Effects of drought on electrical impedance spectroscopy parameters in stems of Pinus bungeana Zucc. seedlings

Abstract

The effects of drought during preplanting (three treatments: soil relative water content (RWC) 75%–80%, 55%–60%, 35%–40%; B1, 2, and 3, respectively) and postplanting (four treatments: RWC 75%–80%, 55%–60%, 35%–40%, 15%–20%; A1, 2, 3, and 4, respectively) on electrical impedance spectroscopy (EIS) parameters in the stems of Pinus bungeana Zucc. seedlings were investigated by using 4-year-old container seedlings. Stem impedance spectra were modeled by a distributed circuit element model (2-DCE), which showed the extracellular and intracellular resistance (r_e and r_i), relaxation time (τ₁ and τ₂), and distribution coefficient (ψ₁ and ψ₂) of relaxation time. After preplanting B3 drought treatment, r_e and r_i increased significantly with the increase of soluble sugar of the stem, measured by enthronlsulphuric acid method. After four weeks postplanting A4 drought treatment, relative conductivity, and soluble sugar of stem increased significantly, and r_e of stem decreased significantly and continually, indicating that the cell membrane of stem cells was disrupted by severe drought. After five weeks drought treatment, τ₁ of stem under A4 treatment decreased significantly, and ψ₂ of stem under A2, A3, and A4 treatments was higher than that of A1 treatment. Briefly, drought made r_e, r_i, τ₁, and ψ₂ of stem change regularly, but r_e was found to be the most informative and useful parameter measured if used as a single index to assess the drought resistance of P. bungeana Zucc. seedlings.

Keywords

drought / Pinus bungeana Zucc. / stem / extracellular resistance / intracellular resistance / relaxation time

Introduction

Water scarcity will be the first restrictive factor for the world’s development in the future. Effects of drought on worldwide agriculture and forestry are more severe than any other kind of natural disaster (Yu and Tang, 1996). Almost 50% of China’s landmass is located in drought areas, where the growth of trees is restrained by drought. In recent years, the development of drought research, study of the mechanism of drought resistance, index of drought resistance, and techniques of how to identify drought resistance have become popular. Although many morphological and physiologic indexes have been developed, so far, we do not have complete knowledge on any drought resistance mechanism (Luo and Guo, 2008).

Electrical impedance spectroscopy (EIS) is a quick and practicable physical method, which has been used to study cell membrane injury caused by stress factors, such as heat and frost (Zhang and Willison, 1992; Zhang et al., 1993; Repo et al., 1994, 2000). In China, at present, the EIS method has been mainly applied to cold hardiness (Li et al., 2008; Zhang et al., 2009) studies. Other kinds of environment stresses like soil salinity (Liu et al., 2009a), heavy metals (Liu et al., 2009b), and drought (Liu et al., 2007) have also been studied. However, EIS studies in woody plant species under drought stress have not been reported. In the present experiment, we measured EIS parameters in stems of Pinus bungeana Zucc. seedlings under different preplanting and postplanting drought treatments. We sought to find some parameters that change regularly with the drought level and find one or several useful parameters that can be used as an index to assess drought resistance of P. bungeana Zucc. seedlings.

Materials and methods

Experiment design

One thousand four-year-old P. bungeana Zucc. seedlings (diameter, 0.48±0.1 cm; height, 20.98±3.2 cm) were taken from Beijing Ming Tombs Nursery (40°13′N, 116°13′E) and transplanted in the specimen garden of the Agricultural University of Hebei (38°50′N, 115°26′E) on March 25, 2008. Eight hundred seedlings were replanted in 18€cm×18€cm pots and managed in a plastic greenhouse for a month.

Preplanting and postplanting drought treatments were included in this experiment. Pot seedlings were divided into three parts for preplanting drought treatments on May 5. They were B1: 75%-80% soil relative water content (RWC), B2: 55%-60%, and B3: 35%-40%. The first sampling was on May 14th for measuring EIS and other physiologic indexes.

Preplanting and postplanting drought treatments were included in this experiment. Pot seedlings were divided into three parts for preplanting drought treatments on May 5. They were B1: 75%–80% soil relative water content (RWC), B2: 55%–60%, and B3: 35%–40%. The first sampling was on May 14 for measuring EIS and other physiologic indexes.

Surface soil (20 cm) of the specimen garden of the Agricultural University of Hebei and sand (2∶1) was used as container soil. Field moisture capacity was 22.61%, and bulk density was 1.24. Soil water content (volume) was measured at 18:00 every day by a soil moisture meter (TDR100, U.S.A.). Relative water content was obtained by formula transformation. Water was added according to the amount of the soil water content to make each treatment retain the given amount of water. Postplanting control water treatments continued for five weeks. A total of five sampling times were used on May 31, June 8, June 15, June 22, and June 29 with one week intervals during the control water period.

Electrical impedance spectroscopy of stem

Eight seedlings were taken from each treatment at each sampling time. Every two seedlings came from one replicate. Two 15 mm sections were cut from the middle of the stems. Impedance spectra were measured with HP4284A meter (Agilent, U.S.A.). Ag/AgCl electrodes (RC 1, WPI, Ltd., Sarasota, FL, U.S.A.) were set in horizontal tubes in contact with HP4284A meter and electrode gel. A 15 mm stem section was placed between electrode gels. Before measurement, open and short calibrations were needed. The real and imaginary values of impedance were measured at 42 frequencies between 80 Hz and 1 MHz. According to the shape of the spectra, the two distributed circuit element model (2-DCE) was suitable for these woody plant stems (Hurme et al., 1997). The total complex impedance of the model is

(1)Z=R+{\displaystyle \frac{R_{\mathrm{1}}}{\mathrm{1}+{(i·{\tau }_{\mathrm{1}}·\omega )}^{{\psi }_{\mathrm{1}}}}}+{\displaystyle \frac{R_{\mathrm{2}}}{\mathrm{1}+{(i·{\tau }_{\mathrm{2}}·\omega )}^{{\psi }_{\mathrm{2}}}}}

where Z is total impedance, ω is angular velocity (ω = 2πf), and i is imaginary unit. There are seven parameters that can be obtained in the 2-DCE model: three resistances (R, R₁ and R₂, Ω), two relaxation times (τ₁ and τ₂, µs), and two distribution coefficients (ψ₁ and ψ₂) of the relaxation times. The mathematical interpretation of the 2-DCE model parameters is shown in Repo et al. (2000). The resistors (R₁ and R₂) of the model are obtained from the interceptions of the circles with the X-axis. The relaxation times (τ₁ and τ₂) are obtained from the apex of the arcs. At the apex, (2πf) × τ = 1, where f refers to a characteristic frequency. The centers of two circles located under the X axis are defined as ψ₁ and ψ₂. The parameters of the 2-DCE model are estimated by means of a complex nonlinear least squares (CNLS) program LEVMv 8.06 (Macdonald, 1987).

Extracellular resistance (R_e) is obtained as

(2)R_{\mathrm{e}}=R+R_{\mathrm{1}}+R_{\mathrm{2}}

Intracellular resistance (R_i) is obtained as

(3)R_i=R·\left(\mathrm{1}+{\displaystyle \frac{R}{R_{\mathrm{1}}+R_{\mathrm{2}}}}\right)

The resistance parameters were normalized with respect to the cross-sectional area (As= π × d²/4) and the length of the sample (l = 15 mm) in order to obtain the specific resistances. Lower case letters will be used to indicate the normalized values.

(4)r_x={\displaystyle \frac{A}{l}}·R_x

Physiologic indicators measurements

Water content of stems was measured according to Zhang et al. (2005). Electrolyte leakage of stems was measured by relative conductivity method (Ryyppö et al., 1998). Soluble sugar of stems was measured by enthronlsulphuric acid method (Zhang et al., 2003).

Statistical analysis

EIS parameters were fitted by LEVMv 8.06 software. One-way ANOVA (SPSS 11.0) was used to analyze the differences of parameters in different preplanting drought treatments. General Linear Model (SPSS 11.0) was used to analyze the differences between A treatments, B treatments, and A × B interaction effects in postplanting drought period. In this article, the effects of postplanting drought treatments on EIS parameters and other physiologic indexes are only shown by using seedlings (B1) of preplanting normal irrigation.

Results

EIS parameters of stem after preplanting drought treatments

Extracellular resistance (r_e) increased significantly when soil RWC decreased to 35%-40% (P<0.05). Intracellular resistance (r_i) of B2 and B3 treatments was higher than that of B1 treatment, but no significant difference was found between B2 and B3 treatment. Relaxation time (τ₁ and τ₂) had no significant differences among three treatments. Distributed coefficient (ψ₁) of B2 treatment was lower than that of B1 and B3, while distributed coefficient (ψ₂) of B2 treatment was higher than that of B1 and B3 (Fig. 1).

Physiologic indicators of stem after preplanting drought treatments

Water content of stems with the temporary soil RWC reduction had no difference among B1, B2, and B3 treatments (Fig. 2). Soluble sugar concentration of stems increased significantly when soil RWC decreased to 35%-40% (P<0.05) as r_e did (Fig. 3). Relative electrolyte leakage of stems increased with the drought; however, no difference between three treatments existed (Fig. 4).

EIS parameters of stems after postplanting drought treatments

At the beginning of the postplanting drought treatment, r_e of stems under A1 and A4 treatments was lower than that of A2 and A3 treatments. Afterward, r_e of all treatments was raised with higher r_e values at the second and third weeks. After four weeks drought phase, r_e of A2, A3, and A4 treatments reduced greatly, but r_e of A1 increased continually. At this time, the tendency of r_e was A4<A2<A1 (P<0.05), whereas A3 did not have a difference with that of A4 and A2 treatments. After five weeks drought phase, r_e of A4 treatment reduced continually, and r_e of A4 treatment was lower than that of A1, A2, and A3 (P<0.05) (Fig. 5).

Intracellular resistance r_i of stems in all postplanting drought treatments had no significant differences at the first two and the fourth sampling times. The r_i of A4 treatment was lower than that of A2 (P<0.05), and r_i of A1 and A3 did not differ significantly from that of A2 and A4 at the third week. At the last week, r_i of A4, A3, and A2 was lower than that of A1 (P<0.05) (Fig. 6).

The decrease in τ₁ reflects the change of cell membrane composition, ionic mobility in apoplast and symplast of cell and their response to frequency change. There were no differences in relaxation time (τ₁ and τ₂) in stems between postplanting drought treatments at the first and fourth weeks. At the second week, τ₁ of A4 was higher than that of A1 and A2, whereas τ₁ of A3 had no difference with that of A2 and A4; τ₂ of A3 and A4 was higher than that of A1 and A2 (P<0.05). At the third week, τ₁ and τ₂ of the stem of the A2 treatment were higher than those of A1, A3, and A4. At the last week, τ₁ of A1, A2, and A3 treatments were higher than that of A4 treatment (P<0.05); τ₂ of A2, A3, and A4 treatments were higher than that of A1 treatment (P<0.05) (Fig. 7).

In the second sampling time, the distribution coefficient ψ₁ of stem of A1 treatment was higher than that of A2, A3, and A4 treatments (P<0.05). No significant differences between postplanting drought treatments existed in other times. There were no differences in distribution coefficient ψ₂ at the third and fourth weeks. At the first week, ψ₂ of A2 treatment was lower than that of A1, A3, and A4 treatments. At the second and fifth weeks, ψ₂ of A1 was lower than A2, A3, and A4 treatments (Fig. 8).

Physiologic indicators of stems after postplanting drought treatments

In the beginning, the water content of A3 treatment was lower than that of the A1 treatment (P<0.05); however, the water content of A2 and A4 treatments had no difference with that of A1 and A3. There were no significant differences between treatments at the second and third sampling times. From the fourth week, water content of stems in A4 was lower significantly than that of A1 and A2 treatments (P<0.05), while water content of A3 treatment had no difference with A4, A1, and A2. Afterward, the water content of A4 decreased significantly, and the water content of A4 was lower than that of A1, A2, and A3 at the end of the experiment (Fig. 9).

The soluble sugar concentration between treatments did not differ after the first two weeks treatments. From the third week, soluble sugar concentration of A4 and A3 treatments rose significantly and that of A1 and A2 decreased with the trend of A3 and A4>A1 and A2 at that time (P<0.001). Afterward, soluble sugar concentration in all treatments increased and that of A3 and A4 treatments was still higher than that of A1 and A2 after four weeks. In the end of the experiment, soluble sugar concentration in A1 and A4 treatments was lower than that of A2 and A3 treatments (Fig. 10).

No difference in the relative conductivity of stems between treatments was found at the first three drought weeks. After four weeks drought treatment, the relative conductivity of A4 treatment was higher than the other treatments (Fig. 11).

Discussion

In present experiment, extracellular resistance (r_e) increased significantly under 35%–40% soil RWC preplanting drought treatments. This result was in accordance with the result of Liu et al. (2007) that r_e of wheat leaves increased in drought research. In addition, during frost hardening of Scots pine (Pinus sylvestris L.), r_e of stem increased (Repo et al., 1994). All of the above suggested that during the early stress phase, r_e increased. In contrast, after four weeks, postplanting drought treatments in this experiment, r_e of A2, A3, and A4 treatments were lower significantly than that of A1 treatment. The decrease of r_e of stem under postplanting drought treatments was similar to the decrease of that under cellular injuries caused by frost and heat (Repo et al., 1994).

The increase in soluble sugar concentration measured in the stems during preplant drought treatment is likely responsible for the increase in r_e. Plant stem sugar, mainly sucrose, is a nonelectrolyte fluid, and an increase in such solutes will lead to an increase in resistance, as was found in this research. A probable reason for the decrease in stem r_e, in A2, A3, and A4 during postplanting drought was the increase of relative electrolyte leakage, possibly from damaged cells, after four weeks of drought.

Intracellular resistance (r_i) increased significantly under 55%–60% and 35%–40% soil RWC preplanting drought treatments. Similarly, r_i of wheat leaves was higher in severe drought treatment than normal irrigation (Liu et al., 2007). Repo et al. (2004) found that when cytoplasmic matrix is diluted or symplastic ionic mobility decrease as a result of stress, r_i increases. However, in postplanting drought period in this experiment, there was no difference in r_i between treatments at most sampling times. At the last sampling time, the change of r_i under severe drought treatments did not accord with preplanting drought period and previous research.

Relaxation time (τ₁ and τ₂) had no significant difference between preplanting drought treatments. However, after two weeks postplanting drought treatments, τ₁ of A3 and A4 treatments was higher than that of A1 treatment. The τ₁ of drought-resistant varieties in wheat leaves in severe drought treatment was higher than the normal irrigation (Liu et al., 2007). According to the report of Liu, the increase of τ₁ after two weeks in severe drought might be one kind of adaption to drought condition as drought-resistant species. At the last week, τ₁ of A4 treatment was lower than the others; correspondingly, relative electrolyte leakage of A4 treatment increased, r_e of A4 decreased. Therefore, we think that the decrease of τ₁ is related to the cell membrane injury. In addition, the τ₁ of the stem of Scots pine decreased in cellular injuries caused by frost and heat (Repo et al., 1994).

The distribution coefficient of the relaxation time (ψ₁ and ψ₂) differed between preplanting drought treatments. Postplanting drought treatments already had effects on ψ₁ at the second sampling time, but the effects on ψ₂ was observed at the third time. In the present research, ψ₂ of stem under A2, A3, and A4 treatments was higher compared with A1 at the last week. This is also according to the study of Repo et al. (1994) who report that ψ₂ of the stems of Scots pine increased clearly with frost damage.

In conclusion, EIS parameters r_e, r_i, τ₁, and ψ₂ changed regularly under drought treatments. Therefore, they could be used as index for evaluating drought resistance of P. bungeana Zucc. seedlings. Among them, whether during the preplanting or postplanting drought phase, r_e changed regularly and can be explained clearly. Therefore, r_e is supposed to be the best one. In addition, those useful parameters in other species need to be verified further.

References

[1] Hurme P, Repo T, Savolainen O, Pääkkönen T (1997). Climatic adaptation of bud set and frost hardiness in Scots pine (Pinus sylvestris). Can J For Res, 27(5): 716–723

[2] Li Y Q, Zhang G, Que S P, Zhu L, Di B, Jin X M (2008). Relation between electrical impedance spectroscopy parameters and frost hardiness in stems and needles of Pinus bungeana. Scientia Silvae Sinicae, 44(4): 28–34 (in Chinese)

[3] Liu X H, Wang G D, Zhang G (2007). The effect of impedance parameters of wheat leaves of different varieties in water stress. Journal of Northwest A&F University (Nat. Sci. Ed.), 35(2): 210–214 (in Chinese)

[4] Liu X H, Huang T L, Wang G D, Zhang G (2009a). Effect of salt stress on electrical impedance spectroscopy parameters of wheat (Triticum aestivum L.) leaves. Journal of Zhejiang University (Agric1 & Life Sci1), 35(5): 564–568 (in Chinese)

[5] Liu X H, Huang T L, Wang G D, Zhang G (2009b). Response of electrical impedance spectroscopy parameters of wheat (Triticum aestivum L.) leaves to the stress of heavy meatals. Acta Scientiarum Naturalium Universitatis Sunyatseni, 48(5): 114–119 (in Chinese)

[6] Luo Y, Guo W (2008). Development and problems of crop models. Transactions of the CSAE, 24(5): 307–312 (in Chinese)

[7] Macdonald J R (1987). Impedance Spectroscopy. New York: John Wiley & Sons, 346

[8] Repo T, Zhang M I N, Ryyppö A, Vapaavuori E, Sutinen S (1994). Effects of freeze-thaw injury on parameters of distributed electrical circuits of stems and needles of Scots pine seedlings at different stages of acclimation. J Exp Bot, 45(6): 823–833

[9] Repo T, Zhang G, Ryyppö A, Rikala R (2000). The electrical impedance spectroscopy of Scots pine (Pinus sylvestris L.) shoots in relation to cold acclimation. J Exp Bot, 51(353): 2095–2107

[10] Repo T, Oksanen E, Vapaavuori E (2004). Effects of elevated concentrations of ozone and carbon dioxide on the electrical impedance of leaves of silver birch (Betula pendula) clones. Tree Physiol, 24(7): 833–843

[11] Ryyppö A, Repo T, Vapaavuori E (1998). Development of freezing tolerance in roots and shoots of Scots pine seedlings at nonfreezing temperatures. Can J For Res, 28(4): 557–565

[12] Yu S W, Tang Z C (1996). Phant Physiology and Molecular Biology. 2nd ed. Beijing: Science Press (in Chinese)

[13] Zhang M I N, Willison J H M (1992). Electrical impedance analysis in plant tissues: in vivo detection of freezing injury. Can J Bot, 70(11): 2254–2258

[14] Zhang M I N, Willison J H M, Cox M A, Hall S A (1993). Measurement of heat injury in plant tissue by using electrical impedance analysis. Can J Bot, 71(12): 1605–1611

[15] Zhang G, Ryyppö A, Vapaavuori E, Repo T (2003). Quantification of additive response and stationarity of frost hardiness by photoperiod and temperature in Scots pine. Can J For Res, 33(9): 1772–1784

[16] Zhang G, Liu M, Ren Y X (2005). Dehardening and rehardening of Pinus bungeana Zucc. seedlings in spring. Plant Physiology Communications, 41(6): 761–763 (in Chinese)

[17] Zhang J, Zhao H J, Zhang G, Yang M S (2009). Cold hardiness assessment of germplasm resources in Robinia pseudoacacia using electrical impedance spectroscopy. Journal of Plant Genetic Resources, 10(3): 419–425 (in Chinese)

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg