A Trihelix DNA Binding Protein Counterbalances Hypoxia-Responsive Transcriptional Activation in Arabidopsis

Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule–insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein–protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops.

植物在低氧胁迫下的转录激活过程由乙烯响应因子VII家族（ethylene-responsive factor group VII, ERF-VII）转录因子统筹调控。这类转录因子在低氧条件下保持稳定，但在常氧环境中会通过靶向选择性蛋白水解的N端规则途径（N-end rule pathway）发生降解。在水稻中，条件性表达的ERF-VII基因可赋予植株有效的淹涝耐受策略；而拟南芥（Arabidopsis thaliana）中不依赖N端规则的ERF-VII因子RAP2.12组成型过量积累则会产生适应不良反应。这提示，介导厌氧代谢的低氧条件下转录激活过程需要受到精细调控。然而目前尚不清楚是否存在针对RAP2.12的制衡机制。全基因组转录组分析筛选到一个未被表征的三螺旋转录因子基因，我们将其命名为低氧响应衰减因子1（HYPOXIA RESPONSE ATTENUATOR1, HRA1），该基因的表达在低氧条件下显著上调。HRA1能够拮抗RAP2.12对核心低氧响应基因的诱导作用，以及对低氧响应启动子的转录激活活性。通过酵母双杂交实验（yeast-two-hybrid assay）与染色质免疫共沉淀（chromatin immunoprecipitation），我们证实HRA1可与RAP2.12蛋白结合，但仅能结合少数低氧调控基因的基因组DNA区域，这表明HRA1通过蛋白质-蛋白质相互作用的方式调控RAP2.12的功能。对具有不同代谢性低氧水平的组织（即茎尖区域与成熟莲座叶）的低氧响应进行比较分析后发现，RAP2.12与HRA1之间的拮抗互作可使植物对波动的低氧环境产生灵活响应，这对植株的胁迫存活至关重要。在拟南芥中，有效的低氧感知响应需要先稳定RAP2.12，随后诱导HRA1的表达，通过对RAP2.12的负反馈调控来调节厌氧响应的强度。这一机制在亚优氧条件下对植物存活具有关键作用。植物氧感知机制反馈调控环路的发现，为耐涝作物的遗传育种开辟了全新的研究方向。