The vacuolar Ca2+ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana.

Flooding represents a major threat to global agricultural productivity and food security, but plants are capable of deploying a suite of adaptive responses that can lead to short- or longer-term survival to this stress. One cellular pathway thought to help coordinate these responses is via flooding-triggered Ca2+ signaling. We have mined publicly available transcriptomic data from Arabidopsis subjected to flooding or low oxygen stress to identify rapidly upregulated, Ca2+ -related transcripts. We then focused on transporters likely to modulate Ca2+ signals. Candidates emerging from this analysis included AUTOINHIBITED Ca2+ ATPASE 1 and CATION EXCHANGER 2. We therefore assayed mutants in these genes for flooding sensitivity at levels from growth to patterns of gene expression and the kinetics of flooding-related Ca2+ changes. Knockout mutants in CAX2 especially showed enhanced survival to soil waterlogging coupled with suppressed induction of many marker genes for hypoxic response and constitutive activation of others. CAX2 mutants also generated larger and more sustained Ca2+ signals in response to both flooding and hypoxic challenges. CAX2 is a Ca2+ transporter located on the tonoplast, and so these results are consistent with an important role for vacuolar Ca2+ transport in the signaling systems that trigger flooding response.

Ethylene Receptors Signal via a Noncanonical Pathway to Regulate Abscisic Acid Responses.

Ethylene is a gaseous plant hormone perceived by a family of receptors in Arabidopsis (Arabidopsis thaliana) including ETHYLENE RESPONSE1 (ETR1) and ETR2. Previously we showed that etr1-6 loss-of-function plants germinate better and etr2-3 loss-of-function plants germinate worse than wild-type under NaCl stress and in response to abscisic acid (ABA). In this study, we expanded these results by showing that ETR1 and ETR2 have contrasting roles in the control of germination under a variety of inhibitory conditions for seed germination such as treatment with KCl, CuSO4, ZnSO4, and ethanol. Pharmacological and molecular biology results support a model where ETR1 and ETR2 are indirectly affecting the expression of genes encoding ABA signaling proteins to affect ABA sensitivity. The receiver domain of ETR1 is involved in this function in germination under these conditions and controlling the expression of genes encoding ABA signaling proteins. Epistasis analysis demonstrated that these contrasting roles of ETR1 and ETR2 do not require the canonical ethylene signaling pathway. To explore the importance of receptor-protein interactions, we conducted yeast two-hybrid screens using the cytosolic domains of ETR1 and ETR2 as bait. Unique interacting partners with either ETR1 or ETR2 were identified. We focused on three of these proteins and confirmed the interactions with receptors. Loss of these proteins led to faster germination in response to ABA, showing that they are involved in ABA responses. Thus, ETR1 and ETR2 have both ethylene-dependent and -independent roles in plant cells that affect responses to ABA.

Identification of Regions in the Receiver Domain of the ETHYLENE RESPONSE1 Ethylene Receptor of Arabidopsis Important for Functional Divergence.

Ethylene influences the growth and development of Arabidopsis (Arabidopsis thaliana) via five receptor isoforms. However, the ETHYLENE RESPONSE1 (ETR1) ethylene receptor has unique, and sometimes contrasting, roles from the other receptor isoforms. Prior research indicates that the receiver domain of ETR1 is important for some of these noncanonical roles. We determined that the ETR1 receiver domain is not needed for ETR1's predominant role in mediating responses to the ethylene antagonist, silver. To understand the structure-function relationship underlying the unique roles of the ETR1 receiver domain in the control of specific traits, we performed alanine-scanning mutagenesis. We chose amino acids that are poorly conserved and are in regions predicted to have altered tertiary structure compared with the receiver domains of the other two receptors that contain a receiver domain, ETR2 and ETHYLENE INSENSITIVE4. The effects of these mutants on various phenotypes were examined in transgenic, receptor-deficient Arabidopsis plants. Some traits, such as growth in air and growth recovery after the removal of ethylene, were unaffected by these mutations. By contrast, three mutations on one surface of the receiver domain rendered the transgene unable to rescue ethylene-stimulated nutations. Additionally, several mutations on another surface altered germination on salt. Some of these mutations conferred hyperfunctionality to ETR1 in the context of seed germination on salt, but not for other traits, that correlated with increased responsiveness to abscisic acid. Thus, the ETR1 receiver domain has multiple functions where different surfaces are involved in the control of different traits. Models are discussed for these observations.

The Ethylene Receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 Have Contrasting Roles in Seed Germination of Arabidopsis during Salt Stress.

In Arabidopsis (Arabidopsis thaliana), ethylene responses are mediated by a family of five receptors that have both overlapping and nonoverlapping roles. In this study, we used loss-of-function mutants for each receptor isoform to determine the role of individual isoforms in seed germination under salt stress. From this analysis, we found subfunctionalization of the receptors in the control of seed germination during salt stress. Specifically, loss of ETHYLENE RESPONSE1 (ETR1) or ETHYLENE INSENSITIVE4 (EIN4) leads to accelerated germination, loss of ETR2 delays germination, and loss of either ETHYLENE RESPONSE SENSOR1 (ERS1) or ERS2 has no measurable effect on germination. Epistasis analysis indicates that ETR1 and EIN4 function additively with ETR2 to control this trait. Interestingly, regulation of germination by ETR1 requires the full-length receptor. The differences in germination between etr1 and etr2 loss-of-function mutants under salt stress could not be explained by differences in the production of or sensitivity to ethylene, gibberellin, or cytokinin. Instead, etr1 loss-of-function mutants have reduced sensitivity to abscisic acid (ABA) and germinate earlier than the wild type, whereas etr2 loss-of-function mutants have increased sensitivity to ABA and germinate slower than the wild type. Additionally, the differences in seed germination on salt between the two mutants and the wild type are eliminated by the ABA biosynthetic inhibitor norflurazon. These data suggest that ETR1 and ETR2 have roles independent of ethylene signaling that affect ABA signaling and result in altered germination during salt stress.

Analysis of plant flooding response.

Flooding represents an environmental stress that has widespread effects on plants in natural ecosystems as well as causing major crop losses. As climate change leads to more severe weather extremes, severe flooding events are likely to become even more frequent. Thus, there is intense interest in understanding how flooding affects plants and in identifying cellular and molecular targets for engineering flood resilience into crop species. Such research requires well controlled, highly reproducible flooding protocols for use in the laboratory. However, there are many ways that a plant can be flooded. For example, waterlogging of the soil, where water levels reach the soil surface, generally generates a hypoxic environment around the root system. In contrast, full submergence of the plant adds effects on the aerial organs such as impaired photosynthesis from the combination of lowered CO2 availability and the reduced light penetration into often turbid flood waters. In this chapter, approaches to imposing controlled flooding conditions to the model plant Arabidopsis thaliana are discussed. A series of straight-forward assays are then described to document the effects of stress-related changes in growth patterns, pigment accumulation and levels of oxidative stress. These assays are complemented by monitoring the expression of a series of molecular markers of flood response by qPCR.

A touchy subject: Ca2+ signaling during leaf movements in Mimosa.

Mimosa pudica, the sensitive plant, responds to stimuli such as touch and wounding with leaf movements that propagate throughout the plant. The motion is driven by changes in the turgor of specialized cells in a set of motor organs called pulvinae. By imaging cellular Ca2+ levels as the wave of movement propagates through the leaf, Hagihara and colleagues now show that Ca2+ signals precede and predict the pulvinar movements. These results provide compelling support for a model where Mimosa uses a Ca2+-related response system to trigger its leaf movements. These researchers then used CRISPR to delete a critical genetic regulator of pulvinar development, producing plants with immobile leaves. These plants experienced more herbivory than wild type, suggesting that the Ca2+-triggered leaf movements are an adaptation to deter herbivory.

Ethylene-triggered subcellular trafficking of CTR1 enhances the response to ethylene gas.

The phytohormone ethylene controls plant growth and stress responses. Ethylene-exposed dark-grown Arabidopsis seedlings exhibit dramatic growth reduction, yet the seedlings rapidly return to the basal growth rate when ethylene gas is removed. However, the underlying mechanism governing this acclimation of dark-grown seedlings to ethylene remains enigmatic. Here, we report that ethylene triggers the translocation of the Raf-like protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a negative regulator of ethylene signaling, from the endoplasmic reticulum to the nucleus. Nuclear-localized CTR1 stabilizes the ETHYLENE-INSENSITIVE3 (EIN3) transcription factor by interacting with and inhibiting EIN3-BINDING F-box (EBF) proteins, thus enhancing the ethylene response and delaying growth recovery. Furthermore, Arabidopsis plants with enhanced nuclear-localized CTR1 exhibited improved tolerance to drought and salinity stress. These findings uncover a mechanism of the ethylene signaling pathway that links the spatiotemporal dynamics of cellular signaling components to physiological responses.

Ethylene causes transcriptomic changes in Synechocystis during phototaxis.

Ethylene is well known as a plant hormone, but its role in bacteria is poorly studied. We recently showed that Synechocystis sp. Strain PCC 6803 has a functional receptor for ethylene, ethylene response 1 (Etr1), that is involved in various processes such as phototaxis in response to directional light and biofilm formation. Here, we use RNA sequencing to examine the changes in gene transcripts caused by ethylene under phototaxis conditions. Over 500 gene transcripts across many functional categories, of approximately 3700 protein-encoding genes, were altered by application of ethylene. In general, ethylene caused both up- and downregulation of genes within a functional category. However, the transcript levels of amino acid metabolism genes were mainly upregulated and cell envelope genes were mostly downregulated by ethylene. The changes in cell envelope genes correlate with our prior observation that ethylene affects cell surface properties to alter cell motility. Ethylene caused a twofold or more change in 62 transcripts with the largest category of upregulated genes annotated as transporters and the largest category of downregulated genes annotated as glycosyltransferases which sometimes are involved in changing the composition of sugars on the cell surface. Consistent with changes in cell envelope, glycosyltransferase, and transporter gene transcripts, application of ethylene altered the levels of specific sugar moieties on the surface of cells. Light signaling from Etr1 involves two proteins (Slr1213 and Slr1214) and a small, noncoding RNA, carbon stress-induced RNA1 (csiR1). Application of ethylene caused a rapid, but transient, decrease in the transcript levels of etr1, slr1213, and slr1214 and a rapid and prolonged decrease in csiR1 transcript. Deletion of Slr1214 caused a large increase in csiR1 transcript levels and ethylene lowered csiR1 transcript. These data combined with prior reports indicate that ethylene functions as a signal to affect a variety of processes altering the physiology of Synechocystis cells.

Loss of the ETR1 ethylene receptor reduces the inhibitory effect of far-red light and darkness on seed germination of Arabidopsis thaliana.

When exposed to far-red light followed by darkness, wild-type Arabidopsis thaliana seeds fail to germinate or germinate very poorly. We have previously shown that the ethylene receptor ETR1 (ETHYLENE RESPONSE1) inhibits and ETR2 stimulates seed germination of Arabidopsis during salt stress. This function of ETR1 requires the full-length receptor. These roles are independent of ethylene levels and sensitivity and are mainly mediated by a change in abscisic acid (ABA) sensitivity. In the current study we find that etr1-6 and etr1-7 loss-of-function mutant seeds germinate better than wild-type seeds after illumination with far-red light or when germinated in the dark indicating an inhibitory role for ETR1. Surprisingly, this function of ETR1 does not require the receiver domain. No differences between these mutants and wild-type are seen when germination proceeds after treatment with white, blue, green, or red light. Loss of any of the other four ethylene receptor isoforms has no measurable effect on germination after far-red light treatment. An analysis of the transcript abundance for genes encoding ABA and gibberellic acid (GA) metabolic enzymes indicates that etr1-6 mutants may produce more GA and less ABA than wild-type seeds after illumination with far-red light which correlates with the better germination of the mutants. Epistasis analysis suggests that ETR1 may genetically interact with the phytochromes (phy), PHYA and PHYB to control germination and growth. This study shows that of the five ethylene receptor isoforms in Arabidopsis, ETR1 has a unique role in modulating the effects of red and far-red light on plant growth and development.