Ethylene influences many aspects of plant growth and development. The biosynthesis of ethylene is highly regulated by a variety of internal and external cues. A key target of this regulation is 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), generally the rate-limiting step in ethylene biosynthesis, which is regulated both transcriptionally and post-transcriptionally. Prior studies have demonstrated that cytokinin and brassinosteroid (BR) act as regulatory inputs to elevate ethylene biosynthesis by increasing the stability of ACS proteins. Here, we demonstrate that several additional phytohormones also regulate ACS protein turnover. Abscisic acid, auxin, gibberellic acid, methyl jasmonic acid, and salicylic acid differentially regulate the stability of ACS proteins, with distinct effects on various isoforms. In addition, we demonstrate that heterodimerization influences the stability of ACS proteins. Heterodimerization between ACS isoforms from distinct subclades results in increased stability of the shorter-lived partner. Together, our study provides a comprehensive understanding of the roles of various phytohormones on ACS protein stability, which brings new insights into crosstalk between ethylene and other phytohormones, and a novel regulatory mechanism that controls ACS protein stability through a heterodimerization of ACS isoforms.

Of five functional ethylene biosynthesis genes in the rice genome, OsACS1 plays a more important role in regulating Pi deficiency-responsive root architecture modulation, Pi uptake, and Pi starvation responses

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.

Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination.

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Protein kinases are central components of signal transduction pathways in the cell. They catalyze the phosphorylation of substrate proteins, resulting in changes of the activity, localization, stability, and protein interactions of the substrates, ultimately coordinating the activity of important cellular processes. CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) is a Raf-like protein kinase that functions as a negative regulator in the phytohormone ethylene signaling pathway. CTR1 physically interacts with ethylene receptors via its N-terminal domain at the endoplasmic reticulum, and is involved in suppressing ethylene signaling in the absence of ethylene. Recent studies demonstrated that CTR1 directly interacts with and differentially phosphorylates the positive regulator ETHYLENE INSENSITIVE 2 (EIN2), therefore regulating the movement of EIN2 into the nucleus. Here, we describe protocols for determining the kinase activity of CTR1 by calculating the incorporated radiolabeled phosphate [γ-P] from ATP into its physiological substrate, EIN2 protein.

Ethylene gas controls plant growth and stress responses. Ethylene-exposed dark-grown seedlings exhibit a dramatic growth reduction, yet the seedlings rapidly return to the basal growth rate when ethylene gas is removed. However, the underlying mechanism governing this remarkable reversible acclimation of dark-grown seedlings to ethylene remains enigmatic. Here, 5 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 inhibits the ETHYLENE-INSENSITIVE3 (EIN3) transcription factor via the EIN3-BINDING F-box Proteins, resulting in rapid suppression of the ethylene response, thus promoting fast growth recovery. These findings uncover a 10 mechanism of the ethylene signaling pathway that links the spatiotemporal dynamics of cellular signaling components to organismal responses. 15 20 25 3 Introduction:The ability of organisms to respond to and integrate environmental signals leading to an appropriate response is critical for optimal growth and development, particularly for plants, which are non-motile. Plants adapt to a wide variety of abiotic stresses, and upon removal of stress, they need to rapidly restore basal cellular homeostasis. One key signal for abiotic stress 5 is the plant hormone ethylene. Ethylene is involved in multiple aspects of growth and development, including fruit ripening, leaf and floral senescence, cell elongation, seed germination, and root hair formation, as well as responses to biotic and abiotic stress 1-3 .Ethylene-mediated stress acclimation includes, but is not limited to, the rapid elongation of rice internodes in response to flooding, drought responses, salt tolerance, heavy metal tolerance, 10 and morphological changes of roots in response to nutrient deficiency 4-8 . How ethylene regulates such remarkable plasticity of plant stress adaptation is poorly understood, however.Extensive molecular genetic studies have elucidated the basic ethylene signaling pathway 2, 9-12 . In the absence of ethylene, the endoplasmic reticulum (ER)-localized ethylene receptors activate the CTR1 protein kinase, which in turn phosphorylates Ethylene-Insensitive 2 15 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. CTR1-mediated phosphorylation of EIN2 leads to its ubiquitination and proteolysis by the 26S proteasome [11][12][13][14][15][16][17][18] . In response to ethylene, the receptors, and hence CTR1, are inactivated, leading to reduced phosphorylation and increased accumulation of EIN2. EIN2 is then proteolytically cleaved, releasing the C-terminal domain (EIN2-CEND) which translocates 20 into the nucleus where it activates the Ethylene-Insensitive 3 (EIN3) and EIN3-Like (EIL) paralogs which function as central transcription factors in ethylene signaling [16][17][18] . EIN2-CEND also associates with the EIN3-Binding F-box 1 (EBF1) and EBF2 mRNAs and represses their translation, thus ...