TheGreening after Extended Darkness1Is an N-End Rule Pathway Mutant with High Tolerance to Submergence and Starvation  

Abstract

Plants respond to reductions in internal oxygen concentrations with adaptive mechanisms (for example, modifications of metabolism to cope with reduced supply of ATP). These responses are, at the transcriptional level, mediated by the group VII Ethylene Response Factor transcription factors, which have stability that is regulated by the N-end rule pathway of protein degradation. N-end rule pathway mutants are characterized by a constitutive expression of hypoxia response genes and abscisic acid hypersensitivity. Here, we identify a novel proteolysis6 (prt6) mutant allele, named greening after extended darkness1 (ged1), which was previously discovered in a screen for genomes uncoupled-like mutants and shows the ability to withstand long periods of darkness at the seedling stage. Interestingly, this ethyl methanesulfonate-derived mutant shows unusual chromosomal rearrangement instead of a point mutation. Furthermore, the sensitivity of N-end rule pathway mutants ged1 and prt6-1 to submergence was studied in more detail to understand previously contradicting experiments on this topic. Finally, it was shown that mutants for the N-end rule pathway are generally more tolerant to starvation conditions, such as prolonged darkness or submergence, which was partially associated with carbohydrate conservation.During their life cycle, plant cells encounter periods of oxygen deficiency, generally termed as hypoxia stress. This stress depends on the ratio between oxygen consumption and availability. In plants, oxygen diffusion can be constrained by anatomical and morphological features (i.e. densely packed cells [e.g. meristems] and a low surface-to-volume ratio [e.g. fruits and seeds]) or external conditions that hamper gas diffusion, such as complete submergence of plants or waterlogging of soils (root flooding). Because of the requirement of oxygen for biosynthetic processes and more importantly, mitochondrial respiration, a shortage in oxygen leads to an energy deficit in hypoxic cells. To cope with this stress, plants reprogram their metabolism involving downregulation of energy-demanding processes (e.g. protein biosynthesis) and up-regulation of genes important for glycolysis, fermentation, and protection against reactive oxygen species (Bailey- Serres and Voesenek, 2008;

Oxygen Sensing via the Ethylene Response Transcription Factor RAP2.12 Affects Plant Metabolism and Performance under Both Normoxia and Hypoxia

“…In Licausi et al (2011c), 35S::D13RAP2.12 overexpressors as well as ate1ate2 and prt6 mutants of the N-end-rule pathway were grown on soil without sugars and revealed decreased anoxic resistance, while in Gibbs et al (2011) ate1ate2 and prt6 mutants were grown on agar plates including sugars and revealed increased anoxic resistance. However, this does not rule out that also other reasons, such as light conditions and air humidity, can account for the contrasting effects in anoxic resistance of these two different studies (Riber et al, 2015). Taken together, these data support the suggestion that hypoxic tolerance depends to a great extent on the availability of carbohydrate reserves for fermentative metabolism.…”

“…Moreover, studies with N-end-rule knockout mutants such as deficient in arginyl-tRNA protein transferases (ate1ate2) and deficient in the ubiquitin ligase PROTEOLYSIS6 (prt6) showed that constitutive expression of RAP2.12 in the context of these mutants leads to increased anoxic survival rates (Gibbs et al, 2011), which is contradicting the results of Licausi et al (2011c). Several options were suggested to explain the discrepancy between the studies, such as differences in the developmental stage and growth conditions such as the day/night cycle, air humidity, and the presence/ absence of Suc in the medium (Sasidharan and Mustroph, 2011;Bailey-Serres et al, 2012;Riber et al, 2015). It remained unclear, however, how regulated and constitutive expression of RAP2.12 effect plant metabolism under normoxic and hypoxic conditions, and how this works out on low-oxygen tolerance of these plants.…”

Redundant ERF-VII Transcription Factors Bind to an Evolutionarily Conserved cis-Motif to Regulate Hypoxia-Responsive Gene Expression in Arabidopsis

“…The A. thaliana ERF-VII transcription factor family has been investigated in terms of their roles in gene regulation, low-oxygen sensing, and low-oxygen survival (Hinz et al, 2010;Licausi et al, 2010Licausi et al, , 2011aGibbs et al, 2011;Weits et al, 2014;Riber et al, 2015), as well as their roles in germination and skotomorphogenesis (development in the dark) Abbas et al, 2015). Despite this, the quantitative impact of each member and hierarchical mechanisms underlying their redundancy are not satisfyingly understood.…”

“…As external oxygen levels fall below 10%, RAP2.12 migrates into the nucleus concomitant with induction of a major portion of the core HRGs, including ADH1 and PDC1 (Licausi et al, 2011a;Kosmacz et al, 2015). Of the 49 core HRGs, only seven might not be regulated by the N-end rule pathway, as their expression is unchanged in the prt6, ate1 ate2, and ged1 mutants (Gibbs et al, 2011;Riber et al, 2015). During reoxygenation, RAP2.12 becomes rapidly destabilized (Licausi et al, 2011a;Kosmacz et al, 2015), presumably aided by PCO1/2 catalysis of NH 2 -Cys 2 oxidation (Weits et al, 2014).…”

Group VII Ethylene Response Factors Coordinate Oxygen and Nitric Oxide Signal Transduction and Stress Responses in Plants

“…Arabidopsis N-end rule pathway mutants have altered responses to hypoxia or flooding, either enhancing or negatively impacting survival rates, depending upon the context of the stress and recovery conditions (Gibbs et al, 2011;Licausi et al, 2011;Riber et al, 2015). This indicates that the N-end rule pathway is a promising target for manipulating flooding tolerance in crops.…”