Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

BACKGROUND: In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1) is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS), in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits. RESULTS: Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE) did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type. CONCLUSION: These results suggest that ETO1 family proteins specifically interact with and negatively regulate type 2 ACC synthases. Our data also show that Arabidopsis ETO1 can regulate type 2 ACS in a heterologous plant, tomato

Tooth Loss and Carotid Intima-Media Thickness in Relation to Functional Atherosclerosis: A Cross-Sectional Study

Structural arterial stiffness can be evaluated with carotid intima-media thickness (CIMT). Functional arterial stiffness can be evaluated with cardio-ankle vascular index (CAVI). A positive association between CIMT and tooth loss has been reported, but no studies have evaluated the association between CIMT and tooth loss in relation to functional arterial stiffness (functional atherosclerosis). A cross-sectional study of 1235 Japanese individuals aged 40–89 years was conducted. Tooth loss was defined as being in the lowest tertile for the number of remaining teeth (≤20 in men and ≤19 in women). Functional atherosclerosis was defined as CAVI ≥ 9.0. Independent of known confounding factors, CIMT was positively associated with tooth loss only in participants without functional atherosclerosis. Adjusted odds ratios for tooth loss and a 1 standard deviation increment in CIMT were 1.27 (1.04–1.55) for participants without functional atherosclerosis and 0.99 (0.77–1.26) for participants with functional atherosclerosis. CIMT and functional atherosclerosis had a significant effect on tooth loss; the fully adjusted p-value for the interaction on tooth loss was 0.019. Independent of known confounding factors, CIMT is positively associated with tooth loss only in participants without functional atherosclerosis. This finding helps clarify the influence of the progression of arterial stiffness on tooth loss because the progression of structural atherosclerosis might have a beneficial influence on the maintenance of the microcirculation

Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated

To investigate the regulatory mechanism(s) of ethylene biosynthesis in fruit, transgenic tomatoes with all known LeEIL genes suppressed were produced by RNA interference engineering. The transgenic tomato exhibited ethylene insensitivity phenotypes such as non-ripening and the lack of the triple response and petiole epinasty of seedlings even in the presence of exogenous ethylene. Transgenic fruit exhibited a low but consistent increase in ethylene production beyond 40 days after anthesis (DAA), with limited LeACS2 and LeACS4 expression. 1-Methylcyclopropene (1-MCP), a potent inhibitor of ethylene perception, failed to inhibit the limited increase in ethylene production and expression of the two 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) genes in the transgenic fruit. These results suggest that ripening-associated ethylene (system 2) in wild-type tomato fruit consists of two parts: a small part regulated by a developmental factor through the ethylene-independent expression of LeACS2 and LeACS4 and a large part regulated by an autocatalytic system due to the ethylene-dependent expression of the same genes. The results further suggest that basal ethylene (system 1) is less likely to be involved in the transition to system 2. Even if the effect of system 1 ethylene is eliminated, fruit can show a small increase in ethylene production due to unknown developmental factors. This increase would be enough for the stimulation of autocatalytic ethylene production, leading to fruit ripening

Structure of a Precursor to the Blue Components Produced in the Blue Discoloration in Japanese Radish (Raphanus sativus) Roots

The internal blue discoloration in Japanese radish (Raphanus sativus L.) roots has been reported to be a physiological phenomenon after harvest and poses a significant problem for farmers. To avoid this discoloration, the fundamental development of new radish cultivars that do not undergo discoloration and/or improved cultivation methods is required. Elucidating the chemical mechanism leading to this discoloration could help overcome these difficulties. To determine the mechanism underlying this discoloration, this study was designed to probe the structure of a precursor to the blue components generated during the discoloration process. Soaking fresh roots in aqueous H₂O₂ resulted in rapid blue discoloration, similar to the natural discoloration. Using a H₂O₂-based blue discoloration assay, the precursor was extracted and isolated from the fresh roots and identified as the glucosinolate, 4-hydroxyglucobrassicin, via spectroscopy and chemical synthesis

Mechanism Underlying the Onset of Internal Blue Discoloration in Japanese Radish (Raphanus sativus) Roots

The internal blue discoloration observed in Japanese radish (Raphanus sativus L.) roots is a physiological phenomenon caused by storage following harvest at approximately 20 °C and poses a serious problem for farmers. Here, we describe the mechanism underlying the onset of internal blue discoloration of three cultivars: Hukuhomare, SC8-260, and Yuto. Each cultivar was maintained under the same conditions. Additionally, Hukuhomare radish roots were maintained at three different cultivation conditions in a related experiment. The blue discoloration in radish roots was caused by the oxidation of 4-hydroxyglucobrassicin as a result of an increase in oxidative stress involving peroxidase. Thus, the extent of blue discoloration was influenced by the chemical balance involving 4-hydroxyglucobrassicin content, antioxidant capacity, and oxidation activity