植物自行遠離逆境環境的能力，必須直面各種生物和非生物逆境，因此植物演化出了完善的分子機制來應對，當環境對植物不利時就會產生不同類型的細胞內脅迫反應來忍受與適應環境。核仁脅迫是核糖體生合成或功能缺陷誘發的逆境反應，由於核醣體對生物的重要性，核仁脅迫反應往往非常劇烈，在動物研究中，營養缺乏、缺氧或突變都會誘發核仁脅迫。受益於癌症生物學的研究，核仁脅迫在動物中已經被廣泛的研究，動物細胞核仁脅迫反應的主要途徑涉及腫瘤抑制因子 p53 及其不穩定因子 MDM2，但在植物中沒有 p53 和 MDM2 的同源物，唯一被發表過的關鍵調節因子NAC 轉錄家族成員 ANAC082，然而這一植物特異性通路的詳細分子機制在很大程度上是未知的。
為了分析核仁脅迫對植物細胞增殖的影響，我們使用突變體root initiation defective 3 (rid3) 與兩種化學試劑5-fluorouracil（5-FU）和puromycin（Puro）作為核仁脅迫的誘導劑。我首先測試了化學誘導劑，rRNA 前處理抑制劑 5-FU 和阻斷翻譯的化學物質 Puro 會抑制植物主根的生長和癒傷組織的形成，並且此兩種誘導劑都能上調 ANAC082p::GUS 的表達水平。在化學誘導組別的qPCR結果中顯示了三種NAC轉錄因子家族成員的大量表達包括ANAC044、ANAC085和ANAC103，以及三種在DNA受損脅迫中上調的CDK抑制因子包括SMR5、SMR7和KRP6也在核仁脅迫條件下上調，而DNA修復相關基因在表達量的調控則並未觀察到。
為了深入了解ANAC082調控的下游基因，我們制作了β-estradiol誘導的ANAC082過表達植株 (ANAC082-OX)，從表型觀察在誘導了ANAC082表達量後，根生長、芽再生和癒傷組織形成均在核仁脅迫條件下受到抑制。為了進一步確定核仁脅迫過程中調控的下游基因，我對過表達株進行RNA測序以及 RT-qPCR檢查過表達株的 DNA 受損脅迫和細胞周期調控相關基因表達的變化。RNA-seq和RT-qPCR分析顯示，在ANAC082-OX中，多個NAC轉錄因子和CDK抑制因子基因的表達水平升高，這與化學物質處理（5-FU和Puro）以及與核糖體相關的突變（rid2和rid3）中觀察到的表達模式類似。此外，在ANAC082-OX中，DNA修復相關基因的表達水平未顯著上調，這與其他核仁脅迫條件下的情況相同。
綜上所述，雖然ANAC082介導的核仁脅迫路徑和DNA損傷脅迫路徑部分共享調控細胞周期的下游事件，但它們可能是獨立的信號傳遞途徑。

Plants are sessile organisms that face various biotic and abiotic stress conditions. To adapt to the fluctuating environment, plants have developed unique stress response mechanisms. Various types of intracellular stresses occur when conditions are unfavorable for plants. Nucleolar stress is one of the most effective stresses induced by defects in ribosome biogenesis or function. In animals, nucleolar stress is induced by nutritional starvation, hypoxia, or some ribosome-related mutations, which is widely studied in cancer biology. The main pathway of the nucleolar stress response in animal cells involves the tumor suppressor p53 and its destabilizer MDM2. In plants, although there is no ortholog for p53 and MDM2, ANAC082, a member of the NAC transcription family of Arabidopsis thaliana, was identified as a pivotal regulator in the nucleolar stress response pathway. However, the detailed molecular mechanism of this plant-specific pathway is largely unknown.
To analyze the effects of nucleolar stress on plant cell proliferation, we used one mutation, root initiation defective 3 (rid3), and two chemical reagents, 5-fluorouracil (5-FU) and puromycin (Puro), as stress inducers. I first focused on these chemicals. 5-FU, an inhibitor of pre-rRNA processing, and Puro, a chemical that blocks translation, repressed root growth, and callus formation. These drugs upregulated the expression level of ANAC082p::GUS, which means they work as nucleolar stress inducers in plants. The qPCR analysis showed that, in the treatment with chemicals, the expression levels of three genes encoding NAC transcription factors, ANAC044, ANAC085, and ANAC103, and three genes of CDK inhibitors, SMR5, SMR7, and KRP6, which are all upregulated in DNA damage stress, were also elevated, whereas almost no alteration in expression of DNA repair-related genes was observed.
For insight into the downstream of ANAC082, we made the β-estradiol-inducible overexpression line of ANAC082 (ANAC082-OX). The phenotypic analysis showed repression of primary root growth, shoot regeneration, and callus formation in the inductive conditions. To identify the downstream genes regulated under nucleolar stress, we carried out RNA sequencing (RNA-seq) analysis with this inducible line. Also, I performed RT-qPCR and checked the genes related to DNA damage response and cell cycle regulation. Both RNA-seq and RT-qPCR showed similar gene expression patterns, such as elevated levels of genes of several NAC transcription factors and CDK inhibitors, in ANAC082-OX to those in the treatment with chemicals (5-FU and Puro), in the ribosome-related mutations (rid2 and rid3). Furthermore, the expression levels of DNA repair-related genes were not significantly elevated in ANAC082-OX, which is also the same as in other nucleolar stress conditions.
In summary, the ANAC082-mediated nucleolar stress pathway and the DNA damage stress pathway are possibly independent signaling pathways although they partially share the downstream events to regulate the cell cycle.

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