水稻ERFVIIs對於淹水反應的功能性分析

Abstract

隨著氣候變遷，極端的天氣現象越發嚴重與頻繁，而劇烈降雨所造成的淹水事件會造成植物浸沒於水中，進而影響植物生長與生存。第七類乙烯轉錄因子(The group VII of ethylene response factors, ERFVIIs)可以作為氧氣感應者，其N端保守序列可以作為N-end rule的受質，使其蛋白質在一般環境下不穩定而降解，而在淹水以及低氧環境下，穩定的蛋白質能協調下游基因以降低淹水所造成的傷害。水稻對於淹水環境有著天然耐受性，但只有少數品系在完全浸泡於水中超過2週後能恢復生長。水稻帶有18個ERFVII基因，其中的SUB1A-1作為重要調控因子，只存在於特定品系，可以調控許多代謝反應並抑制植物生長，降低能量負擔。此外，SUB1A-1可以調控ERF66和ERF67表現，可共同促進淹水耐受性。除了SUB1A相關機制外，水稻ERFVIIs對於淹水耐受性的調控是未知的。 本篇報導透過轉錄量分析，發現數個基因(ERF65、ERF70、ERF71與ERF72)持續表現，並且在不同遺傳背景下亦然，代表其功能可能是保守並且與淹水時快速反應有關。透過在水稻原生質體暫時表達，ERF65、ERF70和ERF72可以激活ERF67啟動子，而ERF65/70/72與SUB1A的共表達可以促進ERF67的轉錄，多個ERFVII蛋白佔據於ERF67啟動子上的GCC box可能是造成共協調的主因。有趣的是，雖然SUB1A-1與SUB1A-2均可與ERF65/70/72共同激活ERF67表現，但SUB1A-2無法有效誘導內生的ERF67表現，這突顯了SUB1A的磷酸化在調控功能上的重要性。此外，ERFVIIs可以調控非共生血紅蛋白(non-symbiotic hemoglobin, nsHBs)的表現，其作為一氧化氮清除者，在缺氧期間可調節細胞內一氧化氮含量。ERF65、ERF70與ERF71有較高能力促進HB1和HB2表現，ERF72為中等，SUB1A-1與ERF67則有較低能力促進HB2表現，而當ERF67與ERF71共表達時，可能產生競爭關係而微調HB2表現量。 總之，我們提出了一條ERFVII的調控路徑，有助於水稻對於淹水的基礎耐受性；而在耐淹水品系中，SUB1A的參與可以增加對ERF67的調控，進而延長浸沒的生存時間。

With climate change, extreme weather phenomena are more severe and frequent. Flooding events by severe rainfall cause plants to be submerged in water, which will affect the growth and survival of plants. The group VII of ethylene response factors (ERFVIIs) are the substrates of N-end rule which make their protein unstable under normal oxygen conditions. Under submergence and low oxygen, the stabilized ERFVII proteins could coordinately regulate downstream genes to reduce the damages caused by flooding. Rice (Oryza sativa) is naturally tolerant to flooding, but only a few cultivars can survive after fully submerged in water for a prolonged period of time. There are eighteen ERFVIIs in rice. SUB1A-1 that only exists in specific cultivars is a master regulator to coordinate metabolic responses and repress plant growth during submergence which can reduce energy burden. In addition, SUB1A-1 could regulate the expression of ERF66 and ERF67, which could promote flooding tolerance. Except for SUB1A-dependent mechanisms, the regulation of ERFVIIs in rice for flooding tolerance is unknown. In this study, several ERFVII genes including ERF65, ERF70, ERF71 and ERF72 were found constitutively expressed in different genetic backgrounds via transcription analysis, indicating their function might be conserved and associated with rapid response to flooding. Through transient expressions in rice protoplasts, ERF65, ERF70 and ERF72 could activate the promoter of ERF67. Co-expression of ERF65/70/72 with SUB1A could enhance the transcript of ERF67. The occupancy of GCC boxes on the ERF67 promoter by multiple ERFVII proteins might be the main reason for synergistic transcriptional activation. Interestingly, SUB1A-1 and SUB1A-2 respectively with ERF65/70/72 could co-activate the expression of the ERF67-Luc reporter gene, but the group of SUB1A-2 failed to efficiently induce the endogenous ERF67 expression. It highlighted the importance of the phosphorylation of SUB1A for the regulatory function. In addition, ERFVIIs could regulate the expression of non-symbiotic hemoglobins (nsHBs), as nitric oxide (NO) scavengers to modulate NO content during hypoxia. ERF65, ERF70 and ERF71 had better capabilities to activate the expression of HB1 and HB2. SUB1A-1 and ERF67 showed low abilities to induce the expression of HB2. Co-expression of ERF67 and ERF71 might compete for the binding sites and fine-tune the expression of HB2. In summary, we proposed a regulatory pathway of ERFVIIs which contributed to the basal tolerance of flooding for rice. In tolerant cultivars, the involvement of SUB1A-1 would enhance the expression of ERF67, which would prolong the survival during submergence.