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Abstract：
BACKGROUND: 6 - 4 photoproducts are the second most common UV-induced DNA lesions after cyclobutane pyrimidine dimers. In plants, they are mainly repaired by photolyases in a process called photoreactivation. While pyrimidine dimers can be deleterious, leading to mutagenesis or even cell death, 6 - 4 photoproducts can activate specific signaling pathways. Therefore, their removal is particularly important, especially for plants exposed to high UV intensities due to their sessile nature. Although photoreactivation in nuclear DNA is well-known, its role in plant organelles remains unclear. In this paper we analyzed the activity and localization of GFP-tagged AtUVR3, the 6 - 4 photoproduct specific photolyase.
RESULTS: Using transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 - 4 photoproduct removal under blue light. Measurements of 6 - 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein.
CONCLUSIONS: Photolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 - 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 - 4 photoproducts. The efficient photoreactivation of 6 - 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.
RESULTS: Using transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 - 4 photoproduct removal under blue light. Measurements of 6 - 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein.
CONCLUSIONS: Photolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 - 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 - 4 photoproducts. The efficient photoreactivation of 6 - 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.
摘要：
背景：6-4光产物是仅次于环丁烷嘧啶二聚体的第二常见的紫外线诱导的DNA损伤。在植物中,它们主要由光解酶在一个称为光活化的过程中修复。虽然嘧啶二聚体可能是有害的，导致诱变甚至细胞死亡，6-4个光产物可以激活特定的信号通路。因此,他们的移除尤为重要，特别是对于由于其固着性质而暴露于高紫外线强度的植物。尽管核DNA中的光活化是众所周知的，其在植物细胞器中的作用尚不清楚。在本文中，我们分析了GFP标记的AtUVR3的活性和定位，该6-4光产物特异性光解酶。
结果：使用具有不同AtUVR3表达水平的转基因拟南芥，我们证实了这些水平与蓝光下6-4光产物去除率之间的正趋势。野生型叶绿体和核DNA中6-4种光产物水平的测定,光解酶突变体，和过表达AtUVR3的转基因植物表明，光活化是负责去除两个细胞器中这些病变的主要修复途径。GFP标记的AtUVR3主要位于细胞核中，一小部分存在于转基因拟南芥和烟草品系的叶绿体和线粒体中。在叶绿体中，这种光解酶与质体包膜DNA结合蛋白标记的类核共定位。
结论：光解酶主要位于植物细胞核，叶绿体和线粒体中只有一小部分存在。尽管分布不平衡,光激活是导致从成年叶片的核和叶绿体DNA中去除6-4种光产物的主要机制。AtUVR3光解酶的量是影响6-4个光产物光活化率的限制因素。35S：AtUVR3-GFP拟南芥和烟草中6-4种光产物的有效光活化是一个有希望的起点，可以评估过度生产这种光解酶的转基因作物是否更耐受高紫外线照射，以及它们如何响应其他非生物和生物胁迫在田间条件下。
结果：使用具有不同AtUVR3表达水平的转基因拟南芥，我们证实了这些水平与蓝光下6-4光产物去除率之间的正趋势。野生型叶绿体和核DNA中6-4种光产物水平的测定,光解酶突变体，和过表达AtUVR3的转基因植物表明，光活化是负责去除两个细胞器中这些病变的主要修复途径。GFP标记的AtUVR3主要位于细胞核中，一小部分存在于转基因拟南芥和烟草品系的叶绿体和线粒体中。在叶绿体中，这种光解酶与质体包膜DNA结合蛋白标记的类核共定位。
结论：光解酶主要位于植物细胞核，叶绿体和线粒体中只有一小部分存在。尽管分布不平衡,光激活是导致从成年叶片的核和叶绿体DNA中去除6-4种光产物的主要机制。AtUVR3光解酶的量是影响6-4个光产物光活化率的限制因素。35S：AtUVR3-GFP拟南芥和烟草中6-4种光产物的有效光活化是一个有希望的起点，可以评估过度生产这种光解酶的转基因作物是否更耐受高紫外线照射，以及它们如何响应其他非生物和生物胁迫在田间条件下。
