Semi-lethal high temperature and heat tolerance of eight Camellia species

Resumen. Se utilizaron segmentos foliares anuales de ocho especies de Camellia para estudiar la tolerancia al calor por un método de conductividad eléctrica, en combinación con una ecuación logística para dilucidar la temperatura alta semi-letal ajustando la curva de tasa de daño celular. La relación entre la temperatura y la tasa de daño celular en Camellia mostró una curva con forma de “S’’ típica, siguiendo el modelo logístico. El coeficiente de correlación estuvo por encima de 0,95. La temperatura alta semi-letal LT50 de las ocho especies de Camellia, determinada por el punto de inflexión en la curva, varió entre 50 y 57 LT50 / °C de acuerdo con el siguiente orden descendente: Camellia oleifera > C. japónica > C. polyodonta > C. semiserrata > C. nitidissima > C. gigantocarpa > C. nanyongensis > C. vietnamensis. La temperatura alta semi-letal de C. oleifera fue 56,8 °C, y la de C. vietnamensis 50,6 °C. Los resultados mostraron que C. oleifera parece ser una especie más adecuada para introducir en zonas donde se registren altas temperaturas. Camellia vietnamensis parece más adecuada para ser plantada en regiones templadas, a temperaturas más bajas. Estos resultados pueden proveer una base teórica en programas de cruzamiento genético e introducción de cultivares de aceite-té tolerantes al calor.


INTRODUCTION
Camellia, mainly distributed in south China, and a small amount distributed in South Asia, is a species with a production value of edible oil, which belongs to Camellia spp. of Theaceae.Camellia is a small evergreen tree or shrub, which is one of the world's four major woody oil plants together with olive, oil palm and coconut.The planting area of Camellia in China is about 400 million hm 2 , which accounts for more than 80% of our cultivation area of edible-oil woody species (Zhuang, 2008).Camellia oleifera, the most common Camellia species, is shown in Figure 1.
Experimental methods.This study was conducted in July 2011.A selected number of consistent, healthy, pest-free annual functional leaves from the tested Camellia species were washed with deionized water.Leaves were first dried with absorbent paper, and small, uniform 0.5 cm 2 -size discs were subsequently cut, avoiding the main vein.They were accurately weighed and 0.5 g were placed into a test tube.Twenty five mL of deionized water were added into the tubes so that leaves remained immersed.Thereafter, the leaf discs were put into a water bath at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C or 70 °C, respectively, for 20 min.Then, they were taken out of the water bath and cooled at room temperature for 2 h.Conductivity (R) (μs•cm 2 ) was then measured by a HI-9033 conductivity Meter.Then, all leaves were placed into a boiling water bath for 15 min to kill the plant tissue.Leaves were taken out and allowed to cool.Conductivity (R 0 ) was then determined in the same way at 25 °C.All studies were repeated three times.The control (CK) was the conductivity of the leaf discs at 25 °C (room temperature).
The relative electrolyte leakage was calculated following Zhang et al. (1989) as follows: Cell injury rate (%) = (R-CK) / (R 0 -CK) × 100‰ Statistical analysis.The leaf cell injury rate of Camellia species at the different temperatures was based on the Logistic Equation by SPSS 17.The logistic regression equation was y = K / (1+ae -bx ), where (1) y represents the rate of cell damage, (2) x represents temperature, (3) K represents the maximum rate of cell damage saturation capacity, due to elimination of background interferences in this experiment, so it takes the 100 K value, and (4) a and b are the equation parameters.According to regression analysis, the turning point of temperature in the equation is precisely the high-temperature, halflethal temperature of Camellia.

MATERIALS AND METHODS
Experimental materials.Camellia material sources and planting sites are shown in Table 1.These Camellia species were all planted in 2003 in Nanning, Guangxi.

RESULTS AND DISCUSSION
Effect on leaf cell injury rate of Camellia species at different temperature.The membrane system is the center of resistance to heat injury and heat; the thermal stability of the cell membrane reflects the ability of plant heat-resistance (Wang et al., 2010).When plant cells are subjected to stress injury, cell membrane structure is destroyed, and cell membrane permeability is increased.This results into leakage of the intracellular solution; the greater the degree of cell membrane damage, the greater the conductivity of the electrolyte leakage.
When treatment temperature was below 45 °C, the leaf cell injury rate of the eight study Camellia species increased slowly as the temperature rose (Fig. 2).When the treatment temperature reached 50 °C, leaf cell injury rates of the eight study Camellia species increased sharply.However, the rate of increase varied according to the species.When the temperature was higher than 60 °C, leaf cell injury rates were stable (Fig. 2), which proved that the membrane system of each study leaf was almost completely destroyed.The whole process of electrolyte leakage with increasing temperatures was a typical "S"-shaped type of growth.The eight study Camellia species have the same variation of the curve (Fig. 2).Leaf cell injury rate of Camellia increased sharply from 50 °C to 60 °C.This proved that Camellia leaves were most sensitive to damage by high temperature at this temperature range.Thereafter, we can initially infer the critical temperature that might cause an irreversible damage to the membrane system of Camellia leaves when going from 50 °C to 60 °C.
Determining Logistic equation parameters and the lethal temperature of Camellia species.Relative electrolyte leakage values of eight Camellia species were modeled and predicted applying nonlinear Regression using SPSS 17.0 software.The results were fitted by the Logistic regression equation.After iteration, the parameter values of the equations were obtained.The procedure is as follows: (1) solve the second derivative of the Logistic equation, and presume it is equal to zero, (2) get the curve inflection point t = ln a/b, which is the semi-lethal temperature; K/2 is the knee injury rate W; bW/2 is the maximum growth rate of cell damage.Logistic equation parameters of the Camellia species, and its growth parameters, are shown in Table 2.
Results showed that semi-lethal temperatures of the eight Camellia species ranged from 50 °C to 57 °C, and correlation coefficients that fitted by the Logistic equation were above 0.95 (Table 2).The higher the semi-lethal high temperatures, the stronger the capability to resist those high temperatures, whereas the lower the half-lethal high temperatures, the weaker the capability to resist those high temperatures (Li et al., 2010).The descending order of the capacity to resist high   Camellia nitidissima had the maximum growth rate of cell damage (5.89%) followed by Camellia japonica (5.85%), which showed that the extent of cell damage increased faster when temperature increased 1 °C.Camellia nanyongensis showed a 2.81% growth rate of leaf damage, which showed that this species had the stronger ability to resist high temperatures.

Species
Tolerance of plants to adverse environments is one of the key factors to determine whether its introduction and cultivation will be successful or not (Zhao et al., 2003).The ability of resistance to adverse environmental conditions varies according to the habitat and the species.Different plant materials have different quantitative resistance indicators: the seeds are usually identified by germination rate (Liang & Tan, 1997), cell suspension solutions by its protein content ( Jugawara & Sakai, 1978), and visual methods to survey plant survival (Yelenosky, 1979).The adaptability of plants to high temperature stress is mainly reflected by the cell membrane permeability changes (Yu & Tang, 1998).Cell membrane structure under high temperature conditions is subject to different degrees of damage; leakage of intracellular solution is caused by membrane permeability changes.Severity of the injury and high temperature tolerance of plants can be often reflected by the conductivity of the cell electrolyte leakage rate.Rajashekevar et al. (1979) fitted the relationship of cell injury rate of the wheat and Kentucky bluegrass leaves versus temperature using the Logistic regression equation.They reported that the inflection point of the equation determined the half lethal temperature of plants.Zhu et al. (1986) determined the temperature when the leakage rate of the cells was 50% (i.e., the half-lethal temperature: LT 50 ).Currently, conductivity methods have been widely used in Taxus (Liang & Tan, 1987), plums (Liu et al., 1999), apple (Gao et al., 2000), sweet cherry (Liu et al., 2005) and eucalyptus (Liu et al., 2009) to determine the plant tolerance to adverse environments.However, the heat-resistance of Camellia has been rarely reported.This study found that as temperature increased, the leaf cell injury of all study Camellia species also increased (i.e., there was a positive correlation between temperature and cell membrane permeability).
The relationship between the Camellia leaf cell injury rate and temperature was a typical "S" type curve, which was in line with the Logistic equation.We determined that the half-lethal temperature ranged from 50 °C to 57 °C in the eight Camellia species using the conductivity method and the Logistic equation.Camellia oleifera appeared as the best species for introduction into high heat zones, and Camellia vietnamensis appeared as more suitable for planting in temperate regions of lower temperatures.

Fig. 2 .
Fig. 2. Leaf cell injury rate of Camellia species under different temperatures.

Table 1 .
Source and planting sites of Camellia species.Origen y sitios de plantación de especies de Camellia.
Note: * represents a significant level at p<0.05, ** represents a significant level at p<0.01.