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.

The evidence base for risk assessment tools used in U.S. child protection investigations: A systematic scoping review.

BACKGROUND: Child protective services (CPS) agencies use risk assessment tools to augment decision making about alleged child maltreatment. Under the Family First Prevention Services Act, states and tribes are permitted to claim federal reimbursement for prevention services for children at imminent risk of entering foster care based on assessment tools and protocols. In this context, existing tools are being repurposed. It is critical to reassess the evidence supporting their use. OBJECTIVE: We aimed to synthesize the evidence pertaining to validity and reliability of specific risk assessment tools designed for CPS agencies, summarize how this work has been carried out, and review the conceptual dimensions of risk included in each tool. PARTICIPANTS AND SETTING: We included United States-based, quantitative evaluations of risk assessment tools published between 1990 and May 2021. METHODS: We carried out a scoping review using a protocol in alignment with PRISMA-ScR. We used a multiphase selective screening approach with at least two screeners. RESULTS: In total, 25 studies met inclusion criteria. Overall, research about the validity and reliability of risk assessment tools is dated and heterogeneous in methodology. The conceptualizations of risk assessment and the operationalization of risk also varied widely. There was a general dearth of evidence that supported the use of tools across demographic subgroups. CONCLUSIONS: Heterogeneity of studies assessing tool validity and reliability suggests a lack of agreement about how to assess tools and makes it difficult to interpret findings across studies. Agencies should be cautious about overreliance on tools for which evidence is limited.

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.

Improving Parenting Competency and Permanency Awareness for Kinship Foster Parents Through In-Service Licensure Training.

Child welfare jurisdictions increasingly place foster children with kinship foster parents as a means of meeting their need for stability, family connection, and behavioral and emotional support. However, the lack of financial and educational assistance provided to kin by child welfare authorities often undermines these caregivers' ability to provide effective and lasting care for the children in their homes. This study uses a mixed-methods approach to understand how formal training and licensure processes can aid kinship foster parents in facilitating positive outcomes for children and youth in the foster care system. Specifically, we investigated the barriers experienced by kinship foster parents while trying to access existing licensure-based training and supports, as well as the initial outcomes of a kin-tailored licensure training curriculum alternatingly administered in in-person and virtual delivery formats. Participants reported that incomplete or inaccurate communication about licensing processes, practical difficulties in attending training, irrelevant session content, and stringent licensing requirements acted as barriers to accessing these resources. However, participants in the kin-specific licensure training administered in this study reported high levels of learning related to key parenting competencies and increased awareness of kinship permanency supports, although these outcomes appeared to be less pronounced among those receiving the training in a virtual format. These findings suggest that researchers and policymakers should consider developing, implementing, and evaluating further initiatives to provide accessible and tailored supports to kinship foster parents as a means of improving outcomes for the children in their care.

A stable start: cotranslational Nt-acetylation promotes proteome stability across kingdoms.

Two recent studies show that cotranslational N-terminal protein acetylation (NTA) promotes proteome stability in humans (Mueller et al.) and plants (Linster et al.) by masking nonacetylated N-degrons that would otherwise destabilise proteins. This is in contrast to previous findings linking NTA to degradation, suggesting that this widespread mark has complex and context-specific functions in regulating protein half-lives.

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.

Network analysis of Arabidopsis mitochondrial dynamics reveals a resolved tradeoff between physical distribution and social connectivity.

Mitochondria in plant cells exist largely as individual organelles which move, colocalize, and interact, but the cellular priorities addressed by these dynamics remain incompletely understood. Here, we elucidate these principles by studying the dynamic "social networks" of mitochondria in Arabidopsis thaliana wildtype and mutants, describing the colocalization of individuals over time. We combine single-cell live imaging of hypocotyl mitochondrial dynamics with individual-based modeling and network analysis. We identify an inevitable tradeoff between mitochondrial physical priorities (an even cellular distribution of mitochondria) and "social" priorities (individuals interacting, to facilitate the exchange of chemicals and information). This tradeoff results in a tension between maintaining mitochondrial spacing and facilitating colocalization. We find that plant cells resolve this tension to favor efficient networks with high potential for exchanging contents. We suggest that this combination of physical modeling coupled to experimental data through network analysis can shed light on the fundamental principles underlying these complex organelle dynamics. A record of this paper's transparent peer review process is included in the supplemental information.

The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation.

Chloroplast function requires the coordinated action of nuclear- and chloroplast-derived proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) proteins that regulate plastid mRNA metabolism. Despite their large number and importance, regulatory mechanisms controlling PPR expression are poorly understood. Here we show that the Arabidopsis NOT4A ubiquitin-ligase positively regulates the expression of PROTON GRADIENT REGULATION 3 (PGR3), a PPR protein required for translating several thylakoid-localised photosynthetic components and ribosome subunits within chloroplasts. Loss of NOT4A function leads to a strong depletion of cytochrome b6f and NAD(P)H dehydrogenase (NDH) complexes, as well as plastid 30 S ribosomes, which reduces mRNA translation and photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. Quantitative transcriptome and proteome analysis of the not4a mutant reveal it lacks PGR3 expression, and that its molecular defects resemble those of a pgr3 mutant. Furthermore, we show that normal plastid function is restored to not4a through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring robust photosynthetic function during development and stress-response, through promoting PGR3 production and chloroplast translation.

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.

Disruption of the Nα-Acetyltransferase NatB Causes Sensitivity to Reductive Stress in Arabidopsis thaliana.

In Arabidopsis thaliana, the evolutionary conserved N-terminal acetyltransferase (Nat) complexes NatA and NatB co-translationally acetylate 60% of the proteome. Both have recently been implicated in the regulation of plant stress responses. While NatA mediates drought tolerance, NatB is required for pathogen resistance and the adaptation to high salinity and high osmolarity. Salt and osmotic stress impair protein folding and result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER-membrane resident E3 ubiquitin ligase DOA10 targets misfolded proteins for degradation during ER stress and is conserved among eukaryotes. In yeast, DOA10 recognizes conditional degradation signals (Ac/N-degrons) created by NatA and NatB. Assuming that this mechanism is preserved in plants, the lack of Ac/N-degrons required for efficient removal of misfolded proteins might explain the sensitivity of NatB mutants to protein harming conditions. In this study, we investigate the response of NatB mutants to dithiothreitol (DTT) and tunicamycin (TM)-induced ER stress. We report that NatB mutants are hypersensitive to DTT but not TM, suggesting that the DTT hypersensitivity is caused by an over-reduction of the cytosol rather than an accumulation of unfolded proteins in the ER. In line with this hypothesis, the cytosol of NatB depleted plants is constitutively over-reduced and a global transcriptome analysis reveals that their reductive stress response is permanently activated. Moreover, we demonstrate that doa10 mutants are susceptible to neither DTT nor TM, ruling out a substantial role of DOA10 in ER-associated protein degradation (ERAD) in plants. Contrary to previous findings in yeast, our data indicate that N-terminal acetylation (NTA) does not inhibit ER targeting of a substantial amount of proteins in plants. In summary, we provide further evidence that NatB-mediated imprinting of the proteome is vital for the response to protein harming stress and rule out DOA10 as the sole recognin for substrates in the plant ERAD pathway, leaving the role of DOA10 in plants ambiguous.

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.

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.

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.

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.

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.

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.

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.