Gyeong Mee Yoon

CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis

The gaseous phytohormone ethylene C2H4 mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis. Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

Protein phosphatase 2A controls ethylene biosynthesis by differentially regulating the turnover of ACC synthase isoforms.

The gaseous hormone ethylene is one of the master regulators of development and physiology throughout the plant life cycle. Ethylene biosynthesis is stringently regulated to permit maintenance of low levels during most phases of vegetative growth but to allow for rapid peaks of high production at developmental transitions and under stress conditions. In most tissues ethylene is a negative regulator of cell expansion, thus low basal levels of ethylene biosynthesis in dark-grown seedlings are critical for optimal cell expansion during early seedling development. The committed steps in ethylene biosynthesis are performed by the enzymes 1-aminocyclopropane 1-carboxylate synthase (ACS) and 1-aminocyclopropane 1-carboxylate oxidase (ACO). The abundance of different ACS enzymes is tightly regulated both by transcriptional control and by post-translational modifications and proteasome-mediated degradation. Here we show that specific ACS isozymes are targets for regulation by protein phosphatase 2A (PP2A) during Arabidopsis thaliana seedling growth and that reduced PP2A function causes increased ACS activity in the roots curl in 1-N-naphthylphthalamic acid 1 (rcn1) mutant. Genetic analysis reveals that ethylene overproduction in PP2A-deficient plants requires ACS2 and ACS6, genes that encode ACS proteins known to be stabilized by phosphorylation, and proteolytic turnover of the ACS6 protein is retarded when PP2A activity is reduced. We find that PP2A and ACS6 proteins associate in seedlings and that RCN1-containing PP2A complexes specifically dephosphorylate a C-terminal ACS6 phosphopeptide. These results suggest that PP2A-dependent destabilization requires RCN1-dependent dephosphorylation of the ACS6 C-terminus. Surprisingly, rcn1 plants exhibit decreased accumulation of the ACS5 protein, suggesting that a regulatory phosphorylation event leads to ACS5 destabilization. Our data provide new insight into the circuitry that ensures dynamic control of ethylene synthesis during plant development, showing that PP2A mediates a finely tuned regulation of overall ethylene production by differentially affecting the stability of specific classes of ACS enzymes.

14-3-3 Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Protein Turnover in Arabidopsis

14-3-3 proteins function in many cellular processes. Here, we show that 14-3-3s regulate the stability of proteins involved in ethylene biosynthesis. 14-3-3 directly interacts with and regulates the turnover of ACC synthase, a key ethylene biosynthesis enzyme, and the ETO1/EOLs E3 ubiquitin ligases, which regulate ACS protein turnover, thus playing a key role in regulating ethylene biosynthesis. 14-3-3 proteins are a family of conserved phospho-specific binding proteins involved in diverse physiological processes. Plants have large 14-3-3 gene families, and many binding partners have been identified, though relatively few functions have been defined. Here, we demonstrate that 14-3-3 proteins interact with multiple 1-aminocyclopropane-1-carboxylate synthase (ACS) isoforms in Arabidopsis thaliana. ACS catalyzes the generally rate-limiting step in the biosynthesis of the phytohormone ethylene. This interaction increases the stability of the ACS proteins. 14-3-3s also interact with the ETHYLENE-OVERPRODUCER1 (ETO1)/ETO1-LIKE (EOLs), a group of three functionally redundant proteins that are components of a CULLIN-3 E3 ubiquitin ligase that target a subset of the ACS proteins for rapid degradation by the 26S proteasome. In contrast with ACS, the interaction with 14-3-3 destabilizes the ETO1/EOLs. The level of the ETO1/EOLs in vivo plays a role in mediating ACS protein turnover, with increased levels leading to a decrease in ACS protein levels. These studies demonstrate that regulation of ethylene biosynthesis occurs by a mechanism in which 14-3-3 proteins act through a direct interaction and stabilization of ACS and through decreasing the abundance of the ubiquitin ligases that target a subset of ACS proteins for degradation.

1-Aminocyclopropane-1-carboxylic acid as a signalling molecule in plants

This review summarizes and discusses the role of ACC synthase in plants. The classic role of ACC synthase is to act as the key enzyme in the biosynthetic pathway for the plant hormone ethylene. Several recent papers have converged on the notion that ACC, the immediate product of ACC synthase, acts as a novel signaling molecule in plants independent of its conversion to ethylene. The evidence for this hypothesis from these papers and potential roles for ACC is summarized and discussed.

Expression patterns of theNPP1 protein phosphatase gene and biochemical activity of its encoded protein

TheNPP1 cDNA encoding the catalytic subunit of a type 1 serine/threonine protein phosphatase (PP1) was previously cloned and characterized inNicotiana tabacum (Plant Mol Biol 36, 315–322,1998). In this study, the expression patterns ofNPP1 mRNA in response to various stimuli were examined to gain insight on the cellular function of the NPP1 protein.NPP1 mRNA accumulation was stimulated by Ca++, wounding, fungal elkitiors, and chitosan in leaves. However, with abscisic acid treatment, no change in transcript levels was observed. The recombinant NPP1 protein was catalytically active and could dephosphorylate the autophosphorylated recombinant NtCDPK1, a calcium-dependent protein kinase from tobacco.

ACC synthase and its cognate E3 ligase are inversely regulated by light

1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the key enzyme in ethylene biosynthesis, catalyzing the conversion of S-adenosylmethionine (AdoMet) to ACC, which is the immediate precursor of ethylene. The regulation of ACS protein stability plays an important role in controlling ethylene biosynthesis. We have recently shown that 14-3-3 positively regulates ACS protein stability by both a direct effect and via downregulation of the stability of the E3 ligases regulating its turnover, Ethylene Overproducer1 (ETO1)/ETO1-like (EOL). Here, we report that treatment of etiolated Arabidopsis seedlings with light rapidly increases the stability of ACS5 protein. In contrast, light destabilizes the ETO1/EOLs proteins, suggesting that light acts to increase ethylene biosynthesis in part through a decrease in the level of the ETO1/EOL proteins. This demonstrates that the ETO1/EOLs are regulated in response to at least one environmental cue and that their regulated degradation may represent a novel input controlling ethylene biosynthesis.

A novel dual‐specificity protein kinase targeted to the chloroplast in tobacco1

The NtDSK1 cDNA encoding a novel chloroplast‐targeted protein kinase was identified in Nicotiana tabacum. It contains the kinase domain at the C‐terminus and a putative regulatory domain at the N‐terminus. The recombinant NtDSK1 underwent autophosphorylation of serine, threonine, and tyrosine residues, indicating that NtDSK1 encodes a functional dual‐specificity protein kinase. The NtDSK1–green fluorescent protein fusion protein was targeted to chloroplasts. Furthermore, the NtDSK1 protein was immunodetected in chloroplast fractions isolated from tobacco seedlings. The NtDSK1 mRNA expression was developmentally regulated in different tissues, including anthers and germinating seeds, and strongly stimulated by gibberellin. The mRNA was rapidly light responsive during seedling growth. NtDSK1 may play a role in a light‐regulated signaling process in tobacco.

PiSCP1 and PiCDPK2 Localize to Peroxisomes and Are Involved in Pollen Tube Growth in Petunia Inflata

Petunia inflata small CDPK-interacting protein 1 (PiSCP1) was identified as a pollen expressed PiCDPK1 interacting protein using the yeast two hybrid system and the interaction confirmed using pull-down and phosphorylation assays. PiSCP1 is pollen specific and shares amino acid homology with uncharacterized proteins from diverse species of higher plants, but no protein of known function. Expression of PiSCP1-GFP in vivo inhibited pollen tube growth and was shown to localize to peroxisomes in growing pollen tubes. As PiCDPK1 is plasma membrane localized, we investigated the localization of a second isoform, PiCDPK2, and show that it co-localizes to peroxisomes with PiSCP1 and that the two proteins interact in the yeast 2 hybrid interaction assay, suggesting that interaction with the latter CDPK isoform is likely the one of biological relevance. Both PiCDPK2 and PiSCP1 affect pollen tube growth, presumably by mediating peroxisome function, however how they do so is currently not clear.