The Abnormal Response Of Calcium Signalsystem In Consecutive Monoculture Of Rehmanniaglutinosa L.

Rehmannia glutinosa L is a perennial herbaceous species belonging to the Scrophulariaceae family. It is a traditional Chinese medicine and one of the famous "Four Huai Medicines". This specie has been extensively cultivated for a long history. However, a serious consecutive monoculture problem is identified in the R. glutinosa production process. The R. glutinosa continuously cultivated grows poorly, and tuberous root can not normally expand or even without yield. This phenomenon can sustain for a period of 8-10 years.The consecutive monoculture problem has severely obstructed the sustainable production of R. glutinosa. It has become an urgent problem to restrict the development of Chinese medicine agriculture production and regional economy.So far, the researches of R. glutinosa consecutive monoculture problem are mainly focused on the causes of this phenomenon, the changes of soil microorganisms environment and the allelopathic substances in root exudates.The molecular mechanismof R. glutinosa responding to consecutive monoculture and the signal transduction pathways remain unclear.In order to study the molecular mechanism about consecutive monoculture problem of R. glutinosa,based on the early transcriptome of normal and consecutive cultivated R. glutinosa at the earlier tuberous root expansion stage, we selected the R. glutinosa cultivar"Wen 85-5" asexperimental material and conducted the relative researches as follows:Utilizing the Solexa/Illumina sequencing technology, we constructed and analyzed the Digital Gene Expression Profiling (DGE) of the normal and consecutive cultivated R.glutinosa, and the candidates were collected for consecutivecultivatedrelated genes. The expression of genes related to the calcium signal coding protein (calcium channel and "calcium pump") and decoding protein (calcium target protein) were significantly different, the genes of calcium ion channel protein and the intracellular calcium target protein were up-regulated, and "calcium pump" genes were down-regulated. The spatial and temporal expression profiles of key genes in calcium signaling system in the normal and consecutive cultivated R. glutinosa at different growth period were analyzed by qRT-PCR and the data were consistent with that in DGE.Our results indicated that the calcium signal system might be involved in R.glutinosa consecutive monoculture problem responsive processes.To confirm above results, Ca2+ localization and distribution in tuberous root cambial cells in the normal and consecutive cultivated R. glutinosa were investigated with antimonite precipitate-electromicroscopic-cytochemical method at the tuberous root elongating and the early expansion stage. The concentration and the change of free calcium ions in the root tip cells were measured by the calcium fluorescent indicator method combining with confocal fluorescence microscope technology.Results showed that a large amounts of calcium ions precipitation assembled in the consecutive cultivated R. glutinosa tuberous root meristematic cells, whereas it was on the contrary in the normal cultivated R. glutinosa root cells. The concentration of free calcium ions in the consecutive cultivated R. glutinosa root tip cells was higher than that in the normal cultivated, and the distribution of calcium ions was dispersive.The results showed that the consecutive monoculture caused intracellular Ca2+ accumulation and scattered distribution in the R. glutinosacells. It provided an objective and direct evidence for the idea mentioned above. The consecutive monoculture problem caused the concentration changes and abnormal distribution of the intracellular calcium ion, resulting in calcium signal system "disorder" and plants appeared abnormal growth.Moreover, the consecutive cultivated R. glutinosa was treated by calcium signal blocking agents. The results showed that the application of calcium signal blockers had good effect on alleviating symptom of the consecutive monoculture. In particular, the calcium chelator EGTA had most obvious effects, the treated plants grew well and consecutive monoculture poisoning phenomenon was blocked, and the root biomass at maturity was close to the normal cultivated R. glutinosa. The growth of plants treated with calcium channel inhibitor Verapamil and calmodulin antagonist trifluoperazine were significantly improved, and biomass statistics were greatly increased. Quantitative PCR technology was used to verify the influence of the different calcium signal blocking agent on the expression patterns of the related genes which are related to consecutive monoculture problem. The data showed that calcium signal blocker could regulate the abnormally expressed genes caused by consecutive cultivated return to the normal expression. The results demonstrated that the blocking effect of the calcium signal blocker on abnormal calcium signal could effectively relieve the poison caused by the consecutive monoculture problem.According to the analysis of the different genes expression profiles of the normal and consecutive cultivated R. glutinosa tuberous in root, we think that the calcium signal is involved in the response to consecutive monoculture problem of R. glutinosa. This opinion was directly demonstrated through the calcium antimonite precipitate method and the calcium fluorescent indicator technology. We also confirmed the correctness of this opinion according to the effect of calcium signal blocking agents. This research preliminarily revealed the action mechanism of calcium signal in response to consecutive monoculture problem of R, glutinosa. It laid the theoretical foundation for further research on the causes of consecutive monoculture problem. It also was beneficial to reveal the molecular mechanism of consecutive monoculture problem and to develop the effective method of alleviating the harm of consecutive monoculture problem.