RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 and the stress-induced morphogenetic response

Plants exposed to abiotic stress show a range of morphogenetic responses, sometimes termed the stress-induced morphogenetic response (SIMR). SIMR is principally composed of three components: inhibition of cell elongation, alterations in cell differentiation, and stimulus of cell division in localized areas. An explanation proposed for SIMR has been increased accumulation of reactive oxygen species (ROS) and alterations in hormone signaling. Mutations in the Arabidopsis thaliana RADICAL-INDUCED CELL DEATH1 (RCD1) gene have altered abiotic stress responses and ROS accumulation. Even in the absence of exogenous stress, these plants show many morphological changes also seen in SIMR. In the September issue of Plant Physiology we reported an in depth analysis of the phenotype of rcd1-3 plants as well as the phenotype of a mutations in the previously uncharacterized paralog of RCD1, SIMILAR TO RCD ONE1 (SRO1). sro1-1 plants have mild morphological changes and abiotic stress response defects while rcd1-3; sro1-1 double mutant plants have severe developmental defects, including less cell elongation. In this Addendum, we hypothesize that rcd1, sro1 and rcd1; sro1 mutant plants are under constitutive stress, and that this stress is responsible for at least some of the developmental defects seen in these plants.Key words: RCD1, SRO1, Arabidopsis thaliana, reactive oxygen species, stress-induced morphogenetic response, PARPPlants as sessile organisms cannot move upon environmental change. Therefore, plants have evolved a diverse repertoire of responses in order to lower stress exposure, limit the damage caused or repair such damage. Chronic mild stress, from a variety of abiotic stresses, can cause a morphogenetic response that has been termed the stress-induced morphogenetic response (SIMR).¹ This response involves growth inhibition through suppression of cell elongation, changes in cell differentiation status, and localized stimulation of cell division. Typical SIMR responses include decreased elongation of the primary root accompanied by increased formation of lateral roots, decreased stem height, decreased leaf area and increased branching. Importantly, plants do not cease growth, rather they redistribute the areas undergoing active growth.Although the molecular and cellular network underlying SIMR has not been completely worked out, several key elements have been identified.² The importance of the phytohormone auxin in morphogenetic changes seen in SIMR has been noted by several groups. Changes in auxin distribution and metabolism are induced by many stresses and correlates well with phenotypes induced by stress, such as increased lateral root growth, suggesting that changes in auxin signaling may be a causative agent in SIMR. Furthermore, reactive oxygen species (ROS) are known to accumulate in plants upon stress of many types, especially those that have been linked with SIMR. Once ROS accumulates, plants upregulate ROS scavenging systems, which can subsequently provide protection against a range of further environmental assaults. Extensive interactions between the auxin signaling pathway and ROS have been documented, suggesting that these two pathways may act in concert during SIMR.Mutations in the Arabidopsis thaliana gene RADICAL-INDUCED CELL DEATH1 (RCD1) were originally isolated in a screen for plants hypersensitive to ozone.³ This gene encodes a putative poly(ADP-ribose) polymerase (PARP).⁴ PARPs attach ADP-ribose subunits from NAD⁺ to proteins post-translationally and are found across the eukaryotes. Although members of this enzyme family share the PARP catalytic domain, other regions of the proteins can vary dramatically, reflecting the diversity of functions these proteins have acquired. RCD1 belongs to a group of PARPs found only in land plants (Citarelli, Teotia S and Lamb RS, submitted) and contains a WWE domain N-terminal to the PARP catalytic domain. RCD1 has been shown to have complex roles in abiotic stress and development. rcd1 mutants are known to accumulate, even under non-inducing conditions, ROS³ and nitric oxide,⁵ suggesting that it normally works, directly or indirectly, to negatively regulate the accumulation of these compounds. Further evidence that rcd1 plants may be under stress include upregulation in the mutant of AOX1a and UPOX, two markers of oxidative stress.⁶ Complicating any interpretation of defects seen in rcd1 single mutants is the fact that, in addition to RCD1, Arabidopsis also encodes a paralog, SIMILAR TO RCD ONE1 (SRO1).In our recent publication,⁷ we describe in detail phenotypes of mutations in RCD1 and SRO1 and double mutants between the two. The developmental defects seen in the single mutants are similar to those associated with SIMR, although defects in RCD1 generally cause more severe defects. Both rcd1-3 and sro1-1 plants have an increased number of lateral roots (increase in local cell division and redirected growth), while rcd1-3 plants also have shorter primary roots. rcd1-3 plants are shorter with smaller leaves (growth inhibition). Examination of double mutant plants further support the hypothesis that many phenotypes seen when these genes are malfunctioning are due to deregulated SIMR. Most rcd1-3; sro1-1 plants die during embryogenesis; however, those that survive have severe defects. These plants are extremely short, due, at least in part, to reduced cell elongation in the stem. The leaves are small for similar reasons. In addition, these plants are bushy due to arrest of the shoot apical meristem and activation of axillary meristems. All of these phenotypes are extreme examples of phenotypes seen in plants under stress from a variety of sources, including UV-B, heavy metals and salt.¹In order to determine if rcd1-3; sro1-1 seedlings grown under normal conditions are under stress, we examined molecular markers of stress in this background. The small ubiquitin-like modifier (SUMO) is a ubiquitin-like polypeptide attached covalently to proteins. In Arabidopsis it has been demonstrated that sumoylated proteins accumulate under a variety of abiotic stresses such as heat shock and H₂O₂.⁸ We examined the accumulation of SUMO-modified proteins in rcd1-3; sro1-1 seedlings in comparison to wild type and two mutant backgrounds (nuclear pore anchor (nua)-1 and -2) in which such proteins have previously been shown to accumulate (Fig. 1A; western done according to⁹). The double mutant seedlings accumulate more sumoylated proteins, not only in comparison to wild type but also in comparison to the nua mutants. The accumulation of modified proteins supports the hypothesis that rcd1-3; sro1-1 seedlings are exhibiting constitutive stress. The expression of PARP2, which encodes a so-called classical PARP enzyme involved in DNA repair,¹⁰ has been shown to go up under a number of stress conditions.^11–15 We used RT-PCR to examine expression of this gene in our mutant backgrounds. PARP2 expression is increased in rcd1-3; sro1-1 seedlings and may also be higher than wild type in rcd1-3 and sro1-1 single mutants (Fig. 1B). This further supports our contention that loss of function in RCD1 and SRO1 results in constitutive stress and morphogenetic defects similar to those seen in SIMR.Open in a separate windowFigure 1rcd1-3; sro1-1 plants are under constitutive stress. (A) rcd1-3; sro1-1 seedlings accumulate sumoylated proteins. The upper panel shows a western blot with anti-SUM O antibody according to Xu et al.⁹ Asterisk indicates sumoylated proteins, while the lower bands are free SUM O. The lower panel shows a coomassie-stained gel showing total protein as a loading control. Lane 1, wild type; lane 2, rcd1-3; sro1-1; lane 3, nua-1; lane 4, nua-2. (B) Expression of the stress-inducible gene PARP2 is increased in rcd1-3; sro1-1 seedlings. RT -PCR done from two biological replicates with RNA extracted from seedlings is shown. Primers used to amplify PARP2 were as follows: PAR P2RT F (GCA AGC CCA CAT AA G CC G TGG AGG) and PAR P2RTR (TGC CT G CTC TT G AAT TT G TTT AC G TGC). Actin expression was used as a control; primers as in Teotia and Lamb.⁷ Lane 1, wild type; lane 2, rcd1-3; lane 3, sro1-1; lane 4, rcd1-3; sro1-1.In conclusion, we hypothesize that RCD1 and SRO1 are negative regulators of ROS and/or nitric oxide. When their function is compromised, these compounds accumulate, even in the absence of stress conditions. This causes the plant to develop as if under constitutive abiotic stress, leading to a SIMR phenotype even under ideal growth conditions. Further experimentation will be required to test this hypothesis.     

Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses

Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.     

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast‐derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo‐oxidative stress and display EDS1‐dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1‐regulated SA and ROS by examining gene expression profiles, photo‐oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA‐biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast‐derived O₂^?? that lead to SA‐assisted H₂O₂ accumulation as part of a mechanism limiting cell death. A combination of EDS1‐regulated SA‐antagonized and SA‐promoted processes is necessary for resistance to host‐adapted pathogens and for a balanced response to photo‐oxidative stress. In contrast to SA, the apoplastic ROS‐producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo‐oxidative stress. Thus, chloroplastic O₂^?? signals are processed by EDS1 to produce counter‐balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O₂^?? or O₂^??‐generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.     

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.     

Chloroplast Signaling and LESION SIMULATING DISEASE1 Regulate Crosstalk between Light Acclimation and Immunity in Arabidopsis

Plants are simultaneously exposed to abiotic and biotic hazards. Here, we show that local and systemic acclimation in Arabidopsis thaliana leaves in response to excess excitation energy (EEE) is associated with cell death and is regulated by specific redox changes of the plastoquinone (PQ) pool. These redox changes cause a rapid decrease of stomatal conductance, global induction of ASCORBATE PEROXIDASE2 and PATHOGEN RESISTANCE1, and increased production of reactive oxygen species (ROS) and ethylene that signals through ETHYLENE INSENSITIVE2 (EIN2). We provide evidence that multiple hormonal/ROS signaling pathways regulate the plant''s response to EEE and that EEE stimulates systemic acquired resistance and basal defenses to virulent biotrophic bacteria. In the Arabidopsis LESION SIMULATING DISEASE1 (lsd1) null mutant that is deregulated for EEE acclimation responses, propagation of EEE-induced programmed cell death depends on the plant defense regulators ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We find that EDS1 and PAD4 operate upstream of ethylene and ROS production in the EEE response. The data suggest that the balanced activities of LSD1, EDS1, PAD4, and EIN2 regulate signaling of programmed cell death, light acclimation, and holistic defense responses that are initiated, at least in part, by redox changes of the PQ pool.     

Analysis of Putative Apoplastic Effectors from the Nematode,Globodera rostochiensis,and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.     

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial‐associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti‐microbial secondary metabolites. Mitogen‐activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor‐induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4^DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4^DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4^DD‐induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP‐triggered defense responses.     

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.     

Populus cathayana males are less affected than females by excess manganese: Comparative proteomic and physiological analyses

The comprehension of sexually different responses in dioecious plants to excess manganese (Mn) stress requires molecular explanation. Physiological and proteomic changes in leaves of Populus cathayana males and females were analyzed after 4 wk of exposure to Mn stress. Under excess Mn conditions, shoot height and photosynthesis decreased more in females than in males. Females also showed severe browning and subcellular damage, higher Mn²⁺ absorption, and different antioxidant enzyme activities compared with males. There were ten differently regulated protein spots induced by excess Mn stress. They were mainly related to photosynthesis, ROS cleaning, and cell signaling associated to ROS, plant cell death, heat shock, cell defense and rescue, and gene expression and regulation. Variation in protein expression between the sexes clearly showed that males have evolved more efficient photosynthesis capacity, more stable gene expression and regulation, and better cell defense and rescue to prevent further injury under excess Mn stress.     

RCD1 and SRO1 are necessary to maintain meristematic fate in Arabidopsis thaliana

The radical-induced cell death1 and similar to RCD ONE1 genes of Arabidopsis thaliana encode members of the poly(ADP-ribose) polymerase (PARP) superfamily and have pleiotropic functions in development and abiotic stress response. In order to begin to understand the developmental and molecular bases of the defects seen in rcd1-3; sro1-1 plants, this study used the root as a model. Double mutant roots are short and display abnormally organized root apical meristems. However, acquisition of most cell fates within the root is not significantly disrupted. The identity of the quiescent centre is compromised, the zone of cell division is smaller than in wild-type roots and abnormal divisions are common, suggesting that RCD1 and SRO1 are necessary to maintain cells in a division-competent state and to regulate division plane placement. In addition, differentiation of several cell types is disrupted in rcd1-3; sro1-1 roots and shoots, demonstrating that RCD1 and SRO1 are also necessary for proper cell differentiation. Based on the data shown in this article and previous work, we hypothesize that RCD1 and SRO1 are involved in redox control and, in their absence, an altered redox balance leads to abnormal development.     

Oxidative burst and cell death in ozone-exposed plants

The phytotoxic air pollutant ozone spontaneously generates reactive oxygen species (ROS) in the leaf apoplast, provokes hypersensitive response-like lesions and induces defence reactions that significantly overlap with pathogen and other oxidative stress responses. Consequently, ozone has been used as a tool to unravel in planta ROS-induced plant defence and cell death mechanisms. Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide or superoxide anions in distinct species. Accumulation of these ROS precedes the induction of cell death, and both responses co-occur spatially in the periveinal regions of the leaves. The review summarizes some of the recent results that have been obtained concerning the molecular basis of apoplastic ROS production in monocot and dicot species. Signal molecules, in particular ethylene and salicylic acid, control and potentiate the oxidative burst and subsequent cell death in its initiation and propagation phases while jasmonate leads to lesion containment. Amplification mechanisms that result in the production of excess ROS and hypersensitive cell death are discussed as major factors in ozone sensitivity of plant species and cultivars.