The Research of Cold Stress on Three Revetment Plants

In order to select proper revetment plant to apply in the rock slope protection, we used Cynodon dactylon as control to investigate the effect of cold stress on two wild revetment plants (Pogonatherum paniceum and Eriophorum comosum) in current study. With the dropping of temperature (0°C, -5°C, -10°C, -15°C, -20°C), the content of soluble sugar, soluble protein, and free proline in these three plants rose first, and then decreased. Meanwhile, the chlorophyll content declined, but the electrolyte osmotic rate increased. Under the five different temperatures, the content of soluble sugar and soluble protein were highest (P < 0.05) in E. comosum, the lowest (P < 0.05) in C. dactylon, and P. paniceum in the middle. The free proline contents in E. comosum was similar to P. paniceum at -15°C, and -20°C, but both of them were significant greater than that in C. dactylon (P < 0.05). Additionally, C. dactylon had the highest electrolyte osmotic rate, and E. comosum was the lowest. Meanwhile, the semilethal low temperature was the lowest (-13.6°C) in E. comosum as compared to C. dactlylon (-6.86°C) and P. paniceum (-9.37°C). Both the contents of Chlorophyll a and Chlorophyll b were highest in P. paniceum, lowest in C. dactylon, and those in E. comosum were medium. These suggested that E. comosum may have the strongest cold resistance capacity, and that in C. dactylon was the weakest. The results showed that E. comosum was the best option to the rock slope protection due to their strongest anti-cold ability among these three grasses.


Introduction
The development in socio-economic cannot achieve without infrastructure development, especially in the construction of road and railway.However, which also brought inevitable destruction on the ecological balance, leading to bareness in surface, decreasing the soil anti-erosion capacity, and aggravating the soil erosion (Wang & Peng, 1997).Thus, it is very necessary to carry out vegetation restoration in bare rock slope.The key in which is not the slope protection technique and soil matrixes, but the selection of proper revetment plant (Zhen & Fang, 2009).
However, research in eco-engineering for the rock slope protection is still at the starting stage in China (Xiong et al., 2011).At present, single pasture or lawn grass is selected for rock slope protection, but which is often followed by inadaptability.When the temperature is lower, the water stress occurs, and the survival rate decreases (He et al., 2012).Nevertheless, wild lithophytes can effectively solve this problem due to their advantageous traits (Liu & Han, 2007).It has been reported that lithophytes can directly grow in steep rock and cemented masonry stone, and they have greater resistance to drought, cold, disease and barren, and fecundity than other grasses.Therefore, the selection of proper lithophytes has important significance in the rock slope protection (Li et al., 2004b).According to the survey by Li et al. (2006), Pogonatherum paniceum and Eriophorum comosum can be used as revetment plants because of the characteristics in strong deep roots, and well tolerant to barrenness, drought and water erosion.They can grow in a crack between rocks, and form symbiotic relationships with lower organisms, such as algae, lichen, moss, etc (Li & Luo, 2006).
At present, systemic researches have been performed in seed certification, drought adaptability, microbial ecological distribution in rhizosphere soil, plant tissue culture, and genetic diversity of Pogonatherum paniceum and Eriophorum comosum (Li et al., 2004a;Wang et al., 2005;Zhuang et al., 2010).However, the study on anti-cold ability of these two grasses has not been reported yet.In the current research, we used Pogonatherum paniceum and Eriophorum comosum as experimental materials, and Cynodon dactylon as control to study the cold adaptability of these two plants.Results of this study could provide scientific theoretical basis for selection of revetment plant.

Site for Experiment
The experiment was carried out at the teaching practice base of the college of Forestry, Sichuan Agricultural University, Ya'an, Sichuan province, China.Average altitude, mean annual precipitation, annual temperature and relative air humidity in this area is 620 m, 1800 mm, 16.2 ºC, and 79%, respectively.The annual cumulative temperature (≥10 ºC) is 5231 ºC, annual sunshine is 1039.6 h, and frost-free period is 298 d.

Experimental Design
Wild Pogonatherum paniceum, Eriophorum comosum and Cynodon dactylon were collected from the rock slope at the town of Duoying, Ya'an, and the slope gradient was steeper than 85%.Pot experiment was used in this study.The experimental soil was purple sandy shale soil, and which was the heavy clay soil with a pH value of 6.2.
The collected seedlings were planted on 10 th May 2012.Six seedlings were planted in each pot with 20 duplicates in each grass.Fresh leaves were collected from three grasses at 7 am on 15 th November 2012.The collected leaves were stored in ice bags, and taken back to the lab rapidly.Leaves were cleaned with deionized water, and dried by the filter paper.Then, five different temperatures include 0°C, -5°C, -10°C, -15°C, and -20°C were employed to treat leaves, and 10 duplicates in each temperature treatment.

Indexes Measuring
The membrane permeability was measured by conductimeter (DDS-11A).The soluble protein content was tested with staining of coomassie brilliant blue R 250, and followed the described by Zhang (Zhang, 2006).The soluble sugar content was measured by sulfuric anthrone reaction based on the method of Li (Li, 2000).The Chlorophyll content was determined according to Wintermans and De Motes (Wintermans & De Mots, 1965).The free proline was measured directly using the ninhydrin colorimetric method of Troll and Lindsley (Troll & Lindsley, 1955)

Statistical Analysis
One-way ANOVA was used to compare the effect of treatments and, Tukey's test was used for the multiple comparison.Statistics analysis was performed using SPSS 17.0 software, and the P-value for significance was set at P < 0.05.

Effect of Low Temperature Stress on Membrane Permeability of Leaf Cells
As shown in the Figure 1, the electrolyte osmotic rate of three grasses went up with the decrease of temperature.The electrolyte osmotic rate of C. dactlylon increased quickly from 0 to -15°C, and slower growth was found after -15°C.However, the rising trend was slowly before -10°C and then increased sharply in E. comosum and P. paniceum.Additionally, the electrolyte osmotic rate was lowest in E. comosum, greatest in C. dactlylon, and P. paniceum was medium at all temperatures.
The semilethal low temperatures of three plants were calculated based on the fitted regression equation of the electrolyte osmotic rate (Table 1).The semilethal low temperature was lowest in E. comosum (-13.6°C),highest in C. dactlylon (-6.86°C), and that in P. paniceum (-9.37°C) in the middle, which were consistent with the electrolyte osmotic rate of three plants.It has been demonstrated that the free proline content is closely related with cold resistance in plant, the accumulation of which is considered an adaptation to the cold stress (Dörffling et al., 2008).The increase of proline can decrease the water potential and raise the water-retaining capacity under the low temperature.Thus, its accumulation can as a measure of anti-cold ability (Ashraf & Foolad, 2007).In this study, there were varying degrees of growth with decreasing temperature found in the free proline of three grasses.Meanwhile, the free proline content of E. comosum was highest at all temperatures.These results indicated that these three grasses can improve anti-cold ability through accumulating the proline content, and the anti-cold ability was best in E. comosum.The study of Li et al. (2011) also found that free proline content of different Medicago sativa species showed upward trends under low temperature stress (Li, 2011).
The central role of chlorophyll in photosynthesis is to harvest light energy, and converse it to chemical energy.The chlorophyll content direct affects the photosynthesis capability (Mauzerall, 1976).Chlorophyll a alone exists in the core complex of photosystem.Its organization and composition have highly conservation.Chlorophyll b is an ubiquitous accessory pigment, whose biosynthesis plays a key role in the adaptation to various light environments (Tanaka et al., 1998).In current study, as the temperature dropping, both Chlorophyll a and Chlorophyll b contents in measured plants decreased.It meant that photosynthesis in all of the plants was expected to be negatively affected by these changes.Under the different temperatures, C. dactlylon had the lowest contents of Chlorophyll a and Chlorophyll b suggested that the photosynthesis of C. dactlylon was the weakest among these three grasses.

Conclusion
In conclusion, plants can adapt to low temperature through adjusting the content of osmotic adjustment materials.E. comosum had the highest content of soluble sugar, soluble protein and free proline, higher Chlorophyll contents among three plants, and lowest electrolyte osmotic rate.Additionally, the semilethal low temperature in E. comosum was also the lowest.From the results of the present experiment, it was concluded that cold resistance of these three revetment plants was decreased in the order of E. comosum, P. paniceum, and in C. dactlylon.

Table 1 .
The semilethal temperature of three plants