Jinhui YANG, Lixiong HE, Wan JIANG, Yong SONG,

1. College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, China;

2. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China;

3. Hunan Provincial Engineering Research Center for Potato, Changsha 410128, China

Potato (Solanum tuberosum)is an annual herb belonging to the genus Solanum of family Solanaceae, which is an important grain and economic crop with high nutritional value, strong adaptability and high yield. Low temperature stress may cause great harm to the growth of potato plants,resulting in a serious decline in yield. Potato tubers can be damaged at temperatures below 2 ℃;potato seedlings are susceptible to chilling damages at -0.5-0.8 ℃and freezing damages at -2 ℃; potato plants will die at-4 ℃[1].Low temperatures below the critical temperature of potato will cause chilling and freezing damages to plants.Under low temperature stress, physiological mechanisms of SPDS transgenic potato plants may vary to reduce the damage of low temperature to potato plants.Therefore, investigating the changes in physiological indexes of potato plants under low temperature stress to screen effective prevention measures is of important significance for the production of potatoes and breeding of cold-resistant germplasms.

In this study, using SPDS transgenic potato lines 01-6, 01-47 and 01-49 as experimental materials and Atlantic as the control, changes in chlorophyll content, malondialdehyde(MDA) content and superoxide dismutase (SOD) activity of potato leaves under low temperature stress were determined, aiming at providing the basis for the prevention of chilling injury in potatoes.

Materials and Methods

Materials

Potato lines overexpressing spermidine synthase(SPDS)gene,including 01 -6, 01 -47 and 01 -49, were used as experimental materials, with Atlantic as the control. The above materials were provided by Hunan Provincial Engineering Research Center for Potato, College of Horticulture and Landscape, Hunan Agricultural University.

Methods

Potato lines 01-6, 01-47, 01-49 and Atlantic were planed in the greenhouse of Hunan Agricultural University.At the seedling stage,potato plants were transferred into low temperature climatic chamber at 4 ℃. Before the transfer, adequate leaves were collected, ground with liquid nitrogen and persevered in ultra-low temperature refrigerator. After the transfer, potato leaves were collected from each line at 1, 3, 5 d post-treatment, ground with liquid nitrogen and persevered in ultralow temperature refrigerator. After the sampling,various physiological indexes were determined. Chlorophyll content was determined with the method proposed by Zhang et al.[2];MDA content was determined with thiobarbituric acid reaction method; SOD activity was determined using the kit produced by Nanjing Jiancheng Bioengineering Institute.

Data processing

All the experimental data were analyzed using Excel and SPSS statistical software.

Results and Analysis

Changes in chlorophyll content of potato leaves under low temperature stress

As shown in Fig.1, under low temperature stress, leaves of potato plants exhibited no significant change in the color, and chlorophyll content of potato leaves was declined compared with that before treatment. To be specific, chlorophyll content of 01-6 and 01-49 decreased rapidly; at 5 d posttreatment, chlorophyll content of 01-49, 01-6 and Atlantic was reduced by 74%, 57% and 48% compared with that before treatment, respectively;chlorophyll content of 01-47 declined gently, which was only reduced by 25% at 5 d post-treatment compared with that before treatment, indicating that chlorophyll content of 01-47 fluctuated minimally under low temperature stress.

Changes in MDA content of potato leaves under low temperature stress

As shown in Table 1, changes in MDA content of 01-6,01-47 and 01-49 varied significantly compared with Atlantic; before treatment, MDA content of 01-47 and 01-49 varied significantly compared with Atlantic; at 1 d post-treatment,MDA content of 01-47 and 01 -6 varied significantly compared with Atlantic; at 3 d post-treatment, MDA content of 01-47 and 01-49 varied significantly compared with Atlantic; at 5 d post-treatment, MDA content of 01-47 varied significantly compared with Atlantic, while MDA content of 01-6 and 01-49 exhibited no significant change compared with Atlantic. Therefore, MDA content of SPDS-overexpressing potato line 01-47 varied significantly compared with Atlantic under low temperature stress.Moreover, among these four potato lines, MDA content of 01-6 increased first and then declined,which was lower than that of Atlantic under low temperature stress;MDA content of 01-47 showed an upward trend, which was remarkably higher than that of Atlantic under low temperature stress, indicating that 01-47 exhibited more serious membrane damages than Atlantic,while 01 -6 had slighter membrane damages than Atlantic under low temperature stress.

Table 1 Changes in MDA content of potato leaves under low temperature stress

Changes in SOD activity of potato leaves under low temperature stress

The changes in SOD activity of experimental materials under low temperature stress were shown in Fig.2.To be specific, under low temperature stress,SOD activity of Atlantic showed a decreasing trend; SOD activity of 01-6 and 01-47 increased first and then declined and increased subsequently; SOD activity of 01 -49 increased first and then declined. Furthermore, under low temperature stress, SOD activity of 01-6 and 01-47 was significantly higher than that of Atlantic.

Conclusion and Discussion

Low temperature stress exerts various effects on plants and causes a series of physiological and biochemical changes. Under low temperature stress, plants produce excessive reactive oxygen species that damage antioxidant system of plants, thereby resulting in chilling injury[3]. Studies have shown that changes in chlorophyll content, MDA content and SOD activity are responses of plants to lowtemperature stress[4-5].

Chlorophyll is an important pigment involved in photosynthesis in plants. In general, the minimum temperature, maximum temperature and optimal temperature for the formation of total chlorophyll is 2-4 ℃,around 30℃and 40 ℃,respectively. The results indicate that chlorophyll content of experimental varieties shows a decreasing trend at 4 ℃, suggesting that chlorophyll formation in potatoes is inhibited in low temperature environments, which is not conducive to the growth of potatoes under low temperature stress[6-7].

MDA is the final decomposition product of plasma membrane peroxidation, and its content can reflect the extent of damage to plants. Higher MDA content indicates more serious plasma membrane peroxidation and greater damages to plant cell membrane systems[8]. In this study, MDA content of 01-47 is significantly higher than that of Atlantic; MDA content of 01-6 and 01-49 is lower than that of Atlantic after treatment, indicating that cold resistance of 01-6 and 01-49 is slightly higher than that of Atlantic.

Low temperature conditions can not only improve the level of reactive oxygen species, but also induce the establishment of plant defense systems.SOD is a very important antioxidant enzyme in plants, which can effectively remove O2-to reduce or prevent damages of reactive oxygen species to plants[9-11]. According to the results, SOD activity of 01-6 and 01-47 increases first and then declines,which is higher than that of Atlantic after treatment, suggesting that 01 -6 and 01-47 are more adaptive to low temperature stress than Atlantic.

In this study, SPDS transgenic potato lines 01-6 and 01-47 exhibit superior physiological performance to Atlantic under low temperature stress.Therefore, 01-6 and 01-47 can be further cultivated to breed new coldresistant potato varieties.

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