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为揭示核桃的抗寒机理,研究了低温逆境下不同抗寒性核桃(Juglans regiaL.)品种展叶期叶片细胞中第二信使Ca2+的分布变化。试验材料为抗寒性较强的核桃品种‘哈特雷’和抗寒性较差的‘晋龙2号’展叶期1年生枝的顶端幼叶,将枝叶于1℃低温下分别水培处理3、12、24、48和72h,以未经低温处理的叶片作为对照,采用电镜细胞化学方法,定位观察叶肉细胞中的Ca2+分布。结果表明,展叶期核桃幼叶细胞中Ca2+主要分布于液泡和细胞间隙中,线粒体、叶绿体和细胞核中也有较多Ca2+分布,基础细胞质中Ca2+含量较少。1℃低温可使细胞质和细胞核中的Ca2+浓度迅速升高,在低温处理24h时,抗寒性较强的‘哈特雷’细胞中游离Ca2+浓度开始下降,处理72h时基本恢复到静息态水平,细胞超微结构变化不大,叶片无明显冷害症状;而抗寒性较差的‘晋龙2号’叶片的细胞质、线粒体及细胞核中Ca2+浓度始终处于较高水平,到处理72h时细胞超微结构冷害明显,部分幼叶叶缘呈水浸状。可见,低温逆境下抗寒性较强的品种叶肉细胞质中出现短时间的Ca2+高峰,随后能恢复到静息态水平;而抗寒性差的品种难以恢复,不能完成第二信使的信号传导过程,最终导致细胞和组织冷害。因此,低温逆境下能否恢复第二信使Ca2+的稳定平衡与核桃的抗寒性密切相关。此外,低温处理12h后核桃幼叶的叶绿体被膜上普遍出现Ca2+沉淀,推测此时叶绿体可能起着临时贮存Ca2+的作用,以促进细胞质中Ca2+浓度回落到静息态水平;线粒体可能也具有此作用。
In order to reveal the mechanism of cold resistance of walnut, we studied the distribution of Ca2 +, a second messenger in leaf cells of different cold-resistant walnut cultivars (Juglans regia L.) under low temperature stress. The experimental materials were top hatchlings of 1-year-old branches in walnut variety ’Hadley’ and cold-hardy ’Jinlong 2’ with strong cold resistance. The branches and leaves were individually cultured at 1 ℃ After treated for 3, 12, 24, 48 and 72h, the leaves without hypothermia were used as controls, and the distribution of Ca2 + in mesophyll cells was observed by electron microscopy. The results showed that Ca2 + mainly distributed in the vacuoles and intercellular spaces in young leaves of walnut during the leaf-opening stage. There were also more Ca2 + distribution in mitochondria, chloroplast and nucleus and less Ca2 + in the cytoplasm. Low temperature of 1 ℃ increased the concentration of Ca2 + in cytoplasm and nucleus rapidly. When treated at low temperature for 24h, the free Ca2 + concentration in ’Hardy’ cells began to decrease, and returned to resting state at 72h Level, the ultrastructure of the cells did not change much, and the leaves had no symptoms of chilling injury. However, the Ca2 + concentrations in the cytoplasm, mitochondria and nucleus of ’Jinlong 2’ with poor cold resistance were always at higher level, The ultrastructure of chilling injury obvious, some young leaves edge was flooded. It can be seen that a short period of Ca2 + peak appears in the cytoplasm of leaves with stronger cold resistance under low temperature stress, and then returns to the resting state level. However, the varieties with poor cold tolerance are difficult to recover and can not complete the signal transduction process of the second messenger, Eventually leading to chilling damage to cells and tissues. Therefore, it is closely related to whether the second messenger Ca2 + can restore the stable balance under low temperature stress and the cold resistance of walnut. In addition, Ca2 + precipitation was generally observed on chloroplasts of young leaves of walnut leaves after 12 h of low temperature treatment, suggesting that chloroplast may play a role of temporary storage of Ca2 + to promote the concentration of Ca2 + in the cytoplasm to retreat to a resting level; mitochondria may also have this effect .