外源一氧化氮提高一年生黑麦草抗冷性机制

Studies on mechanism of enhancing the chilling resistance of annual ryegrass by exogenous nitric oxide

用不同浓度的一氧化氮(NO)供体硝普纳(sodiumnitroprusside,SNP)处理低温胁迫下1年生黑麦草幼苗,探讨外源NO对提高黑麦草幼苗抗冷性的作用。结果表明外源NO能减缓低温胁迫下黑麦草幼苗质膜相对透性的增加,促进脯氨酸(Pro)的积累,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)保护酶活性,其中POD酶活性的提高尤为显著。恢复生长时,经SNP处理的幼苗膜透性、脯氨酸和保护酶活性恢复较快,其中0.5mmol/LSNP处理的效果最为明显,0.2、1.0mmol/LSNP处理的效果次之。

The annual ryegrass (Lolioum multiflorum) was used in this study. The seeds of ryegrass were germinated in a growth chamber at 20±1℃, with 60% to 80% of humidity, under 12h light (400 lx)/12h dark. The continuous illumination was provided by cool-white fluorescent lamps (400 lx). When the second or the fourth leaves fully expanded the normal leaves were selected for the experiments. SNP ([Na_2Fe(CN)_5]·NO, Merck, Karmstadt, Germany) was used as NO donor. In the experiments the stock solution of 10mmol/L SNP was prepared and immediately diluted to the demanded concentrations. The following two experiments were carried out: Experiment 1 The SNP spraying treatment: in the growth chamber, when the first leaves fully expanded (3~4cm long) the seedlings were grouped and sprayed with solution of SNP at different concentrations of 0, 0.2, 0.5 and 1.0mmol/L, which contained 2‰ Polysorbate-80 to increase the leaf surface tension. The spray treatments were conducted once a day for three consecutive days. Then the samples with triplicate were taken up for the further determination. Experiment 2 Chilling stress:As soon as the Experiment 1 was finished, some of ryegrass leaves were kept in growth chamber at 0℃ to suffer from chilling stress. Therefore the treatments were designed as follows: A Normal control; B 0℃-chilling stress control; C 0.2mmol/L SNP+0℃-chilling stress; D 0.5mmol/L SNP+0℃-chilling stress and E 1.0mmol/L SNP +0℃-chilling stress. The above chilling stress treatments lasted for one or three days in the growth chamber. Then the growth chamber was reset at 20℃, thereby getting the ryegrass back to a normal growth for three days. During chilling stress period and after a 3-day recovering growth, the leaves were sampled with triplicate. All the samples obtained from the above two experiments were used to measure the membrane permeability, proline content and the activities of SOD, POD and CAT. All the experimental data were expressed as means ±SE, and then analyzed by using EXCEL and DPS tools. Moreover the data from independent samples were comprised by using t-test. The different measurements were subjected to a one-way analysis of variance (ANOVA). In all cases the confidence coefficient was set at 0.05 and 0.01. The results showed that the SNP treatment was able to alleviate the rise of membrane permeability and accelerate the accumulation of proline in ryegrass under chilling stress. Moreover, the activities reduction trends of SOD, POD and CAT in the treated seedlings were apparently slowed compared to those of chilling stress controlled seedlings. After a 3-day recovery, the membrane permeability, proline content and protective enzymes activities of the seedlings treated with SNP got back to the levels of normal seedlings, while those of chilling stress seedlings were not able to get recovery completely due to the chilling damage. These indicated that the SNP had a function of protecting seedlings from being harmed by chilling stress; in particular, the effect of 0.5 mmol/L SNP was much significant. The mechanism that NO is able to enhance cold resistance of ryegrass might be related to the modulation of the activities of protective enzymes and the variation of the membrane permeability and the proline content.