| Riboflavin (vitamin B2) is a water-soluble vitamin. It is phosphorated and combined with nucleotides to form flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are coenzymes of many enzymes, play vital roles in many reactions, especially those characterized by oxidation and reduction.The use of a wide range of riboflavin in vitro plants can promote plant growth, improve crop yields, and enhance the effectiveness of ability to withstand adversity, and can induce a wide range of plant disease resistance, so riboflavin played a role of multi-effect of plant growth regulator.But now the defense responses and signal transduction elicited by riboflavin are not clear. In this study, riboflavin as an elicitor induced tobacco seedlings and tobacco suspension cells, a series of defense reactions and two important soil-borne diseases in tobacco were measured by biochemical, cytochemical, semi-quantative RT-PCR(reverse transcriptase-polymerase chain reaction)and HPLC(high performance liquid chromatography)analysis. The results are as follows:1 Tobacco suspension cells treated with 1 mmol·L-1 riboflavin were used to investigate a series of early defense responses. The results showed riboflavin induces a series of early defense responses, such as an oxidative burst, alkalinization of the extracellular medium. Oxidative burst generated 5 min after treatment. But extracellular pH value increased gradually with extending of time, the pH value increased by 0.57 at 80 min. These results suggest riboflavin triggers early defense reactions in tobacco cell suspensions.In order to illustrate the calcium signal and protein kinase are involved in this signal transduction, tobacco suspension cells treated with different inhibitors (a calcium surrogate La3+, a calcium chelator EGTA and an inhibitor of serine/threonine protein kinases staurosporine) were used to investigate a series of defense responses. The results showed three different inhibitors partially inhibited these defense responses; these suggest calcium signal and protein kinases participate in the elicitation of riboflavin.2 In order to clarify whether riboflavin induced expression of defense-related genes in tobacco suspension cells, four kinds of defense-related genes (PR-1a, PR-1b, PAL and LPO) were measured by RT-PCR. The results showed that the expression of two PR genes reached the highest level at 6 h after treatment, but the PAL gene and LPO gene start slowly, the expression showed a distinct change at 6 h, they achieved the highest level of expression at 24 h.To enucleate the effects of content on phenolic compounds and lignin in tobacco cells treated with riboflavin. The inductions of phenylalanine ammonia-lyase, peroxidase activity, accumulation of scopoletin and lignin were studied by tobacco suspension cells treated with 1 mmol·L-1 riboflavin. The results indicated the activity of PAL and POD increased and reached the highest level at 12 h and 24 h after treatment with riboflavin, respectively; the observation of bright blue fluorescence indicated that more phenolic compounds accumulated in the cells under UV light, and the total phenolics content increased by 1.2 and 1.5 times compared with control; extracts from tobacco cells and their culture filtrate showed strong accumulation of scopoletin within 48 h after treatment, and reached a peak at 12 h after treatment. Lignin deposition strongly increased by 1.5 and 1.8 times respectively at 24 h and 48 h after elicitation when compared with the control. These results showed riboflavin can activate phenylpropanoid metabolism of tobacco cells to defense pathogen infection and expansion.3 Induced resistance of riboflavin was tested for its effect on two soil-borne Pathogens (Phytophthora parasitica var. nicotianae and Ralstonia solanacearum) in tobacco. The disease index was decreased in seven tobacco cultivars treated with 2 mM/L riboflavin, induction efficiencies were different in these cultivars treated with riboflavin, riboflavin treatment resulted in 70.9%和52.4% protection, roots challenged with P. solanacearum after foliar spraying with riboflavin also showed significant reduction of pathogen growth. |
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