Absorption of excess excitation energy induces overproduction of singlet oxygen (1O2) in plants. The major sources of singlet oxygen production are chlorophyll and its intermediates located in the chloroplast. Over-accumulation of the chlorophyll biosynthetic intermediate protochlorophyllide by the exogenous application of 5-aminolevulinic acid (ALA), the precursor of tetrapyrrole, induced singlet oxygen production in the plastidic membranes. Over-expression of protochlorophyllide oxidoreductase C (PORC) in Arabidopsis thaliana resulted in efficient light-induced photo-transformation of protochlorophyllide to chlorophyllide that limited the accumulation of protochlorophyllide. Consequently, the 1O2 generation decreased in the PORC overexpressors (PORCx) and their cell death was minimal. Conversely, porC-2 over-accumulated protochlorophyllide in response to ALA treatment and generated higher amounts of 1O2 in light and had highest cell death as monitored by Evans blue staining. The protoplasts isolated from PORCx plants, when treated with ALA, generated minimal amounts of 1O2 as revealed by singlet oxygen sensor green (SOSG) fluorescence emission from chloroplasts. Conversely, the protoplasts of porC-2 mutants under identical conditions generated the maximum SOSG fluorescence in their chloroplasts and cytosol surrounding the chloroplasts most likely due to the leakage from the organelle. The membrane blebbing, a hallmark of programmed cell death, was clearly visible in WT and porC-2 protoplasts. Similarly, the nick end labelling (TUNEL) assay revealed nicks in the DNA. The TUNEL-positive nuclei after 30 min of light exposure were highest in porC-2 and lowest in PORCx protoplasts. The results demonstrate that higher amounts of singlet oxygen produced in the chloroplasts play an important role in programmed cell death.

中文翻译：

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通过拟南芥叶绿体内产生的单线态氧执行程序性细胞死亡

过量激发能量的吸收会导致植物中单线态氧 (1O2) 的过度产生。单线态氧产生的主要来源是叶绿素及其位于叶绿体中的中间体。通过外源性应用 5-氨基乙酰丙酸 (ALA)（四吡咯的前体），叶绿素生物合成中间体原叶绿素的过度积累会导致质膜中产生单线态氧。拟南芥中原叶绿素酸氧化还原酶 C (PORC) 的过度表达导致原叶绿素内酯有效的光诱导光转化为叶绿素内酯，从而限制了原叶绿素内酯的积累。因此，PORC 过表达子 (PORCx) 中的 1O2 生成减少，并且它们的细胞死亡最少。反过来，porC-2 响应 ALA 处理过度积累原叶绿素，并在光照下产生更多的 1O2，并且通过伊文思蓝染色监测到的细胞死亡最高。从 PORCx 植物中分离的原生质体，当用 ALA 处理时，会产生最少量的 1O2，如叶绿体的单线态氧传感器绿色 (SOSG) 荧光发射所揭示的那样。相反，在相同条件下 porC-2 突变体的原生质体在其叶绿体和叶绿体周围的细胞质中产生最大的 SOSG 荧光，这很可能是由于细胞器的泄漏。膜起泡是程序性细胞死亡的标志，在 WT 和 porC-2 原生质体中清晰可见。类似地，切口末端标记 (TUNEL) 分析显示 DNA 中存在切口。光照 30 分钟后，TUNEL 阳性细胞核在 porC-2 中最高，在 PORCx 原生质体中最低。结果表明，叶绿体中产生的大量单线态氧在程序性细胞死亡中起着重要作用。