The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype.

BACKGROUND: Plant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens. RESULTS: Here, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000. CONCLUSION: We demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses.

Hybrid allele-specific ChIP-seq analysis identifies variation in brassinosteroid-responsive transcription factor binding linked to traits in maize.

BACKGROUND: Genetic variation in regulatory sequences that alter transcription factor (TF) binding is a major cause of phenotypic diversity. Brassinosteroid is a growth hormone that has major effects on plant phenotypes. Genetic variation in brassinosteroid-responsive cis-elements likely contributes to trait variation. Pinpointing such regulatory variations and quantitative genomic analysis of the variation in TF-target binding, however, remains challenging. How variation in transcriptional targets of signaling pathways such as the brassinosteroid pathway contributes to phenotypic variation is an important question to be investigated with innovative approaches. RESULTS: Here, we use a hybrid allele-specific chromatin binding sequencing (HASCh-seq) approach and identify variations in target binding of the brassinosteroid-responsive TF ZmBZR1 in maize. HASCh-seq in the B73xMo17 F1s identifies thousands of target genes of ZmBZR1. Allele-specific ZmBZR1 binding (ASB) has been observed for 18.3% of target genes and is enriched in promoter and enhancer regions. About a quarter of the ASB sites correlate with sequence variation in BZR1-binding motifs and another quarter correlate with haplotype-specific DNA methylation, suggesting that both genetic and epigenetic variations contribute to the high level of variation in ZmBZR1 occupancy. Comparison with GWAS data shows linkage of hundreds of ASB loci to important yield and disease-related traits. CONCLUSION: Our study provides a robust method for analyzing genome-wide variations of TF occupancy and identifies genetic and epigenetic variations of the brassinosteroid response transcription network in maize.

Dissection of Developmental Programs and Regulatory Modules Directing Endosperm Transfer Cell and Aleurone Identity in the Syncytial Endosperm of Barley.

Endosperm development in barley starts with the formation of a multinucleate syncytium, followed by cellularization in the ventral part of the syncytium generating endosperm transfer cells (ETCs) as first differentiating subdomain, whereas aleurone (AL) cells will originate from the periphery of the enclosing syncytium. Positional signaling in the syncytial stage determines cell identity in the cereal endosperm. Here, we performed a morphological analysis and employed laser capture microdissection (LCM)-based RNA-seq of the ETC region and the peripheral syncytium at the onset of cellularization to dissect developmental and regulatory programs directing cell specification in the early endosperm. Transcriptome data revealed domain-specific characteristics and identified two-component signaling (TCS) and hormone activities (auxin, ABA, ethylene) with associated transcription factors (TFs) as the main regulatory links for ETC specification. On the contrary, differential hormone signaling (canonical auxin, gibberellins, cytokinin) and interacting TFs control the duration of the syncytial phase and timing of cellularization of AL initials. Domain-specific expression of candidate genes was validated by in situ hybridization and putative protein-protein interactions were confirmed by split-YFP assays. This is the first transcriptome analysis dissecting syncytial subdomains of cereal seeds and provides an essential framework for initial endosperm differentiation in barley, which is likely also valuable for comparative studies with other cereal crops.

Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana.

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

An antisense noncoding RNA enhances translation via localized structural rearrangements of its cognate mRNA.

A large portion of eukaryotic genes are associated with noncoding, natural antisense transcripts (NATs). Despite sharing extensive sequence complementarity with their sense mRNAs, mRNA-NAT pairs elusively often evade dsRNA-cleavage and siRNA-triggered silencing. More surprisingly, some NATs enhance translation of their sense mRNAs by yet unknown mechanism(s). Here, we show that translation enhancement of the rice (Oryza sativa) PHOSPHATE1.2 (PHO1.2) mRNA is enabled by specific structural rearrangements guided by its noncoding antisense RNA (cis-NATpho1.2). Their interaction in vitro revealed no evidence of widespread intermolecular dsRNA formation, but rather specific local changes in nucleotide base pairing, leading to higher flexibility of PHO1.2 mRNA at a key high guanine-cytosine�(GC) regulatory region inhibiting translation, ∼350-nt downstream of the start codon. Sense-antisense RNA interaction increased formation of the 80S complex in PHO1.2, possibly by inducing structural rearrangement within this inhibitory region, thus making this mRNA more accessible to 60S. This work presents a framework for nucleotide resolution studies of functional mRNA-antisense pairs.

Redox and low-oxygen stress: signal integration and interplay.

Plants are aerobic organisms relying on oxygen to serve their energy needs. The amount of oxygen available to sustain plant growth can vary significantly due to environmental constraints or developmental programs. In particular, flooding stress, which negatively impacts crop productivity, is characterized by a decline in oxygen availability. Oxygen fluctuations result in an altered redox balance and the formation of reactive oxygen/nitrogen species (ROS/RNS) during the onset of hypoxia and upon re-oxygenation. In this update, we provide an overview of the current understanding of the impact of redox and ROS/RNS on low-oxygen signaling and adaptation. We first focus on the formation of ROS and RNS during low-oxygen conditions. Following this, we examine the impact of hypoxia on cellular and organellar redox systems. Finally, we describe how redox and ROS/RNS participate in signaling events during hypoxia through potential post-translational modifications (PTMs) of hypoxia-relevant proteins. The aim of this update is to define our current understanding of the field and to provide avenues for future research directions.

On the move: redox-dependent protein relocation in plants.

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.

Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport.

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli.

HBI1 Mediates the Trade-off between Growth and Immunity through Its Impact on Apoplastic ROS Homeostasis.

Plants continuously need to adapt to their environment and prioritize either growth or defense responses to secure survival and reproduction. Trade-offs between growth and defense are often attributed to the allocation of energy for growth to adaptation responses. Still, the exact mechanisms underlying growth and defense trade-offs are poorly understood. Here, we demonstrate that the growth-related transcription factor HOMOLOG OF BEE2 INTERACTING WITH IBH 1 (HBI1) regulates apoplastic reactive oxygen species (ROS) homeostasis by differentially controlling the expression of NADPH oxidases (NOXs) and peroxidases (POXs). The HBI1 target genes RESPIRATORY BURST OXIDASE HOMOLOG A (RbohA) and RbohC have contrasting effects on the regulation of cell size. In addition, the HBI1-controlled NOXs and POXs oppositely regulate susceptibility toward Pseudomonas syringae. Our findings reveal that the incompatibility between growth and defense programs can be attributed to the way apoplastic ROS homeostasis is modulated during both processes.

The Role and Regulation of Autophagy and the Proteasome During Aging and Senescence in Plants.

Aging and senescence in plants has a major impact on agriculture, such as in crop yield, the value of ornamental crops, and the shelf life of vegetables and fruits. Senescence represents the final developmental phase of the leaf and inevitably results in the death of the organ. Still, the process is completely under the control of the plant. Plants use their protein degradation systems to maintain proteostasis and transport or salvage nutrients from senescing organs to develop reproductive parts. Herein, we present an overview of current knowledge about the main protein degradation pathways in plants during senescence: The proteasome and autophagy. Although both pathways degrade proteins, autophagy appears to prevent aging, while the proteasome functions as a positive regulator of senescence.

Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in Arabidopsis.

Plant response to environmental stimuli involves integration of multiple signals. Upon low-oxygen stress, plants initiate a set of adaptive responses to circumvent an energy crisis. Here, we reveal how these stress responses are induced by combining (i) energy-dependent changes in the composition of the acyl-CoA pool and (ii) the cellular oxygen concentration. A hypoxia-induced decline of cellular ATP levels reduces LONG-CHAIN ACYL-COA SYNTHETASE activity, which leads to a shift in the composition of the acyl-CoA pool. Subsequently, we show that different acyl-CoAs induce unique molecular responses. Altogether, our data disclose a role for acyl-CoAs acting in a cellular signaling pathway in plants. Upon hypoxia, high oleoyl-CoA levels provide the initial trigger to release the transcription factor RAP2.12 from its interaction partner ACYL-COA BINDING PROTEIN at the plasma membrane. Subsequently, according to the N-end rule for proteasomal degradation, oxygen concentration-dependent stabilization of the subgroup VII ETHYLENE-RESPONSE FACTOR transcription factor RAP2.12 determines the level of hypoxia-specific gene expression. This research unveils a specific mechanism activating low-oxygen stress responses only when a decrease in the oxygen concentration coincides with a drop in energy.

Phenotypic Analysis and Molecular Markers of Leaf Senescence.

The process of leaf senescence consists of the final stage of leaf development. It has evolved as a mechanism to degrade macromolecules and micronutrients and remobilize them to other developing parts of the plant; hence it plays a central role for the survival of plants and crop production. During senescence, a range of physiological, morphological, cellular, and molecular events occur, which are generally referred to as the senescence syndrome that includes several hallmarks such as visible yellowing, loss of chlorophyll and water content, increase of ion leakage and cell death, deformation of chloroplast and cell structure, as well as the upregulation of thousands of so-called senescence-associated genes (SAGs) and downregulation of photosynthesis-associated genes (PAGs). This chapter is devoted to methods characterizing the onset and progression of leaf senescence at the morphological, physiological, cellular, and molecular levels. Leaf senescence normally progresses in an age-dependent manner but is also induced prematurely by a variety of environmental stresses in plants. Focused on the hallmarks of the senescence syndrome, a series of protocols is described to asses quantitatively the senescence process caused by developmental cues or environmental perturbations. We first briefly describe the senescence process, the events associated with the senescence syndrome, and the theories and methods to phenotype senescence. Detailed protocols for monitoring senescence in planta and in vitro, using the whole plant and the detached leaf, respectively, are presented. For convenience, most of the protocols use the model plant species Arabidopsis and rice, but they can be easily extended to other plants.

Salt-Related MYB1 Coordinates Abscisic Acid Biosynthesis and Signaling during Salt Stress in Arabidopsis.

Abiotic stresses, such as salinity, cause global yield loss of all major crop plants. Factors and mechanisms that can aid in plant breeding for salt stress tolerance are therefore of great importance for food and feed production. Here, we identified a MYB-like transcription factor, Salt-Related MYB1 (SRM1), that negatively affects Arabidopsis (Arabidopsis thaliana) seed germination under saline conditions by regulating the levels of the stress hormone abscisic acid (ABA). Accordingly, several ABA biosynthesis and signaling genes act directly downstream of SRM1, including SALT TOLERANT1/NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, RESPONSIVE TO DESICCATION26, and Arabidopsis NAC DOMAIN CONTAINING PROTEIN19. Furthermore, SRM1 impacts vegetative growth and leaf shape. We show that SRM1 is an important transcriptional regulator that directly targets ABA biosynthesis and signaling-related genes and therefore may be regarded as an important regulator of ABA-mediated salt stress tolerance.

Living to Die and Dying to Live: The Survival Strategy behind Leaf Senescence.

Senescence represents the final developmental act of the leaf, during which the leaf cell is dismantled in a coordinated manner to remobilize nutrients and to secure reproductive success. The process of senescence provides the plant with phenotypic plasticity to help it adapt to adverse environmental conditions. Here, we provide a comprehensive overview of the factors and mechanisms that control the onset of senescence. We explain how the competence to senesce is established during leaf development, as depicted by the senescence window model. We also discuss the mechanisms by which phytohormones and environmental stresses control senescence as well as the impact of source-sink relationships on plant yield and stress tolerance. In addition, we discuss the role of senescence as a strategy for stress adaptation and how crop production and food quality could benefit from engineering or breeding crops with altered onset of senescence.

Setting the PAS, the role of circadian PAS domain proteins during environmental adaptation in plants.

The per-ARNT-sim (PAS) domain represents an ancient protein module that can be found across all kingdoms of life. The domain functions as a sensing unit for a diverse array of signals, including molecular oxygen, small metabolites, and light. In plants, several PAS domain-containing proteins form an integral part of the circadian clock and regulate responses to environmental change. Moreover, these proteins function in pathways that control development and plant stress adaptation responses. Here, we discuss the role of PAS domain-containing proteins in anticipation, and adaptation to environmental changes in plants.

Transcriptional networks in leaf senescence.

Plant senescence is a natural phenomenon known for the appearance of beautiful autumn colors and the ripening of cereals in the field. Senescence is a controlled process that plants utilize to remobilize nutrients from source leaves to developing tissues. While during the past decades, molecular components underlying the onset of senescence have been intensively studied, knowledge remains scarce on the age-dependent mechanisms that control the onset of senescence. Recent advances have uncovered transcriptional networks regulating the competence to senesce. Here, gene regulatory networks acting as internal timing mechanisms for the onset of senescence are highlighted, illustrating that early and late leaf developmental phases are highly connected.

ROS-mediated redox signaling during cell differentiation in plants.

BACKGROUND: Reactive oxygen species (ROS) have emerged in recent years as important regulators of cell division and differentiation. SCOPE OF REVIEW: The cellular redox state has a major impact on cell fate and multicellular organism development. However, the exact molecular mechanisms through which ROS manifest their regulation over cellular development are only starting to be understood in plants. ROS levels are constantly monitored and any change in the redox pool is rapidly sensed and responded upon. Different types of ROS cause specific oxidative modifications, providing the basic characteristics of a signaling molecule. Here we provide an overview of ROS sensors and signaling cascades that regulate transcriptional responses in plants to guide cellular differentiation and organ development. MAJOR CONCLUSIONS: Although several redox sensors and cascades have been identified, they represent only a first glimpse on the impact that redox signaling has on plant development and growth. GENERAL SIGNIFICANCE: We provide an initial evaluation of ROS signaling cascades involved in cell differentiation in plants and identify potential avenues for future studies. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

A cell type-specific view on the translation of mRNAs from ROS-responsive genes upon paraquat treatment of Arabidopsis thaliana leaves.

Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5'-untranslated region (5'-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.

Transcriptional control of ROS homeostasis by KUODA1 regulates cell expansion during leaf development.

The final size of an organism, or of single organs within an organism, depends on an intricate coordination of cell proliferation and cell expansion. Although organism size is of fundamental importance, the molecular and genetic mechanisms that control it remain far from understood. Here we identify a transcription factor, KUODA1 (KUA1), which specifically controls cell expansion during leaf development in Arabidopsis thaliana. We show that KUA1 expression is circadian regulated and depends on an intact clock. Furthermore, KUA1 directly represses the expression of a set of genes encoding for peroxidases that control reactive oxygen species (ROS) homeostasis in the apoplast. Disruption of KUA1 results in increased peroxidase activity and smaller leaf cells. Chemical or genetic interference with the ROS balance or peroxidase activity affects cell size in a manner consistent with the identified KUA1 function. Thus, KUA1 modulates leaf cell expansion and final organ size by controlling ROS homeostasis.