BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition.

BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5-MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signalling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal (Nt) residue. We crossed a big loss-of-function allele to two N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2) was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and broader processes in Arabidopsis.

Transcriptional analysis in multiple barley varieties identifies signatures of waterlogging response.

Waterlogging leads to major crop losses globally, particularly for waterlogging-sensitive crops such as barley. Waterlogging reduces oxygen availability and results in additional stresses, leading to the activation of hypoxia and stress response pathways that promote plant survival. Although certain barley varieties have been shown to be more tolerant to waterlogging than others and some tolerance-related quantitative trait loci have been identified, the molecular mechanisms underlying this trait are mostly unknown. Transcriptomics approaches can provide very valuable information for our understanding of waterlogging tolerance. Here, we surveyed 21 barley varieties for the differential transcriptional activation of conserved hypoxia-response genes under waterlogging and selected five varieties with different levels of induction of core hypoxia-response genes. We further characterized their phenotypic response to waterlogging in terms of shoot and root traits. RNA sequencing to evaluate the genome-wide transcriptional responses to waterlogging of these selected varieties led to the identification of a set of 98 waterlogging-response genes common to the different datasets. Many of these genes are orthologs of the so-called "core hypoxia response genes," thus highlighting the conservation of plant responses to waterlogging. Hierarchical clustering analysis also identified groups of genes with intrinsic differential expression between varieties prior to waterlogging stress. These genes could constitute interesting candidates to study "predisposition" to waterlogging tolerance or sensitivity in barley.

Complexity of Abiotic Stress Stimuli: Mimicking Hypoxic Conditions Experimentally on the Basis of Naturally Occurring Environments.

Plants require oxygen to respire and produce energy. Plant cells are exposed to low oxygen levels (hypoxia) in different contexts and have evolved conserved molecular responses to hypoxia. Both environmental and developmental factors can influence intracellular oxygen concentrations. In nature, plants can experience hypoxic conditions when the soil becomes saturated with water following heavy precipitation (i.e., waterlogging). Hypoxia can also arise in specific tissues that have poor gas exchange with atmospheric oxygen. In this case, hypoxic niches that are physiologically and developmentally relevant may form. To dissect the molecular mechanisms underlying the regulation of hypoxia response in plants, a wide range of hypoxia-inducing methods have been used in the laboratory setting. Yet, the different characteristics, pros and cons of each of these hypoxia treatments are seldom compared between methods, and with natural forms of hypoxia. In this chapter, we present both environmental and developmental forms of hypoxia that plants encounter in the wild, as well as the different experimental hypoxia treatments used to mimic them in the laboratory setting, with the aim of informing on what experimental approaches might be most appropriate to the questions addressed, including stress signaling and regulation.

Lessons from Comparison of Hypoxia Signaling in Plants and Mammals.

Hypoxia is an important stress for organisms, including plants and mammals. In plants, hypoxia can be the consequence of flooding and causes important crop losses worldwide. In mammals, hypoxia stress may be the result of pathological conditions. Understanding the regulation of responses to hypoxia offers insights into novel approaches for crop improvement, particularly for the development of flooding-tolerant crops and for producing better therapeutics for hypoxia-related diseases such as inflammation and cancer. Despite their evolutionary distance, plants and mammals deploy strikingly similar mechanisms to sense and respond to the different aspects of hypoxia-related stress, including low oxygen levels and the resulting energy crisis, nutrient depletion, and oxidative stress. Over the last two decades, the ubiquitin/proteasome system and the ubiquitin-like protein SUMO have been identified as key regulators that act in concert to regulate core aspects of responses to hypoxia in plants and mammals. Here, we review ubiquitin and SUMO-dependent mechanisms underlying the regulation of hypoxia response in plants and mammals. By comparing and contrasting these mechanisms in plants and mammals, this review seeks to pinpoint conceptually similar mechanisms but also highlight future avenues of research at the junction between different fields of research.

Experimental comparison of two methods to study barley responses to partial submergence.

BACKGROUND: Crop yield is dependent on climate conditions, which are becoming both more variable and extreme in some areas of the world as a consequence of global climate change. Increased precipitation and flooding events are the cause of important yield losses due to waterlogging or (partial) submergence of crops in the field. Our ability to screen efficiently and quickly for varieties that have increased tolerance to waterlogging or (partial) submergence is important. Barley, a staple crop worldwide, is particularly sensitive to waterlogging. Screening for waterlogging tolerant barley varieties has been ongoing for many years, but methods used to screen vary greatly, from the type of soil used to the time at which the treatment is applied. This variation makes it difficult to cross-compare results. RESULTS: Here, we have devised a scoring system to assess barley tolerance to waterlogging and compare two different methods when partial submergence is applied with either water or a starch solution at an early developmental stage, which is particularly sensitive to waterlogging or partial submergence. The use of a starch solution has been previously shown to result in more reducing soil conditions and has been used to screen for waterlogging tolerance. CONCLUSIONS: Our results show that the two methods provide similar results to qualitatively rank varieties as tolerant or sensitive, while also affecting plants differently, in that application of a starch solution results in stronger and earlier symptoms than applying partial submergence with water.

Cutting the line: manipulation of plant immunity by bacterial type III effector proteases.

Pathogens and their hosts are engaged in an evolutionary arms race. Pathogen-derived effectors promote virulence by targeting components of a host's innate immune system, while hosts have evolved proteins that sense effectors and trigger a pathogen-specific immune response. Many bacterial effectors are translocated into host cells using type III secretion systems. Type III effector proteases irreversibly modify host proteins by cleavage of peptide bonds and are prevalent among both plant and animal bacterial pathogens. In plants, the study of model effector proteases has yielded important insights into the virulence mechanisms employed by pathogens to overcome their host's immune response, as well as into the mechanisms deployed by their hosts to detect these effector proteases and counteract their effects. In recent years, the study of a larger number of effector proteases, across a wider range of pathogens, has yielded novel insights into their functions and recognition. One key limitation that remains is the lack of methods to detect protease cleavage at the proteome-wide level. We review known substrates and mechanisms of plant pathogen type III effector proteases and compare their functions with those of known type III effector proteases of mammalian pathogens. Finally, we discuss approaches to uncover their function on a system-wide level.

A simple and efficient Agrobacterium-mediated transient expression system to dissect molecular processes in Brassica rapa and Brassica napus.

The family Brassicaceae is a source of important crop species, including Brassica napus (oilseed rape), Brassica oleracea, and B. rapa, that is used globally for oil production or as a food source (e.g., pak choi or turnip). However, despite advances in recent years, including genome sequencing, a lack of established tools tailored to the study of Brassica crop species has impeded efforts to understand their molecular processes in greater detail. Here, we describe the use of a simple Agrobacterium-mediated transient expression system adapted to B. rapa and B. napus that could facilitate study of molecular and biochemical events in these species. We also demonstrate the use of this method to characterize the N-degron pathway of protein degradation in B. rapa. The N-degron pathway is a subset of the ubiquitin-proteasome system and represents a mechanism through which proteins may be targeted for degradation based on the identity of their N-terminal amino acid residue. Interestingly, N-degron-mediated processes in plants have been implicated in the regulation of traits with potential agronomic importance, including the responses to pathogens and to abiotic stresses such as flooding tolerance. The stability of transiently expressed N-degron reporter proteins in B. rapa indicates that its N-degron pathway is highly conserved with that of Arabidopsis thaliana. These findings highlight the utility of Agrobacterium-mediated transient expression in B. rapa and B. napus and establish a framework to investigate the N-degron pathway and its roles in regulating agronomical traits in these species. SIGNIFICANCE STATEMENT: We describe an Agrobacterium-mediated transient expression system applicable to Brassica crops and demonstrate its utility by identifying the destabilizing residues of the N-degron pathway in B. rapa. As the N-degron pathway functions as an integrator of environmental signals, this study could facilitate efforts to improve the robustness of Brassica crops.

Differential N-end Rule Degradation of RIN4/NOI Fragments Generated by the AvrRpt2 Effector Protease.

In plants, the protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both pattern-triggered immunity and effector-triggered immunity. RIN4 is targeted by several effectors, including the Pseudomonas syringae protease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates potentially unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RESISTANT TO P. SYRINGAE2. Hence, identifying the determinants of RIN4 degradation is key to understanding RESISTANT TO P. SYRINGAE2-mediated effector-triggered immunity, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI domain proteins by AvrRpt2 may contribute to pattern-triggered immunity regulation, the (in)stability of these proteolytic fragments and the determinants regulating their stability remain unexamined. Notably, a common feature of RIN4, and of many NOI domain protein fragments generated by AvrRpt2 cleavage, is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed the N-degron pathway). By contrast, several NOI domain protein fragments are genuine substrates of the N-degron pathway. The discovery of this set of substrates considerably expands the number of known proteins targeted for degradation by this ubiquitin-dependent pathway in plants. These results advance our current understanding of the role of AvrRpt2 in promoting bacterial virulence.

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.

Ubiquitylation in plants: signaling hub for the integration of environmental signals.

A fundamental question in biology is how organisms integrate the plethora of environmental cues that they perceive to trigger a co-ordinated response. The regulation of protein stability, which is largely mediated by the ubiquitin-proteasome system in eukaryotes, plays a pivotal role in these processes. Due to their sessile lifestyle and the need to respond rapidly to a multitude of environmental factors, plants are thought to be especially dependent on proteolysis to regulate cellular processes. In this review, we present the complexity of the ubiquitin system in plants, and discuss the relevance of the proteolytic and non-proteolytic roles of this system in the regulation and co-ordination of plant responses to environmental signals. We also discuss the role of the ubiquitin system as a key regulator of plant signaling pathways. We focus more specifically on the functions of E3 ligases as regulators of the jasmonic acid (JA), salicylic acid (SA), and ethylene hormone signaling pathways that play important roles to mount a co-ordinated response to multiple environmental stresses. We also provide examples of new players in this field that appear to integrate different cues and signaling pathways.

Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway.

Contents Summary 929 I. INTRODUCTION: conservation and diversity of N-end rule pathways 929 II. Defensive functions of the N-end rule pathway in plants 930 III. Proteases and degradation by the N-end rule pathway 930 IV. New proteomics approaches for the identification of N-end rule substrates 932 V. Concluding remarks 932 Acknowledgements 934 References 934 SUMMARY: The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments.

Regulatory interplay between LEAFY, APETALA1/CAULIFLOWER and TERMINAL FLOWER1: New insights into an old relationship.

The gene regulatory network comprised of LEAFY (LFY), APETALA1 (AP1), the AP1 paralog CAULIFLOWER (CAL), and TERMINAL FLOWER1 (TFL1) is a major determinant of the flowering process in Arabidopsis thaliana. TFL1 activity in the shoot apical meristem provides inflorescence identity while the transcription factors LFY and AP1/CAL confer floral identity to emerging floral primordia. It has been thought that LFY and AP1/CAL control the onset of flowering in part by repressing TFL1 expression in flowers. However, in the June issue of Plant Physiology, we reported that LFY and AP1 act antagonistically in the regulation of several key flowering regulators, including TFL1. Specifically, TFL1 transcription was suppressed by AP1 but promoted by LFY. Here, we present additional evidence for the role of LFY as an activator of TFL1 and propose that this regulatory activity is pivotal for the indeterminate growth of the SAM during the reproductive phase of development.

Inducible Promoter Systems for Gene Perturbation Experiments in Arabidopsis.

Assessing molecular changes that occur through altering a gene's activity is often hampered by difficulties that arise due to the typically static nature of the introduced perturbation. This is especially problematic when investigating molecular events at specific stages and/or in certain tissues or organs during Arabidopsis development. To circumvent these issues, we have employed chemically inducible artificial microRNAs (amiRNAs) for the specific knockdown of developmental regulators. For our own research, we have combined this gene perturbation approach with a floral induction system, which allows the simultaneous induction of a large number of flowers on the inflorescence of a single plant, and the ability to knock down a gene's activity at any given stage of development. To enable the plant community to avail of the full benefits of these systems, we describe, in this chapter, strategies for amiRNA-mediated gene perturbations and address some common problems that can be encountered when generating inducible amiRNA constructs, growing these plants, and collecting floral buds for analysis.

Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation.

The transcription factors LEAFY (LFY) and APETALA1 (AP1), together with the AP1 paralog CAULIFLOWER (CAL), control the onset of flower development in a partially redundant manner. This redundancy is thought to be mediated, at least in part, through the regulation of a shared set of target genes. However, whether these genes are independently or cooperatively regulated by LFY and AP1/CAL is currently unknown. To better understand the regulatory relationship between LFY and AP1/CAL and to obtain deeper insights into the control of floral initiation, we monitored the activity of LFY in the absence of AP1/CAL function. We found that the regulation of several known LFY target genes is unaffected by AP1/CAL perturbation, while others appear to require AP1/CAL activity. Furthermore, we obtained evidence that LFY and AP1/CAL control the expression of some genes in an antagonistic manner. Notably, these include key regulators of floral initiation such as TERMINAL FLOWER1 (TFL1), which had been previously reported to be directly repressed by both LFY and AP1. We show here that TFL1 expression is suppressed by AP1 but promoted by LFY. We further demonstrate that LFY has an inhibitory effect on flower formation in the absence of AP1/CAL activity. We propose that LFY and AP1/CAL act as part of an incoherent feed-forward loop, a network motif where two interconnected pathways or transcription factors act in opposite directions on a target gene, to control the establishment of a stable developmental program for the formation of flowers.

The N-end rule pathway regulates pathogen responses in plants.

To efficiently counteract pathogens, plants rely on a complex set of immune responses that are tightly regulated to allow the timely activation, appropriate duration and adequate amplitude of defense programs. The coordination of the plant immune response is known to require the activity of the ubiquitin/proteasome system, which controls the stability of proteins in eukaryotes. Here, we demonstrate that the N-end rule pathway, a subset of the ubiquitin/proteasome system, regulates the defense against a wide range of bacterial and fungal pathogens in the model plant Arabidopsis thaliana. We show that this pathway positively regulates the biosynthesis of plant-defense metabolites such as glucosinolates, as well as the biosynthesis and response to the phytohormone jasmonic acid, which plays a key role in plant immunity. Our results also suggest that the arginylation branch of the N-end rule pathway regulates the timing and amplitude of the defense program against the model pathogen Pseudomonas syringae AvrRpm1.

Molecular and regulatory mechanisms controlling floral organ development.

The genetic and molecular mechanisms that underlie the formation of angiosperm flowers have been studied extensively for nearly three decades. This work has led to detailed insights into the gene regulatory networks that control this vital developmental process in plants. Here, we review some of the key findings in the field of flower development and discuss open questions that must be addressed in order to obtain a more comprehensive understanding of flower formation. In particular, we focus on the specification of the different types of floral organs and on how the morphogenesis of these organs is controlled to give rise to mature flowers. Central to this process are the floral organ identity genes, which encode members of the family of MADS-domain transcription factors. We summarize what is currently known about the functions of these master regulators and discuss a working model for the molecular mechanism that may underlie their activities.

Patterns of gene expression during Arabidopsis flower development from the time of initiation to maturation.

BACKGROUND: The formation of flowers is one of the main model systems to elucidate the molecular mechanisms that control developmental processes in plants. Although several studies have explored gene expression during flower development in the model plant Arabidopsis thaliana on a genome-wide scale, a continuous series of expression data from the earliest floral stages until maturation has been lacking. Here, we used a floral induction system to close this information gap and to generate a reference dataset for stage-specific gene expression during flower formation. RESULTS: Using a floral induction system, we collected floral buds at 14 different stages from the time of initiation until maturation. Using whole-genome microarray analysis, we identified 7,405 genes that exhibit rapid expression changes during flower development. These genes comprise many known floral regulators and we found that the expression profiles for these regulators match their known expression patterns, thus validating the dataset. We analyzed groups of co-expressed genes for over-represented cellular and developmental functions through Gene Ontology analysis and found that they could be assigned specific patterns of activities, which are in agreement with the progression of flower development. Furthermore, by mapping binding sites of floral organ identity factors onto our dataset, we were able to identify gene groups that are likely predominantly under control of these transcriptional regulators. We further found that the distribution of paralogs among groups of co-expressed genes varies considerably, with genes expressed predominantly at early and intermediate stages of flower development showing the highest proportion of such genes. CONCLUSIONS: Our results highlight and describe the dynamic expression changes undergone by a large number of genes during flower development. They further provide a comprehensive reference dataset for temporal gene expression during flower formation and we demonstrate that it can be used to integrate data from other genomics approaches such as genome-wide localization studies of transcription factor binding sites.

Gene network analysis of Arabidopsis thaliana flower development through dynamic gene perturbations.

Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome-wide experiments with stage-specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development.

Flower development: open questions and future directions.

Almost three decades of genetic and molecular analyses have resulted in detailed insights into many of the processes that take place during flower development and in the identification of a large number of key regulatory genes that control these processes. Despite this impressive progress, many questions about how flower development is controlled in different angiosperm species remain unanswered. In this chapter, we discuss some of these open questions and the experimental strategies with which they could be addressed. Specifically, we focus on the areas of floral meristem development and patterning, floral organ specification and differentiation, as well as on the molecular mechanisms underlying the evolutionary changes that have led to the astounding variations in flower size and architecture among extant and extinct angiosperms.