A simple new method, competitive hybridization, for identification of differentially regulated genes was used to isolate novel genes induced by ethylene in citrus (Citrus sinensis [L.] Osbeck cv. Shamouti) leaves. One of the isolated genes, an ethylene-induced esterase gene (EIE), was further characterized. The deduced protein sequence of this gene shows a similarity to those of several plant alpha/beta hydrolase gene family members, which are known to be involved in secondary metabolism. Northern blot analysis demonstrated that EIE mRNA was induced by ethylene within 4 h and accumulated to a very high level 24 h after the initiation of ethylene treatment. Induction of EIE by ethylene could be counteracted by 1-methylcyclopropene, a potent ethylene perception inhibitor, indicating that the expression of EIE is ethylene-dependent. The bacterially expressed protein of EIE was recognized by antiserum against Pir7b, a naphthol AS esterase induced in rice by the non-host pathogen, Pseudomonas syringae pv. syringae. The EIE protein was identified in ethylene-treated leaves using anti-Pir7b antibodies. An alpha-naphthyl acetate esterase accumulated concomitantly with the increase in EIE protein in ethylene-treated citrus leaves. An enzyme activity assay followed by western analysis confirmed that the esterase was EIE.
The mechanisms of negative effects of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP), a pyrazole-derived plant growth regulator used as a citrus abscission agent, were explored in Arabidopsis by integrating transcriptomic, physiological, and ultrastructural analyses. CMNP promoted starch degradation and senescence-related symptoms, such as chloroplast membrane disruption, electrolyte leakage, and decreased chlorophyll and protein content. Symptoms of plant decline were evident 12 h after CMNP treatment. Microarray analysis revealed that CMNP influenced genes associated with stress, including those related to anoxia, senescence, and detoxification. Sucrose treatment arrested CMNP-induced plant decline. The results demonstrate that the plant response to CMNP shares common elements with various stresses and senescence at physiological and molecular levels.
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