Involvement Of Phospholipases C And D In The Defence Responses Of Riboflavin-treated Tobacco Cells

Riboflavin is an activator of defence responses in plants that increases resistance against diseases caused by fungal, oomycete, bacterial and viral pathogens. However, the mechanisms are poorly understood. Phospholipase C (PLC) signal pathway and phospholipase D (PLD) signal pathway play important roles in defence responses of animals and plants. Inositol1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) hydrolysed by PLC, and phosphatidic acid (PA) that can be produced by PLC and PLD, as well as NO which acts as signal molecules, are important second messengers in plant cells in various physiological processes. But now, little is known about whether PLC and PLD have involved in the defence responses of riboflavin-treated tobacco cells.We investigated the signal transduction pathways of PLC and PLD in tobacco(Nicotiana tabacum) suspension cells using a pharmacological approach to confirm whether riboflavin-mediated activation of the defence response is dependent on both PLC and PLD. A series of defense reactions were measured by biochemical, cytochemical, semi-quantative RT-PCR (reverse transcriptase-polymerase chain reaction), Real-time PCR (RT-PCR) and HPLC (high performance liquid chromatography) analysis. The results are as follows:1. In order to investigate expression patterns of all four genes of NtPLC and NtPLD in responses to riboflavin stimuli were studied by applying Real Time-PCR approach. These results showed that multiple members of the gene family were differentially expressed in tobacco suspension cells forriboflavin. NtPLCl are expressed at relatively low levels, while other NtPLCs are expressed at higher levels. NtPLDs also showed differences in expression patterns and induction.2. To elucidate whether PLC and PLD are involved in the production of H2O2induced by riboflavin in tobacco suspension cells, cells treated with U73122(a PLC special inhibitor),1-butanol (a PLD special inhibitor) and their inactive analogues U73343, tert-butanol in riboflavin-induced cells were used to compare the inhibition of the production of H2O2. These results showed that the inhibition of H2O2production rates of U73122(10μmol·L-1) was79.5%, and the inhibition of H2O2production rates of1-butanol (0.1%, v/v) was62.9%, respectively. Tert-butanol had no effect on H2O2production activation by riboflavin. These results suggest that both PLC and PLD are involved in H2O2production activatedd by riboflavin. In addition, riboflavin can stimulate the production of nitric oxide (NO).3. Tobacco suspension cells treated with PLC inhibitors (U73122) and PLD inhibitors (1-butanol) in riboflavin-induced cells were used to study the expression of defense-related genes. Expression of3defense-related genes was partially inhibited by U73122. PLD inhibitors (1-butanol) also partially have inhibited the expression of defense-related genes. While U73343, an inactive analogue of U73122and tert-butanol, an inactive analogue of1-butanol, had no effect on these defense responses induced by riboflavin.4. Scopoletin accumulation was significantly inhibited by addition of U73122and1-butanol in riboflavin-induced cells, respectively. However, U73343and1-butanol had no effect. Moreover, treatment tobacco cells with phosphatidic acid, a signalling molecule produced by phospholipase catalysis, induced the accumulation of the phytoalexin scopoletin and compensated for the suppressive effects of U73122and1-butanol on riboflavin-induced accumulation of the phytoalexin.