Allelophathic Effects And Mechanism Of Chlorogenic Acid And Artemisinin

The phenomenon of allelopathy was discovered for a long time, and wasgradually defined from1930s to1980s. Then, allelopathic research has been attractedmore and more attention, and developed rapidly. Research on allelopathy in Chinastarted later than some countries, but also made a lot of achievements. Currently,allelopathic research are more concentrated in crops and invasive plants, and theresults can provide a theoretical basis for sustainable agricultural development andecological protection.Research of allelopathy can be divided into three areas, first is the analysis ofallelopathic effects and the separation and identification of allelochemicals; thenfurther study is the mechanism of allelochemicals, and finally the ecological effects ofthe released allelochemicals will be researched. In addition, allelochemicals withspecial activities can be developed by combining with organic chemistry and otherdisciplines. According to current research status of allelopathy worldwide, themechanism of action of allelochemicals is one of the difficulties. One allelochemicalcan affect many aspects of plant growth and development, such as gene expression,cell division, hormone levels, and other physiological processes. Therefore, themechanism of action of allelochemicals is very complex.This study focused on the mechanism of allelopathy, and thus two knownallelochemicals-chlorogenic acid and artemisinin were choosed. Phenolicallelochemicals were usually found in crops such as rice, sorghum, corn, cucumbers,alfalfa, etc. Some studies suggestted that phenolic acid may be one of the factors forcrop cropping obstacle. Therefore, mechanism study on the activity of chlorogenicacid can provide a theoretical basis for elucidating the mechanism of croppingobstacle. Artemisinin is a well-known natural product because of its effects on malariatreatment. The excellent stability and strong activities in soil make artemisinin atypical allelochemicals, and thus is an ideal material for study on mechanism ofallelopathic effects.Lettuce is very sensitive to most allelochemicals and thus is often used as arecipient plant in allelopathy research. As a model plant, Arabidopsis has been wellstudied in its genetic background and regulatory pathways of various life processes,which provide convenience to study mode of action of allelochemicals. Therefore,lettuce and Arabidopsis were used as receptors in this study. Methods of plant cellbiology, plant physiology and molecular biology were used to research the mechanismof activity of chlorogenic acid and artemisinin.Bioassays of the two allelochemicals on lettuce were conducted. Results showedthat the effects of chlorogenic acid on root and stem elongation and seedling growthexhibited a low-concentration-promoting and high-concentration-inhibiting mode.The effect on lettuce root growth by chlorogenic acid is most evident, especially athigh concentrations of100and1000?M. Lettuce stem growth is less sensitive tochlorogenic acid in all concentrations used, and the differences between treated and control seedlings were not significant. Therefore, the growth inhibition of lettuceseedlings by chlorogenic acid mainly depended on the inhibition of root elongation.The mode of artemisinin on the growth of lettuce is similar to chlorogenic acid.However, effect of artemisinin is stronger than chlorogenic acid, because it showedinhibitory effects at a lower concentration of1?M and had significant effects whenthe concentrations were10?M and more. Moreover, inhibitory effects on both rootsand stems of artemisinin are very strong, resulting in significantly decreased in freshweight of lettuce seedling.Then, mechanism of action of the two allelochemicals on lettuce growth werestudied using methods of plant cell biology. In addition, the physiological indicatorsof the impact of artemisinin on lettuce were also tested. Schiff's reagent stainingshowed that mitotic index of lettuce root tips was decreased with increasingconcentrations of allelochmemicals used. Moreover, the number of cells in eachdivision process was also reduced. FDA-PI double staining and Evans blue absorptionexperiments show that with the increase of allelochemicals amount, dead cells beganto appear and significantly increased. At the highest concentration (1000?M), deadcells increased significantly and most of cells have lost vitality. Staining with reactiveoxygen species (ROS) specific dye-DHE showed that ROS produced andaccumulated with increasing concentrations of allelochemicals. In addition, MDAcontents were significantly increased after treated with artemisinin. Meanwhile, freeproline content was also rised, while chlorophyll content was significantly reduced.These results indicated that two allelochemicals are able to induce the generation ofROS in acceptor plant, and suppress cell viability and cell division. The difference isthat the impact of artemisinin was stronger.Chlorogenic acid has a variety of biological activities, and artemisinin is used asan antimalarial drug. Therefore, the mechanism of these two compounds were wellstudied in other model organisms, particularly in microorganisms and cancer cells.Many findings are consistent with the results of this study. For example, artemisinincan induce production and accumulation of ROS in cancer cells and thereby causing avariety of changes which ultimately achieve the effect of inhibiting tumor growth. Aswe known, ROS is an oxidative stress marker in plants, and also a signaling moleculeto regulate many life processes, including defense response, apoptosis and stomatalresponse. Presumably, two allelochemicals first induced ROS generation, then causedmembrane damage, abnormal cell division and programmed cell death (PCD). Thephysiological indicators were also changed, resulting in inhibition of plant growth.The response of plants to gravity stimulus can be divided into three steps: gravitysignal reception, signal transduction, and asymmetric growth of the root. Themechanism of gravitropism has been well studied, and results are abundant. However,the effect of allelochemicals on gravity on gravitropism of plant was rarely reported.In this paper, Arabidopsis was used as a receptor to study the mechanism ofartemisininn on plant's gravitropism. Results showed that with the increase of theconcentrations of artemisinin, Arabidopsis root growth was inhibited, and rootgravitropic response was also interfered. Gravitropic angles first decreased and thenincreased with the increasing of artemisinin concentrations. In order to further clarify the effects of artemisinin on Arabidopsis root gravitropism, Arabidopsis seedlingswere stimulated by gravity and results showed that gravitropic angles were alsodifferent with control. These results suggest that artemisinin can significantly inhibitthe growth of Arabidopsis root and affect its gravitropic response in a dose-dependentmanner.The starch-statolith hypothesis proposes that starch grains in plant root tip cellsplayed a key role in gravity sensing process. Therefore, iodine staining were used toobserve the effects of artemisinin on Arabidopsis starch grains. Results showed thatand after24hours with5?M, artemisinin can significantly reduce the amount ofapical starch grains. Cholodny-Went hypothesis proposes that auxin play a key role inasymmetric growth. In this study, two transgenic Arabidopsis lines DR5::GUS andDR5::GFP were observed after treatment with artemisinin. Results show that withincreasing concentrations of artemisinin, auxin distribution is decreased in apical andthe redistribution of auxin after gravity stimulus was also interferred.Results of semi-quantitative RT-PCR showed that the expression of functionalgenes of auxin (PIN1, PIN2, and AUX1) was significantly reduced in Arabidopsis aftetreatments with artemisinin. While, expression level of starch grain related gene(PGM1) did not changed. Based on the findings above, artemisinin may affectdownstream genes of PGM1, leading to reduction in the content of starch grains, andalso affects some auxin polar transport-related genes, resulting in the polar auxintransport dysfunction and abnormal distribution. Finally, the normal response ofArabidopsis root to gravity was influenced by artemisinin.