脯氨酸/P5C循环在拟南芥病原菌诱导的超敏反应中的作用机理的研究

Proline accumulation is a widespread metabolic adaptation in higher plants in response to environmenal stresses, and is believed to be protective for plants under adverse conditions. The exact role of the proline accumulation during stresses, however, is still a matter of detabe. Recently, it has been demonstrated that the Proline/P5C cycle is operating in plant cell, similar to animal counterpart. P5C produced from the oxidation of proline by proline dehydrogenase (PDH), can emerge from mitochondria and be converted back to proline by cytosolic P5C reductase. P5C reductase participates in a metabolic interlock with glucose-6-phosphate dehydrogenase of the pentose phosphate pathway transferring reducing equivalents generated by the oxidation of glucose into mitochondria to generate ATP, or ROS in some circumstances. This finding has attracted many attentions back to the research on the function of proline accumulation. Several reports and the results from our lab indicated that PDH played an important role in avirulent pathogen-induced hypersensitive response (HR), but it is not clear whether the effect of PDH is via Pro/P5C cycle activity. In order to understand whether Pro/P5C cycle is involved in HR, we have isolated a mutant deficient in the activation of Pro/P5C by proline treatment, which also showed hypersensitive to avirulent pathogen infection. In this project, we plan to clone the gene by map-based cloning and further characterize the mechanism underlying the function of Pro/P5C cycle in HR. Our research will shed new light on the metabolic implication of proline accumulation during stresses.

脯氨酸积累是高等植物应答环境胁迫的一种普遍的代谢适应机制，被证明有利于提高植物的抗逆性，但对其作用机理尚无定论。近来，Pro/P5C循环的存在在植物中被证实，即脯氨酸被PDH降解的产物P5C可以被运到细胞质中，重新合成为脯氨酸。该循环通过与其它代谢途径，如Pentose Phosphate Pathway相偶联，将还原力转运到线粒体中用于合成ATP或活性氧。这一发现为研究脯氨酸积累的功能的研究提供了新的切入点。最新报道和我们的结果都表明，烟草和拟南芥中PDH在avirulent病原菌引起的超敏反应中具有重要功能，但是否是通过Pro/P5C循环来完成的尚不清楚。本申请拟深入分析Pro/P5C循环在超敏反应中的作用，并通过对相关突变体的研究，鉴定病原菌侵染调控Pro/P5C循环活性的信号元件，阐明Pro/P5C循环的生物学功能，从新的视角为揭示脯氨酸积累在植物应答胁迫中的作用提供重要的证据。

过去的报道普遍认为胁迫诱导的脯氨酸积累来源于脯氨酸合成的增加和降解的减少，而对脯氨酸转运的作用只知甚少。为了鉴定脯氨酸转运的关键调控元件，我们分离和鉴定了拟南芥突变体pre1-1，该突变体在含脯氨酸的培养基上吸收较少的脯氨酸，因而比较抗脯氨酸。图位克隆的结果表明，PRE1编码一个氨基酸通透酶家族的成员AtAAP1. 在pre1-1突变体中，该基因的编码区有一段19碱基的缺失，造成了翻译的提前终止。该基因的表达水平受盐胁迫和ABA处理的诱导，但不受脯氨酸的诱导。有意思的是，在盐胁迫下，野生型拟南芥和p5cs1突变体对脯氨酸的吸收都有显著增加，说明在环境胁迫下，拟南芥可以通过吸收环境中的脯氨酸使体内脯氨酸含量得到增加。但在pre1-1突变体中，脯氨酸吸收却大大降低了，说明AtAAP1介导了盐胁迫诱导的脯氨酸的吸收。此外，盐胁迫诱导的脯氨酸吸收可以提高拟南芥的耐盐性。因此，我们得出结论，除了脯氨酸代谢，在环境中存在脯氨酸的情况下吸收也可能是脯氨酸积累的来源之一。