Endoplasmic reticulum–bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana

Abstract

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)–anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

The vacuolar Ca2+ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana

“…Indeed, the ER is known to harbor many Ca 2+ ‐dependent processes that are likely affected by hypoxia‐triggered changes in ER Ca 2+ dynamics (e.g., Groenendyk et al ., 2021). For example, hypoxia‐induced ER stress and upregulation of the unfolded protein response may be an important factor across eukaryotes (Bartoszewska & Collawn, 2020) and plants are well documented to show hypoxic effects on features ranging from ER redox dynamics (Ugalde et al ., 2022), to triggering the release of hypoxia‐related transcription factors sequestered at the ER surface (Eysholdt‐Derzsó et al ., 2023). However, mutants in CAX2 were resistant to sustained flooding stress in all the responses we tested, ranging from maintained chlorophyll and carotenoid levels (Fig.…”

‘Against all floods’: plant adaptation to flooding stress and combined abiotic stresses

“…While both ANAC013 and ANAC017 form complexes with rhomboid protease RHOMBIOD LIKE PROTEASE 2 (RBL2), and respond to mitochondrial stress, their participation in MRS differs significantly. Cleavage and nuclear localization of ANAC013 was already observed after 1.5 h of hypoxia stress, whereas ANAC017 did not translocate to the nucleus under short‐term hypoxic treatment (<4 h) and cleavage was so far reported after 24 h submergence (Bui et al., 2020; Eysholdt‐Derzsó et al., 2023). Considering that ANAC017 is also involved in chloroplastic retrograde signalling and reductive stress tolerance (Fuchs et al., 2022; van Aken, de Clercq, et al., 2016), its function might be key for integrating different signal cascades after prolonged stress, while ANAC013 mediates rather rapid transcriptional responses at the onset of stress.…”

“…ERFVII factors are highly conserved in flowering plants (Nakano et al., 2006) and have emerged to be key factors in sensing low‐oxygen stress and subsequent transcriptional reprogramming. In Arabidopsis, the latter is achieved through binding of ERFVII factors to the HYPOXIA‐RESPONSIVE PROMOTER ELEMENT (HRPE) present in about 70% of all HCG promoters (Bui et al., 2015; Eysholdt‐Derzsó et al., 2023; Gasch et al., 2016; Gibbs et al., 2011; Licausi et al., 2011; Zubrycka et al., 2023). Moreover, RAP2.12 induces RBOHD and HYPOXIA RESPONSIVE UNIVERSAL STRESS PROTEIN 1 ( HRU1 ) expression, the latter modulates ROS levels under hypoxia by interacting with RBOHD, linking oxygen sensing with ROS production (Gonzali et al., 2015; Yao et al., 2017).…”

“…Moreover, the ROS burst produced during post‐submergence is inextricably linked to augmented expression and activity of RBOHD (Peláez‐Vico et al., 2023; Yeung et al., 2018). TFs involved in mitochondrial retrograde signalling (MRS) are ER‐tethered NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP‐SHAPED COTYLEDON 13 (ANAC013) and ANAC017, which both positively regulate submergence tolerance (Bui et al., 2020; de Clercq et al., 2013; Eysholdt‐Derzsó et al., 2023; Kim et al., 2018; Meng et al., 2020; van Aken, de Clercq, et al., 2016; van Aken & Pogson, 2017). While both ANAC013 and ANAC017 form complexes with rhomboid protease RHOMBIOD LIKE PROTEASE 2 (RBL2), and respond to mitochondrial stress, their participation in MRS differs significantly.…”

“…Considering that ANAC017 is also involved in chloroplastic retrograde signalling and reductive stress tolerance (Fuchs et al., 2022; van Aken, de Clercq, et al., 2016), its function might be key for integrating different signal cascades after prolonged stress, while ANAC013 mediates rather rapid transcriptional responses at the onset of stress. In ANAC013 knock‐down lines, anoxia and submergence tolerance were diminished through failed induction of HCGs containing the MITOCHONDRIAL‐DYSFUNCTION MOTIF (MDM) cis ‐element (Eysholdt‐Derzsó et al., 2023). Similarly, anac017 knock‐out mutants displayed reduced low‐oxygen tolerance due to impaired transcriptional responses (Bui et al., 2020; Meng et al., 2020).…”