstime/dI0/(counts·s-1)τ/sβR206645.661.2711.7040.99816211.291.6231.7430.99825055.052.4111.7190.99935351.541.7161.5310.99944689.221.8491.5850.99856405.121.9741.5740.999

2.2.1 Effect of PEF on initial photon numberI0

To explain the change of delayed fluorescence curves in figure 2 quantitatively, the change rule of dynamic parameters of delayed fluorescence from leaf cells of maize seedlings in table 1 and table 2 was analyzed.

在整体施工建设当中作业人员是最主要的参与者，他们对于建设的总体效果是有着极为重要的影响的。在实施工程建设时，可以适当通过加大对作业人员安全教育的培训力度，使各个项目施工部门以及建设单位都能够从根本上拥有一定的安全意识，由此提升作业人员安全管理意识，确保整个工程管理的有效进行。在进行安全教育与培训的时候要确保整个过程是具有一定目的的，并且要以此制定出来相应的教育内容，并记录下来，保证各项组织活动的多样性。除此以外还应该要及时组织相关人员进行施工规章以及规范的学习，以此最大程度上提升自我保护意识与安全意识。

The effect of PEF on initial photon numberI0of dynamic parameters of delayed fluorescence was illustrated in figure 3. From figure 3, at the beginning of the experiment, in control group, initial photon numberI0fell rapidly, later, there was fluctuation, and then there was a peak on third day. It also showed that the initial photon numberI0in treated group was higher than that in control group at the beginning of drought stress, and the relative growth rate reached to 24.2%. During the process of drought stress, initial photon numberI0in treated group has the same trend with control group, but the value was always higher, the relative growth rate was 44.87%, 9.03%, 12.09%, 10.20% and 27.84% on 1, 2, 3, 4 and 5 d.

2.2.2 Effect of PEF on coherence timeτ

Effect of PEF on coherence timeτof dynamic parameters of delayed fluorescence was showed in figure 4. Figure 4 showed that coherence timeτgrew quickly after drought stress, and reached peak on the second day then started to fall, but it increased on the fourth day. On Figure 4, coherence timeτin treated group was also plotted, which was higher than that in control group at the beginning of the drought stress, and the relative growth rate reached to 16.3%. The two groups had the same trend, but coherence timeτin treated group was always higher. On the day of 1, 2, 3, 4, 5 d the relative growth rate were 9.2%, 7.7%, 22.5%, 10.6% and 6.1%.

Fig.3 Changes of the initial photon numberI0 of delayed fluorescence

Fig.4 Changes of the coherence time τof delayed fluorescence

2.2.3 Effect of PEF on decay factorβ

Figure 5 showed the influence of PEF on the change of decay factorβ. on this figure,βhad a high value when there was no drought stress, after stress, increased slightly, then decreased and it had a minimal on third day, after this it began to rise.βhad a higher value in treated group without stress, the relative growth rate was 4.4%. After that,βwas always higher in treated group. The relative growth rate were 4.9%, 7.0%, 7.0%, 4.0% and 7.9% on 1, 2, 3, 4 and 5 d respectively.

Fig.5 Changes of the decay factor βof delayed fluorescence

2.3 Effect of PEF on delayed fluorescence integral intensity I(T) of maize seedling leaves under drought stress

Delayed fluorescence integral intensity was defined as the area under the curve of delayed fluorescence, expressed asI(T), and the value of it could be calculated by formula (2). From formula (2),I(T) was decided byI0,τandβ, which could reflect the change of delayed fluorescence synthetically . delayed fluorescence integral intensityI(T) was obtained by Putting the dynamic parameters into the formula (2), and the result was showed on figure 6. According to the graph, the delayed fluorescence integral intensity showed a fluctuation, and theI(T) in treated was always higher than that in control group. On the 0, 1, 2, 3, 4 and 5 d, the relative growth rate were 32.3%, 38.1%, 10.4%, 19.1%, 13.3% and 17.8% respectively.

Fig.6 Changes of the delayed fluorescence integral intensity I(T)

3 Discussion

In the formula of delayed fluorescence dynamics, initial photon numberI0, coherence timeτ, decay factorβwere the characterization of dynamic parameters. When light stopped, the electrons in different parts of the electron transport chain in photosynthesis were back to the photosynthetic reaction center PSⅡ, and produced excited state PSⅡ, the latter generate delayed luminescence when deexcitation[13]. Each component in electron transport chain had different nature and the different ability to storage and release electrons, the time of electron returned to PSⅡ from electron transport chain was different, which caused the a long attenuation of delayed luminescence[14]. Therefore, initial photon numberI0was relevant to the number of PSⅡ in the excited state when lighting, coherence timeτwas related to the time of electronic in electron transport chain back to PSⅡ. The more initial photon numberI0, the more PSⅡ in excited state, and the potential of photosynthesis in photosynthetic cells was better. The reduction of coherence timeτ, revealed that organize sequence of the electron transport chain had been destroyed, and the number of electron back to PSⅡ was reduced. Based on this understanding, in figure 3,I0in control group dropped rapidly at the beginning, and appeared a litter peak in third day, after this recovered on fourth day, which showed that drought stress caused the number of PSⅡ which could stay in excited state decreased, and photosynthesis in leaf cells was also weaken, but on fourth day, with the metabolism regulation and repair of cells, photosynthesis potential recovered . Figure 4 also showed that after drought stress, organize sequence of photosynthetic electron transport system in leaf cells raised quickly, reached to peak on second day, which indicated that photosynthetic electron transport system have been adjusted substantially, which was adaptive response of photoreceptor cell to drought stress.

Decay factorβwas an exponential factor; it was related to the property of the sample, nothing to do with excited action and the amount of sample, represented as the degree of interaction of functional molecules in excited state. Thus,βhad an important biological significance. Figure 5 showedβin control group has a high value without drought stress, with the time being, it rose for a while then reduced gradually, reached to minimal value on the third day, and then increased. Such change indicated that the interactions between functional molecules were stronger, due to drought stress, the interactions between functional molecules increased in the short run, then declined, and it reached minimum on third day, then gradually increased.

Delayed fluorescence integral intensityI(T) could reflect the characteristics of the delayed fluorescence dynamics, that could be regarded as the signature of the ability to photosynthetic capacity[15-16]. According to the biological significance of delayed fluorescence integral intensityI(T), under such stress, Photosynthesis ability first increased, then decreased, after that it increased again (see figure 6).

To sum up, during the process of drought stress, the dynamic parameters of delayed fluorescence from leaf cells of maize seedlings all showed fluctuation, which reflected the reaction, adjustment and adaption of leaf cells . Initial photon numberI0, coherence timeτ, decay factorβcould be regarded as a probe to reflect the system of leaf cells in photosynthesis, they could also reflect the change of photosynthesis system sensitively from different perspectives, besides, these change could decide the functional state and photosynthesis ability, then the condition could be expressed by delayed fluorescence integral intensityI(T).

It was worth mentioning that there was a relatively slow increasing of dry leaf mass between the second day and third day in figure 1, and the dynamic parameters of delayed fluorescence appeared the biggest change during this period, showed the strength of self-regulation of cell at this time, which caused dry leaf mass increasing.

This paper found that, by applying PEF, the dynamic parameters of delayed fluorescence all increased, which indicated that, under drought stress, PEF improved the photosynthesis potential and promoted the organize sequence of photosynthetic electron transport system, and the interaction of functional molecules was also enhanced as well. The change of delayed fluorescence integral intensityI(T) suggested that both functional status of cells and photosynthetic capacity were improved. The reason of this effect maybe that the growth of maize seedling was promoted by the PEF, according to the result of this study, the dry leaf mass and dynamic parameters of delayed fluorescence in treated group was significantly greater than that in control group at the beginning of the experiment, and during the process of experiment, treated group always kept the advantage, thus had a stronger drought resistance.

4 Conclusion

(1) To treated group, dry leaf mass of maize seedling gradually increased under drought stress condition formed by PEG-6000 with -0.1 MPa osmotic potential, indicated that under drought stress, PEF could promote the growth of maize seedling leaf under.

(2) The analysis of delayed fluorescence dynamic about leaf showed that the value of delayed fluorescence dynamic parameters of the leaf cells such as initial photon numberI0, coherence timeτ, decay factorβand integral intensityI(T) rose and fell under drought stress, this change was the adaptive response to drought stress.

(3) The value of delayed fluorescence dynamic parameters and integral intensityI(T) all increased by applying PEF, showing that the photosynthesis potential, organize sequence and interaction between functional molecules in leaf cells were strengthened, besides the leaf photosynthetic capacity was enhanced.

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*通讯联系人

S132

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LED诱导延迟荧光技术评价脉冲电场对玉米幼苗抗旱性的影响

贺瑞瑞，习 岗*，刘 锴，赵燕燕

西安理工大学理学院应用物理系，陕西 西安 710048

脉冲电场(pulsed electric field，PEF)对作物抗旱性的影响是电场生物学效应研究的重要课题，作物叶片延迟荧光动力学参数可以从不同角度灵敏地反映叶片细胞光合系统发生的变化。为了从活体细胞角度揭示脉冲电场对作物幼苗抗旱性的影响及其机理，使用频率为1 Hz、场强为200 kV·m-1、脉宽为80 ms的PEF处理萌发玉米种子，再采用渗透势为-0.1 MPa的PEG-6000溶液形成干旱胁迫，研究了玉米幼苗生长过程中叶片干质量和LED诱导的叶片延迟荧光动力学参数的变化。结果发现，在-0.1 MPa的PEG-6000溶液形成的干旱胁迫下，玉米幼苗叶片干质量逐渐增加，经过PEF处理的玉米幼苗叶片干质量大于对照，相对增长率在5.8%～18.7%之间(p<0.05)。叶片延迟荧光动力学分析显示，干旱胁迫下玉米幼苗叶片延迟荧光动力学参数初始光子数I0、相干时间τ、衰减参数β和延迟荧光积分强度I(T)都发生了波动性的变化，这些变化是叶片细胞对干旱胁迫的适应性反应，PEF处理使玉米幼苗叶片延迟荧光各动力学参数和延迟荧光积分强度均明显大于对照组，表明PEF处理使玉米幼苗叶片细胞的光合潜力、组织序性和功能分子之间的相互作用都有所加强，叶片综合光合能力提高了。研究结果为阐明PEF对作物幼苗抗旱性的影响及其机理提供参考。

延迟荧光； 电场； 干旱胁迫； 动力学参数； 玉米叶片

2015-03-22，

2015-07-04)

Foundation item： the National Natural Science Foundation of China (51277151 and 31471412)

10.3964/j.issn.1000-0593(2016)06-1959-07

Received： 2015-03-22; accepted： 2015-07-04

Biography： HE Rui-rui, female, (1991—), Department of Applied Physics，Institute of Science，Xi’an University of Technology, master e-mail: heruiruijy@163.com *Corresponding author e-mail: xig@xaut.edu.cn