春剑对冷驯化和冰冻处理的响应及其生理机制

The Responses of Cymbidium tortisepalum var. longibracteatum to Cold Acclimation and Freezing Treatment, as well as the Associated Physiological Mechanisms

关键词:  春剑  冷驯化  冰冻处理  半致死温度  低温胁迫‘记忆’

Key words:  Cymbidium tortisepalum var. longibracteatum  cold acclimation  freezing treatment  semi-lethal temperature  low temperature stress ‘memory’

摘要:

通过冷驯化来增强低温耐受性是温带植物应对低温胁迫的重要调控策略，春剑（Cymbidium tortisepalum var. longibracteatum）主要分布在亚热带地区，冷驯化是否能增强其抗冻性尚不清楚。本研究旨在比较广西桂林市兴安县（XA）、永福县（YF）本地种质，与四川地区栽培品种‘隆昌素’（LS）、‘水朱砂’（SZ）等4份材料之间的低温耐受能力是否存在差异，以期筛选出耐寒种质，并初步解析其响应冷驯化和冰冻处理的生理机制。首先利用匀速程控恒温槽设定-1℃至-16℃的低温梯度，测定对照组（CK）与冷驯化组（CA）离体叶片的半致死温度（LT50），然后选择冷驯化略微提高与明显降低了LT50值的XA与SZ，探讨-6℃冰冻处理及恢复培养对其地上部分形态和生理参数的影响。主要结果如下：（1）根据Logistic方程拟合计算出不同叶片样品的LT50值范围在-4.397℃至-10.016℃之间。经历冷驯化后，XA与YF的LT50值升高，而LS与SZ的LT50值降低，且SZ的LT50值降低程度比LS更大；（2）直接冰冻处理对XA与SZ的地上部分均能造成低温损伤，但冷驯化后再冰冻处理只能使XA而非SZ的地上部分表现出冻害症状；（3）生理参数检测结果揭示了冷驯化对XA与SZ的脯氨酸积累无显著影响，但会显著降低类胡萝卜素含量。不论是直接冰冻处理，还是冷驯化后再冰冻处理，均只能诱导SZ而非XA中的脯氨酸合成。与处理前相比，可溶性蛋白含量能够同时受冷驯化、直接冰冻处理及冷驯化后再冰冻处理的扰动，其变化趋势在XA（下降）与SZ（提高）之间存在明显区别，这暗示了脯氨酸与可溶性蛋白的浓度变化可能在SZ抗冻性的建立中发挥了重要作用。综上所述，春剑不同种质之间的冷驯化能力存在差异，逐段降温模式的冷驯化启动了其低温胁迫‘记忆’，通过提高渗透调节物质的含量，最终增强了‘水朱砂’对冰冻胁迫的适应能力。

Abstract:

Enhancing cold tolerance through cold acclimation is a vital regulatory strategy for temperate plants to mitigate low temperature stress. Cymbidium tortisepalum var. longibracteatum is predominantly found in subtropical regions. However, whether cold acclimation can enhance its freezing tolerance remains poorly understood. This study aimed to evaluate the low temperature tolerance of native species from Xing'an County (XA) and Yongfu County (YF) in Guilin City, Guangxi Zhuang Autonomous Region, in comparison to cultivars 'Longchang Su' (LS) and 'Shui Zhusha' (SZ) from Sichuan Province. The objective was to identify cold-tolerant germplasm of C. tortisepalum var. longibracteatum and to conduct a preliminary analysis of its physiological mechanisms underlying responses to cold acclimation and freezing treatment. Initially, a series of low temperature gradients were set, maintaining temperatures between -1℃ and -16℃, utilizing an intelligent multi-stage constant temperature thermostat. The semi-lethal temperature (LT50) of detached leaves was assessed for both the mock group (CK) and the cold acclimation group (CA). Subsequently, XA and SZ, which exhibited slightly increased and significantly decreased in LT50 values due to cold acclimation, were selected to investigate the effects of a -6℃ freezing treatment followed by a recovery culture on the morphological traits of the aboveground portions, as well as physiological parameters. The main results were as follows: (1) According to the Logistic equation fitting, the LT50 values for different leaf samples were determined to range from -4.397℃ to -10.016℃. Following cold acclimation, the LT50 values for XA and YF increased, while those for LS and SZ demonstrated a decrease, with SZ exhibiting a greater reduction compared to LS; (2) Direct freezing treatment resulted in low-temperature damage to the aboveground portions of XA and SZ. While cold acclimation prior to freezing treatment could only make the aboveground parts of XA rather than SZ show symptoms of freezing injury; (3) Physiological assessments indicated that cold acclimation did not significantly affect proline accumulation in either XA or SZ, but lead to a notable decrease in carotenoid content. Both direct freezing treatment and cold acclimation prior to freezing treatment could only induce proline synthesis in SZ rather than XA. Compared with before treatment, the soluble protein contents could be disturbed by cold acclimation, direct freezing treatment, and cold acclimation prior to freezing treatment, yet their trends diverged significantly between XA (decrease) and SZ (increase). These findings suggested that the concentration changes of proline and soluble protein might play critical roles in establishing freezing tolerance in SZ. In summary, there were notable differences in cold acclimation capacity among different germplasms of C. tortisepalum var. longibracteatum. A gradual cooling mode for cold acclimation facilitated the development of the low temperature stress 'memory', which ultimately improved the adaptability of 'Shui Zhusha' to freezing stress by elevating the concentrations of osmotic adjustment substances.