Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan.
Plant Physiol Biochem. 2011 Sep;49(9):937-45. doi: 10.1016/j.plaphy.2011.07.002. Epub 2011 Jul 14.
The lily LLA23 protein is a member of the abscisic acid, stress and ripening-induced (ASR) protein family. Constitutive overexpression of LLA23 under the cauliflower mosaic virus 35S promoter confers cold and freezing tolerance in Arabidopsis. The phenotypical growth and survival percentage of the two transgenic 35S::LLA23 plants showed higher resistance to cold and freezing conditions than those of wild-type (WT) plants. The electrolyte leakage in WT leaves increased by approximately fourfold at -2 °C relative to that at 22 °C whereas both transgenic leaves showed little ion leakage under the same conditions. A microarray analysis of LLA23-overexpressing transgenic line, 35S::LLA23E, under normal growing conditions was previously conducted by Yang et al. (Protoplasma, 2008, 233:241-254). Microarray analysis showed that 12 cold-responsive genes are upregulated and 25 cold-responsive genes are downregulated by lily ASR. Many ASR-regulated genes encode proteins involved in the classes of defense/stress-related, transcription, and metabolism. Quantitative polymerase chain reaction analysis confirms the changes in mRNA levels observed in the microarray analysis. Thus, our results provide in vivo evidence implying that LLA23 mediates cold/freezing stress-responsive signaling. To gain further insight into the functions of LLA23 protein, an in vitro enzyme protection assay was used in which lactate dehydrogenase and malate dehydrogenase were subjected to unfavorable conditions. The assay revealed that both enzyme activities were significantly retained with the addition of LLA23, which was superior to either trehalose or BSA, suggesting that the LLA23 protein can protect enzymatic activities against freeze-thaw cycles. The 35S::LLA23 seedlings also exhibited enzyme activity superior to WT at -4 °C. These results suggest that LLA23 may act as an osmoprotectant as well as a transcription factor to confer 35S::LLA23 plants enhanced cold and freezing resistance.
百合 LLA23 蛋白是脱落酸、胁迫和成熟诱导（ASR）蛋白家族的一员。在花椰菜花叶病毒 35S 启动子的控制下过表达 LLA23 可使拟南芥耐冷和耐冻。与野生型（WT）植物相比，两个 35S::LLA23 转基因植物的表型生长和存活率显示出更高的抗冷和抗冻能力。WT 叶片在-2°C 时的电解质渗漏量比在 22°C 时增加了约四倍，而两个转基因叶片在相同条件下几乎没有离子渗漏。Yang 等人先前对正常生长条件下的 LLA23 过表达转基因系 35S::LLA23E 进行了微阵列分析（原生质体，2008，233：241-254）。微阵列分析显示，12 个冷响应基因上调，25 个冷响应基因下调。许多 ASR 调节的基因编码参与防御/应激相关、转录和代谢的蛋白质。定量聚合酶链反应分析证实了微阵列分析中观察到的 mRNA 水平变化。因此，我们的结果提供了体内证据，表明 LLA23 介导冷/冻胁迫反应信号。为了进一步了解 LLA23 蛋白的功能，使用体外酶保护测定法对乳酸脱氢酶和苹果酸脱氢酶进行了测定。该测定法表明，在添加 LLA23 的情况下，两种酶活性均显著保留，其效果优于海藻糖或 BSA，表明 LLA23 蛋白可以保护酶活性免受冻融循环的影响。35S::LLA23 幼苗在-4°C 时也表现出优于 WT 的酶活性。这些结果表明，LLA23 可能既作为渗透保护剂，又作为转录因子，赋予 35S::LLA23 植物增强的抗冷和抗冻能力。
