Characterization and Functional Analysis of a RING-Type E3 Protein Ligase in Regulating Ovule Development in Arabidopsis thaliana

Abstract

真核生物中其中一種主要的分解蛋白質途徑就是泛素/26S 蛋白酶系統，是一種藉由連接泛素當作標記，形成一種蛋白質降解訊號，在細胞內部此標記的蛋白質得以被辨識，因而控制細胞內蛋白質的生命週期。阿拉伯芥RING finger gene DAF-Like gene 1 (DAFL1) 預測是一個E3 protein ligase，在26S proteosome pathway系統中扮演了重要的角色。在演化樹分析中DAFL1與實驗室前人研究的DAF (DAD1-Activating Factor)基因親緣關係非常接近，但這兩個基因表現的位置卻有明顯的差異。在阿拉伯芥發育早期的花器裡，DAF在雄蕊中表現，而DAFL1專一地表現在雌蕊與胚珠中，推測DAFL1可能與DAF的功能有所不同。異位表現DAFL1 RNAi與antisense的基因轉殖植株可觀察到不孕的性狀，是由於花藥不開裂造成花粉無法正常授粉，異位表現DAFL1-H135A點突變的基因轉殖植株同樣也有此性狀。將野生型阿拉伯芥花粉與不孕植株的雌蕊雜交，能得到正常授粉而延長的果莢，不過果莢中的種子只有野生型的一半左右。另外構築可以專一表現在雌蕊與胚珠的載體，使得基因轉殖植株可以專一地表現DAFL1-H135A點突變蛋白質，其花藥可以正常開裂，果莢中的種子數量同樣只有大約野生型的一半左右。利用酵母菌雙雜交系統，藉由營養篩選方法從阿拉伯芥的花苞cDNA library中去找出能與DAFL1有交互作用的蛋白質，經過高嚴謹度篩選後得到了數個可能是DAFL1的候選受質。其中CAD1經由啟動子活性分析實驗得知其表現位置在胚珠，與DAFL1啟動子表現的部位有重疊，異位表現CAD1的轉基因植株也觀察到種子數目減半的性狀。CAD1已被證實為合成木質素重要成分的關鍵酵素，並參與果莢木質化的過程，與DAFL1同樣表現在雌蕊非木質化的部位。因此推測在雌蕊發育早期的階段，胚珠藉由DAFL1降解CAD1避免木質素過早合成。

In eukaryotes, the ubiquitin/26S proteasome system is one of the major mechanisms that act on the protein degradation. Proteins are tagged with ubiquitin as the signals to be degraded. In the cells, the ubiquitin-tagged proteins can be recognized and to regulate the life cycle of proteins. It was proposed that RING finger gene DAF-Like gene 1 (DAFL1) encodes an E3 protein ligase that plays a key role in 26S proteasome pathway in Arabidopsis thaliana. Based on phylogenetic analysis, DAFL1was closely related to DAF (DAD1-Activating Factor); however, the spatial specificities of DAFL1 and DAF gene expression patterns in Arabidopsis thaliana were different. In the early age of floral organs of Arabidopsis thaliana, DAFL1 specifically accumulated in the pistil and the ovule whereas DAF was expressed in stamen. It was suggested that DAFL1 and DAF might behave divergently in regulating plant development. Transgenic Arabidopsis plants that ectopically expressing DAFL1 RNAi and antisense caused indehiscent anthers and resulted in the unreleased pollen from the anthers. Similarly, ectopic expression of DAFL1-H135A caused indehiscent anthers in transgenic Arabidopsis plants. Pollens of wild-type Arabidopsis were crossed to the stigmas of sterile transgenic plants. At 7 days after pollination, the siliques of sterile transgenic plants developed and elongated. However, the amount of seeds of the transgenic plants is about a half of that of the wild-type Arabidopsis. Furthermore, transgenic plants that specifically expression of DAFL1-H135A in the pistil and the ovule produced dehiscent anthers, but the amount of seeds of the transgenic plants is about a half of that of the wild-type Arabidopsis. To discover the proteins that might interact with DAFL1, yeast-two hybrid system was performed to screen putative ones from flower buds cDNA library of Arabidopsis. One of the candidate genes, CAD1, was applied in the promoter assay. The result indicated that the gene expression patterns of CAD1 and DAFL1 was the same in ovule. In addition, transgenic plants that ectopic expressing CAD1 produced the amount of seeds is about a half of that of the wild-type Arabidopsis. CAD1 is a key enzyme in the process of lignin synthesis and has been thought to be involved in the lignification of siliques. Interestingly, DAFL1 and CAD1 also highly accumulated in the non-lignification site such as in the carpel. Our results revealed that DAFL1 degrades CAD1 and inhibits lignin biosynthesis at the early stage of pistil development.