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.

The lowdown on breakdown: Open questions in plant proteolysis.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases.

The acetylation-dependent (Ac/)N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3 ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multiomics approaches to characterize potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3 ligases in the Nt-acetylation-(NTA)-dependent turnover of proteins at global- and protein-specific scales. Arabidopsis has two endoplasmic reticulum (ER)-localized DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wild type, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localized SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.

Are Geotextiles Silent Contributors of Ultrashort Chain PFASs to the Environment?

We investigated the presence of per- and poly fluoroalkyl substances (PFASs) in woven and nonwoven polypropylene geotextiles and four nonwoven polyester geotextiles commonly used in modern geosynthetic composite lining systems for waste containment facilities such as landfills. Targeted analysis for 23 environmentally significant PFAS molecules and methods for examining "PFAS total" concentrations were utilized to assess their occurrence in geotextiles. This analysis showed that most geotextile specimens evaluated in the current investigation contained the ultrashort chain PFAS compound pentafluoropropionic acid (PFPrA). While the concentrations ranged from nondetectable to 10.84 µg/g, the average measured concentrations of PFPrA were higher in polypropylene than in polyester geotextiles. "PFAS total" parameters comprising total fluorine (TF) and total oxidizable precursors (TOPs) indicate that no significant precursor mass nor untargeted intermediates were present in geotextiles. Therefore, this study identified geotextiles as a possible source of ultrashort PFASs in engineered lined waste containment facilities, which may contribute to the overall PFAS total concentrations in leachates or liquors they are in contact with. The findings reported for the first time herein may lead to further implications on the fate and migration of PFASs in geosynthetic composite liners, as previously unidentified concentrations, particularly of ultrashort-chain PFASs, may impact the extent of PFAS migration through and attenuation by constituents of geosynthetic composite liner systems. Given the widespread use of geotextiles in various engineering activities, these findings may have other unknown impacts. The significance of these findings needs to be further elucidated by more extensive studies with larger geotextile sample sizes to allow broader, generalized conclusions to be drawn.

Psychiatric Residential Treatment Facilities for Child Behavioral Health Services in North Carolina Medicaid.

Background: Psychiatric residential treatment facilities (PRTFs) are non-hospital inpatient treatment settings for children with severe be-havioral health disorders. PRTFs are a restrictive and costly form of care that can potentially be avoided with community-based behavioral health services. Methods: Statewide Medicaid enrollment and claims data for 2015-2022 were used to describe PRTF utilization in North Carolina. We examined annual episodes of care in PRTFs and compared trends before and during the COVID-19 public health emergency. Results: From 2015 to 2022, 10,038 children insured by NC Medicaid entered a PRTF across 10,966 episodes of care. In the past five years (2018-2022), care in PRTFs resulted in Medicaid expenditures of over $550 million total, or over $100 million per year. In 2022, 42% of children who entered PRTFs were in foster care and 44% of children were placed in PRTFs outside of North Carolina. Limitations: The analysis was limited to data collected for administrative purposes. Conclusions: Current trends indicate an ongoing overrepresentation of children in foster care placed in PRTFs and increased out-of-state PRTF placements. Coordinated efforts in future research, policy, and practice are needed to determine the cause of these trends and iden-tify solutions.

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.

Altered collective mitochondrial dynamics in the Arabidopsis msh1 mutant compromising organelle DNA maintenance.

Mitochondria form highly dynamic populations in the cells of plants (and almost all eukaryotes). The characteristics and benefits of this collective behaviour, and how it is influenced by nuclear features, remain to be fully elucidated. Here, we use a recently developed quantitative approach to reveal and analyse the physical and collective 'social' dynamics of mitochondria in an Arabidopsis msh1 mutant where the organelle DNA maintenance machinery is compromised. We use a newly created line combining the msh1 mutant with mitochondrially targeted green fluorescent protein (GFP), and characterize mitochondrial dynamics with a combination of single-cell time-lapse microscopy, computational tracking, and network analysis. The collective physical behaviour of msh1 mitochondria is altered from that of the wild type in several ways: mitochondria become less evenly spread, and networks of inter-mitochondrial encounters become more connected, with greater potential efficiency for inter-organelle exchange-reflecting a potential compensatory mechanism for the genetic challenge to the mitochondrial DNA population, supporting more inter-organelle exchange. We find that these changes are similar to those observed in friendly, where mitochondrial dynamics are altered by a physical perturbation, suggesting that this shift to higher connectivity may reflect a general response to mitochondrial challenges, where physical dynamics of mitochondria may be altered to control the genetic structure of the mtDNA population.

Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors.

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.

Regulatory cascade involving transcriptional and N-end rule pathways in rice under submergence.

The rice SUB1A-1 gene, which encodes a group VII ethylene response factor (ERFVII), plays a pivotal role in rice survival under flooding stress, as well as other abiotic stresses. In Arabidopsis, five ERFVII factors play roles in regulating hypoxic responses. A characteristic feature of Arabidopsis ERFVIIs is a destabilizing N terminus, which functions as an N-degron that targets them for degradation via the oxygen-dependent N-end rule pathway of proteolysis, but permits their stabilization during hypoxia for hypoxia-responsive signaling. Despite having the canonical N-degron sequence, SUB1A-1 is not under N-end rule regulation, suggesting a distinct hypoxia signaling pathway in rice during submergence. Herein we show that two other rice ERFVIIs gene, ERF66 and ERF67, are directly transcriptionally up-regulated by SUB1A-1 under submergence. In contrast to SUB1A-1, ERF66 and ERF67 are substrates of the N-end rule pathway that are stabilized under hypoxia and may be responsible for triggering a stronger transcriptional response to promote submergence survival. In support of this, overexpression of ERF66 or ERF67 leads to activation of anaerobic survival genes and enhanced submergence tolerance. Furthermore, by using structural and protein-interaction analyses, we show that the C terminus of SUB1A-1 prevents its degradation via the N-end rule and directly interacts with the SUB1A-1 N terminus, which may explain the enhanced stability of SUB1A-1 despite bearing an N-degron sequence. In summary, our results suggest that SUB1A-1, ERF66, and ERF67 form a regulatory cascade involving transcriptional and N-end rule control, which allows rice to distinguish flooding from other SUB1A-1-regulated stresses.

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.

How to build an effective research network: lessons from two decades of the GARNet plant science community.

Successful collaborative research is dependent on excellent ideas and innovative experimental approaches, as well as the provision of appropriate support networks. Collaboration requires venues, infrastructures, training facilities, and, perhaps most importantly, a sustained commitment to work together as a community. These activities do not occur without significant effort, yet can be facilitated and overseen by the leadership of a research network that has a clearly defined role to help build resources for their community. Over the past 20 years, this is a role that the UKRI-BBSRC-funded GARNet network has played in the support of the UK curiosity-driven, discovery-led plant science research community. This article reviews the lessons learnt by GARNet in the hope that they can inform the practical implementation of current and future research networks.

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.

Risk factors for admission after shoulder arthroscopy.

BACKGROUND: Shoulder arthroscopy is a common orthopedic procedure typically performed on an outpatient basis. Occasionally, patients require an unplanned hospital admission. An understanding of the incidence and risk factors for admission after shoulder arthroscopy may assist surgeons in determining which patients may be susceptible to unplanned admission after surgery. METHODS: All consecutive shoulder arthroscopy procedures performed during a 10-year period were reviewed. A 2:1 control-case matching technique was used. Univariate analysis was performed to identify differences between patients admitted after surgery and the control group. Multivariate analysis was performed to identify variables associated with admission. RESULTS: There were 5598 arthroscopic shoulder procedures performed, with 233 patients (4.2%) requiring admission. The most common reason for admission was respiratory monitoring. Risk factors for admission by multivariate analysis were chronic obstructive pulmonary disease (odds ratio [OR], 2.73; 95% confidence interval [CI], 1.51-4.95), diabetes (OR, 2.11; 95% CI, 1.28-3.48), obstructive sleep apnea (OR, 1.90; 95% CI, 1.13-3.21), age (OR, 1.02; 95% CI, 1.01-1.04), body mass index (OR, 1.04; 95% CI, 1.01-1.07), and operative time (OR, 1.01; 95% CI, 1.00-1.01). Regional with monitored anesthesia care decreased risk compared with general anesthesia and regional with general anesthesia (OR, 0.44; 95% CI, 0.30-0.63). CONCLUSION: Chronic obstructive pulmonary disease, obstructive sleep apnea, diabetes, increasing age, increasing body mas index, and increasing operative time were all risk factors for admission after shoulder arthroscopy. The absence of general anesthesia was found to decrease the risk of admission.

N-terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N-end rule pathway.

The N-end rule pathway of targeted protein degradation is an important regulator of diverse processes in plants but detailed knowledge regarding its influence on the proteome is lacking. To investigate the impact of the Arg/N-end rule pathway on the proteome of etiolated seedlings, we used terminal amine isotopic labelling of substrates with tandem mass tags (TMT-TAILS) for relative quantification of N-terminal peptides in prt6, an Arabidopsis thaliana N-end rule mutant lacking the E3 ligase PROTEOLYSIS6 (PRT6). TMT-TAILS identified over 4000 unique N-terminal peptides representing c. 2000 protein groups. Forty-five protein groups exhibited significantly increased N-terminal peptide abundance in prt6 seedlings, including cruciferins, major seed storage proteins, which were regulated by Group VII Ethylene Response Factor (ERFVII) transcription factors, known substrates of PRT6. Mobilisation of endosperm α-cruciferin was delayed in prt6 seedlings. N-termini of several proteases were downregulated in prt6, including RD21A. RD21A transcript, protein and activity levels were downregulated in a largely ERFVII-dependent manner. By contrast, cathepsin B3 protein and activity were upregulated by ERFVIIs independent of transcript. We propose that the PRT6 branch of the pathway regulates protease activities in a complex manner and optimises storage reserve mobilisation in the transition from seed to seedling via control of ERFVII action.

Developmental control of hypoxia during bud burst in grapevine.

Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds.

Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants.

Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by roots becoming waterlogged or foliage submergence, triggers changes in gene transcription and messenger RNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production. In contrast to animals, oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen levels in Arabidopsis, through its regulation of key hypoxia-response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia-response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated ethylene response factor group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the amino terminus initiating with Met-Cys. Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice, was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, indicating that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses.

Mechanical constraints imposed by 3D cellular geometry and arrangement modulate growth patterns in the Arabidopsis embryo.

Morphogenesis occurs in 3D space over time and is guided by coordinated gene expression programs. Here we use postembryonic development in Arabidopsis plants to investigate the genetic control of growth. We demonstrate that gene expression driving the production of the growth-stimulating hormone gibberellic acid and downstream growth factors is first induced within the radicle tip of the embryo. The center of cell expansion is, however, spatially displaced from the center of gene expression. Because the rapidly growing cells have very different geometry from that of those at the tip, we hypothesized that mechanical factors may contribute to this growth displacement. To this end we developed 3D finite-element method models of growing custom-designed digital embryos at cellular resolution. We used this framework to conceptualize how cell size, shape, and topology influence tissue growth and to explore the interplay of geometrical and genetic inputs into growth distribution. Our simulations showed that mechanical constraints are sufficient to explain the disconnect between the experimentally observed spatiotemporal patterns of gene expression and early postembryonic growth. The center of cell expansion is the position where genetic and mechanical facilitators of growth converge. We have thus uncovered a mechanism whereby 3D cellular geometry helps direct where genetically specified growth takes place.

Enhanced waterlogging tolerance in barley by manipulation of expression of the N-end rule pathway E3 ligase PROTEOLYSIS6.

Increased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N-end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)-cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N-end rule pathway in vitro. Furthermore, we show that Nt-Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen-sensor reporter protein MCGGAIL-GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N-end rule pathway N-recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia-associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia-related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia-related genes and phenotypes similar to RNAi lines. We conclude that the N-end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals.

From start to finish: amino-terminal protein modifications as degradation signals in plants.

Contents 1188 I. 1188 II. 1189 III. 1190 IV. 1191 V. 1192 1192 References 1192 SUMMARY: The amino- (N-) terminus (Nt) of a protein can undergo a diverse array of co- and posttranslational modifications. Many of these create degradation signals (N-degrons) that mediate protein destruction via the N-end rule pathway of ubiquitin-mediated proteolysis. In plants, the N-end rule pathway has emerged as a major system for regulated control of protein stability. Nt-arginylation-dependent degradation regulates multiple growth, development and stress responses, and recently identified functions of Nt-acetylation can also be linked to effects on the in vivo half-lives of Nt-acetylated proteins. There is also increasing evidence that N-termini could act as important protein stability determinants in plastids. Here we review recent advances in our understanding of the relationship between the nature of protein N-termini, Nt-processing events and proteolysis in plants.

An ancient and conserved function for Armadillo-related proteins in the control of spore and seed germination by abscisic acid.

Armadillo-related proteins regulate development throughout eukaryotic kingdoms. In the flowering plant Arabidopsis thaliana, Armadillo-related ARABIDILLO proteins promote multicellular root branching. ARABIDILLO homologues exist throughout land plants, including early-diverging species lacking true roots, suggesting that early-evolving ARABIDILLOs had additional biological roles. Here we investigated, using molecular genetics, the conservation and diversification of ARABIDILLO protein function in plants separated by c. 450 million years of evolution. We demonstrate that ARABIDILLO homologues in the moss Physcomitrella patens regulate a previously undiscovered inhibitory effect of abscisic acid (ABA) on spore germination. Furthermore, we show that A. thaliana ARABIDILLOs function similarly during seed germination. Early-diverging ARABIDILLO homologues from both P. patens and the lycophyte Selaginella moellendorffii can substitute for ARABIDILLO function during A. thaliana root development and seed germination. We conclude that (1) ABA was co-opted early in plant evolution to regulate functionally analogous processes in spore- and seed-producing plants and (2) plant ARABIDILLO germination functions were co-opted early into both gametophyte and sporophyte, with a specific rooting function evolving later in the land plant lineage.