ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana.

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

An oxygen-sensing mechanism for angiosperm adaptation to altitude.

Flowering plants (angiosperms) can grow at extreme altitudes, and have been observed growing as high as 6,400 metres above sea level1,2; however, the molecular mechanisms that enable plant adaptation specifically to altitude are unknown. One distinguishing feature of increasing altitude is a reduction in the partial pressure of oxygen (pO2). Here we investigated the relationship between altitude and oxygen sensing in relation to chlorophyll biosynthesis-which requires molecular oxygen3-and hypoxia-related gene expression. We show that in etiolated seedlings of angiosperm species, steady-state levels of the phototoxic chlorophyll precursor protochlorophyllide are influenced by sensing of atmospheric oxygen concentration. In Arabidopsis thaliana, this is mediated by the PLANT CYSTEINE OXIDASE (PCO) N-degron pathway substrates GROUP VII ETHYLENE RESPONSE FACTOR transcription factors (ERFVIIs). ERFVIIs positively regulate expression of FLUORESCENT IN BLUE LIGHT (FLU), which represses the first committed step of chlorophyll biosynthesis, forming an inactivation complex with tetrapyrrole synthesis enzymes that are negatively regulated by ERFVIIs, thereby suppressing protochlorophyllide. In natural populations representing diverse angiosperm clades, we find oxygen-dependent altitudinal clines for steady-state levels of protochlorophyllide, expression of inactivation complex components and hypoxia-related genes. Finally, A. thaliana accessions from contrasting altitudes display altitude-dependent ERFVII activity and accumulation. We thus identify a mechanism for genetic adaptation to absolute altitude through alteration of the sensitivity of the oxygen-sensing system.

Allelic shift in cis-elements of the transcription factor RAP2.12 underlies adaptation associated with humidity in Arabidopsis thaliana.

Populations of widespread species are usually geographically distributed through contrasting stresses, but underlying genetic mechanisms controlling this adaptation remain largely unknown. Here, we show that in Arabidopsis thaliana, allelic changes in the cis-regulatory elements, WT box and W box, in the promoter of a key transcription factor associated with oxygen sensing, RELATED TO AP 2.12 (RAP2.12), are responsible for differentially regulating tolerance to drought and flooding. These two cis-elements are regulated by different transcription factors that downstream of RAP2.12 results in differential accumulation of hypoxia-responsive transcripts. The evolution from one cis-element haplotype to the other is associated with the colonization of humid environments from arid habitats. This gene thus promotes both drought and flooding adaptation via an adaptive mechanism that diversifies its regulation through noncoding alleles.

Mitochondrial retrograde signaling through UCP1-mediated inhibition of the plant oxygen-sensing pathway.

Mitochondrial retrograde signaling is an important component of intracellular stress signaling in eukaryotes. UNCOUPLING PROTEIN (UCP)1 is an abundant plant inner-mitochondrial membrane protein with multiple functions including uncoupled respiration and amino-acid transport1,2 that influences broad abiotic stress responses. Although the mechanism(s) through which this retrograde function acts is unknown, overexpression of UCP1 activates expression of hypoxia (low oxygen)-associated nuclear genes.3,4 Here we show in Arabidopsis thaliana that UCP1 influences nuclear gene expression and physiological response by inhibiting the cytoplasmic PLANT CYSTEINE OXIDASE (PCO) branch of the PROTEOLYSIS (PRT)6 N-degron pathway, a major mechanism of oxygen and nitric oxide (NO) sensing.5 Overexpression of UCP1 (UCP1ox) resulted in the stabilization of an artificial PCO N-degron pathway substrate, and stability of this reporter protein was influenced by pharmacological interventions that control UCP1 activity. Hypoxia and salt-tolerant phenotypes observed in UCP1ox lines resembled those observed for the PRT6 N-recognin E3 ligase mutant prt6-1. Genetic analysis showed that UCP1 regulation of hypoxia responses required the activity of PCO N-degron pathway ETHYLENE RESPONSE FACTOR (ERF)VII substrates. Transcript expression analysis indicated that UCP1 regulation of hypoxia-related gene expression is a normal component of seedling development. Our results show that mitochondrial retrograde signaling represses the PCO N-degron pathway, enhancing substrate function, thus facilitating downstream stress responses. This work reveals a novel mechanism through which mitochondrial retrograde signaling influences nuclear response to hypoxia by inhibition of an ancient cytoplasmic pathway of eukaryotic oxygen sensing.

The PRT6 N-degron pathway restricts VERNALIZATION 2 to endogenous hypoxic niches to modulate plant development.

VERNALIZATION2 (VRN2), an angiosperm-specific subunit of the polycomb repressive complex 2 (PRC2), is an oxygen (O2 )-regulated target of the PCO branch of the PRT6 N-degron pathway of ubiquitin-mediated proteolysis. How this post-translational regulation coordinates VRN2 activity remains to be fully established. Here we use Arabidopsis thaliana ecotypes, mutants and transgenic lines to determine how control of VRN2 stability contributes to its functions during plant development. VRN2 localizes to endogenous hypoxic regions in aerial and root tissues. In the shoot apex, VRN2 differentially modulates flowering time dependent on photoperiod, whilst its presence in lateral root primordia and the root apical meristem negatively regulates root system architecture. Ectopic accumulation of VRN2 does not enhance its effects on flowering, but does potentiate its repressive effects on root growth. In late-flowering vernalization-dependent ecotypes, VRN2 is only active outside meristems when its proteolysis is inhibited in response to cold exposure, as its function requires concomitant cold-triggered increases in other PRC2 subunits and cofactors. We conclude that the O2 -sensitive N-degron of VRN2 has a dual function, confining VRN2 to meristems and primordia, where it has specific developmental roles, whilst also permitting broad accumulation outside of meristems in response to environmental cues, leading to other functions.

A Yeast-Based Functional Assay to Study Plant N-Degron - N-Recognin Interactions.

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

Comparative Biology of Oxygen Sensing in Plants and Animals.

Aerobic respiration is essential to almost all eukaryotes and sensing oxygen is a key determinant of survival. Analogous but mechanistically different oxygen-sensing pathways were adopted in plants and metazoan animals, and include ubiquitin-mediated degradation of transcription factors and direct sensing via non-heme iron(Fe2+)-dependent-dioxygenases. Key roles for oxygen sensing have been identified in both groups, with downstream signalling focussed on regulating gene transcription and chromatin modification to control development and stress responses. Components of sensing systems are promising targets for human therapeutic intervention and developing stress-resilient crops. Here, we review current knowledge about the origins, commonalities and differences between oxygen sensing in plants and animals.

The plant N-degron pathways of ubiquitin-mediated proteolysis.

The amino-terminal residue of a protein (or amino-terminus of a peptide following protease cleavage) can be an important determinant of its stability, through the Ubiquitin Proteasome System associated N-degron pathways. Plants contain a unique combination of N-degron pathways (previously called the N-end rule pathways) E3 ligases, PROTEOLYSIS (PRT)6 and PRT1, recognizing non-overlapping sets of amino-terminal residues, and others remain to be identified. Although only very few substrates of PRT1 or PRT6 have been identified, substrates of the oxygen and nitric oxide sensing branch of the PRT6 N-degron pathway include key nuclear-located transcription factors (ETHYLENE RESPONSE FACTOR VIIs and LITTLE ZIPPER 2) and the histone-modifying Polycomb Repressive Complex 2 component VERNALIZATION 2. In response to reduced oxygen or nitric oxide levels (and other mechanisms that reduce pathway activity) these stabilized substrates regulate diverse aspects of growth and development, including response to flooding, salinity, vernalization (cold-induced flowering) and shoot apical meristem function. The N-degron pathways show great promise for use in the improvement of crop performance and for biotechnological applications. Upstream proteases, components of the different pathways and associated substrates still remain to be identified and characterized to fully appreciate how regulation of protein stability through the amino-terminal residue impacts plant biology.

Every Breath You Take: New Insights into Plant and Animal Oxygen Sensing.

Responses to hypoxia are regulated by oxygen-dependent degradation of kingdom-specific proteins in animals and plants. Masson et al. (2019) identified and characterized the mammalian counterpart of an oxygen-sensing pathway previously only observed in plants. Alongside other recent findings identifying novel oxygen sensors, this provides new insights into oxygen-sensing origins and mechanisms in eukaryotes.

Desert crossing strategies of migrant songbirds vary between and within species.

Each year, billions of songbirds cross large ecological barriers during their migration. Understanding how they perform this incredible task is crucial to predict how global change may threaten the safety of such journeys. Earlier studies based on radar suggested that most songbirds cross deserts in intermittent flights at high altitude, stopping in the desert during the day, while recent tracking with light loggers suggested diurnal prolongation of nocturnal flights and common non-stop flights for some species. We analyzed light intensity and temperature data obtained from geolocation loggers deployed on 130 individuals of ten migratory songbird species, and show that a large variety of strategies for crossing deserts exists between, but also sometimes within species. Diurnal stopover in the desert is a common strategy in autumn, while most species prolonged some nocturnal flights into the day. Non-stop flights over the desert occurred more frequently in spring than in autumn, and more frequently in foliage gleaners. Temperature recordings suggest that songbirds crossed deserts with flight bouts performed at various altitudes according to species and season, along a gradient ranging from low above ground in autumn to probably >2000 m above ground level, and possibly at higher altitude in spring. High-altitude flights are therefore not the general rule for crossing deserts in migrant songbirds. We conclude that a diversity of migration strategies exists for desert crossing among songbirds, with variations between but also within species.

Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress.

Timely perception of adverse environmental changes is critical for survival. Dynamic changes in gases are important cues for plants to sense environmental perturbations, such as submergence. In Arabidopsis thaliana, changes in oxygen and nitric oxide (NO) control the stability of ERFVII transcription factors. ERFVII proteolysis is regulated by the N-degron pathway and mediates adaptation to flooding-induced hypoxia. However, how plants detect and transduce early submergence signals remains elusive. Here we show that plants can rapidly detect submergence through passive ethylene entrapment and use this signal to pre-adapt to impending hypoxia. Ethylene can enhance ERFVII stability prior to hypoxia by increasing the NO-scavenger PHYTOGLOBIN1. This ethylene-mediated NO depletion and consequent ERFVII accumulation pre-adapts plants to survive subsequent hypoxia. Our results reveal the biological link between three gaseous signals for the regulation of flooding survival and identifies key regulatory targets for early stress perception that could be pivotal for developing flood-tolerant crops.

The Scope, Functions, and Dynamics of Posttranslational Protein Modifications.

Assessing posttranslational modification (PTM) patterns within protein molecules and reading their functional implications present grand challenges for plant biology. We combine four perspectives on PTMs and their roles by considering five classes of PTMs as examples of the broader context of PTMs. These include modifications of the N terminus, glycosylation, phosphorylation, oxidation, and N-terminal and protein modifiers linked to protein degradation. We consider the spatial distribution of PTMs, the subcellular distribution of modifying enzymes, and their targets throughout the cell, and we outline the complexity of compartmentation in understanding of PTM function. We also consider PTMs temporally in the context of the lifetime of a protein molecule and the need for different PTMs for assembly, localization, function, and degradation. Finally, we consider the combined action of PTMs on the same proteins, their interactions, and the challenge ahead of integrating PTMs into an understanding of protein function in plants.

N-term 2017: Proteostasis via the N-terminus.

N-term 2017 was the first international meeting to bring together researchers from diverse disciplines with a shared interest in protein N-terminal modifications and the N-end rule pathway of ubiquitin-mediated proteolysis, providing a platform for interdisciplinary cross-kingdom discussions and collaborations, as well as strengthening the visibility of this growing scientific community.

The Arabidopsis thaliana N-recognin E3 ligase PROTEOLYSIS1 influences the immune response.

N-degron pathways of ubiquitin-mediated proteolysis (formerly known as the N-end rule pathway) control the stability of substrate proteins dependent on the amino-terminal (Nt) residue. Unlike yeast or mammalian N-recognin E3 ligases, which each recognize several different classes of Nt residues, in Arabidopsis thaliana, N-recognin functions of different N-degron pathways are carried out independently by PROTEOLYSIS (PRT)1, PRT6, and other unknown proteins. PRT1 recognizes type 2 aromatic Nt-destabilizing residues and PRT6 recognizes type 1 basic residues. These two N-recognin functions diverged as separate proteins early in the evolution of plants, before the conquest of the land. We demonstrate that loss of PRT1 function promotes the plant immune system, as mutant prt1-1 plants showed greater apoplastic resistance than WT to infection by the bacterial hemi-biotroph Pseudomonas syringae pv tomato (Pst) DC3000. Quantitative proteomics revealed increased accumulation of proteins associated with specific components of plant defense in the prt1-1 mutant, concomitant with increased accumulation of salicylic acid. The effects of the prt1 mutation were additional to known effects of prt6 in influencing the immune system, in particular, an observed over-accumulation of pipecolic acid (Pip) in the double-mutant prt1-1 prt6-1. These results demonstrate a potential role for PRT1 in controlling aspects of the plant immune system and suggest that PRT1 limits the onset of the defense response via degradation of substrates with type 2 Nt-destabilizing residues.

Distinct branches of the N-end rule pathway modulate the plant immune response.

The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

A Regulatory Module Controlling GA-Mediated Endosperm Cell Expansion Is Critical for Seed Germination in Arabidopsis.

A key component of seed germination is the interplay of mechanical forces governing embryo growth and the surrounding restraining endosperm tissue. Endosperm cell separation is therefore thought to play a critical role in the control of this developmental transition. Here we demonstrate that in Arabidopsis thaliana seeds, endosperm cell expansion is a key component of germination. Endosperm cells expand to accommodate embryo growth prior to germination. We show that this is an actively regulated process supported by spatiotemporal control of the cell expansion gene EXPANSIN 2 (EXPA2). The NAC transcription factors NAC25 and NAC1L were identified as upstream regulators of EXPA2 expression, gibberellin-mediated endosperm expansion, and seed germination. The DELLA protein RGL2 repressed activation of the EXPA2 promoter by NAC25/NAC1L. Taken together, our findings uncover a key role of the GA/DELLA-NAC25/NAC1L-EXPA2 network in regulating endosperm cell expansion to control the seed-to-seedling transition.