Characteristic And Regulatory Mechanism Of The Effects Of New Alkaloid-Narcicilasine From Narcissus Bulb On Plant Root Development

Natural product narciclasine (NCS) is a new Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs and possesses a broad range of biological activities such as antimitotic and antiviral functions. A broad range of inhibitory effects of NCS in plant were found, however, it is still poorly understood about the molecular mechanisms of NCS involvement in plant developmental responses. The plant hormone auxin plays a central role in the regulation of plant growth and development, as well as in responses to environmental stimuli. Basing on the preliminary studies and some special physiology responses of NCS to plant, we hypothesized that NCS may act as an auxin response inhibitor through interfering with auxin signaling pathway or/and auxin transport to affect the auxin-mediated growth and development process. In the present study, the model plant Arabidopsis and rice was used as materials to further study the mechanism of the effects of NCS on the auxin-mediated growth and development process. The main results are as follows:1. NCS inhibits the physiological responses of Arabidopsis root to auxin.The inhibitory effects of NCS on auxin-inducible lateral root and root hair formation by altering the sensitivity of Arabidopsis roots to auxin were demonstrated. The auxin insensitive mutant axr1-3and axr1-12showed a modest resistance to lower concentrations of NCS in primary root elongation compared to WT seedlings, indicating that NCS may indirectly affect theFTIR1/AFB complex-mediated auxin signaling. It was also found that NCS inhibited the expression of primary auxin-inducible genes in Arabidopsis roots using DR5::GUS reporter gene, native auxin promoters (IAA12::GUS, LAA13::GUS) and quantitative reverse transcription PCR analysis. It was further indicated that NCS treatment did not affect the endogenous IAA content in Arabidopsis roots by ELISA assaysis, demonstrating that NCS dose not inhibit auxin biosynthesis. Analysis of Arabidopsis HS::AXR3NT-GUS line showed that NCS relieved the auxin-enhanced degradation of Aux/1AA repressor modulated by the SCFTIR1ubiquitin-proteasome pathway. Biochemical experiments indicated that the impairment of NCS on the degradation of Aux/IAA proteins was not achieved by inhibiting the26S proteasome activity. In addition, the pull-down assay in vitro demonstrated that NCS did not alter the auxin-stimulated interaction between IAA7/AXR2(Aux/IAA proteins) and the F-box protein TIRl activity, suggesting that the inhibition of NCS to auxin signaling maybe by modulating to the some upstream elements of SCFTIR1/AFB complex. In conclusion, NCS inhibits the responses of Arabidopsis roots to auxin by probably affecting on auxin signaling transduction.2. NCS affects auxin transport through modulating the expression of auxin transport proteins in Arabidopsis roots.NCS treatments can cause defects in root gravitropism of Arabidopsis seedlings, which is similar to the characteristic of auxin transport inhibitors TIBA and NPA. The results by DR5::GUS reporter detection showed that the distribution of auxin in Arabidopsis root tips was disturbed by NCS. NCS also affected the auxin-dependent organization of columella cells by starch grain staining. After stimulation of gravity, NCS inhibited asymmetry auxin flow, and no auxin accumulation occurred at the lower side of the root by detecting of auxin response reporter DR5rev::GFP. These results suggested that NCS disturbed asymmetric auxin distribution and root gravitropic responses associated with related to auxin transport in Arabidopsis roots. NCS inhibited both cellular [3H]-IAA import and export by [3H]-IAA radiotracer transport assay in cultured BY-2cells. Using transgenic plants containing the promoters of PINs and AUX1, we found that the expression levels of PIN proteins and AUX1are substantially reduced after12h NCS treatments. qRT-PCR analysis further showed that the expressions of auxin transport genes in Arabidopsis roots was also regulated under NCS treatment. These results indicated that NCS negatively modulates auxin transport in the root is due to a general reduction of auxin carrier protein expression at the transcriptional level and protein level.3. NCS affects the intracellular trafficking of auxin carrier proteins.The analysis to the subcellular localization of auxin carrier proteins in the root tips of Arabidopsis seedlings showed that NCS did not affect the localization of PINs and AUXI proteins in the plasma membrane but PIN2-GFP, PIN3-GFP, PIN4-GFP, PIN7-GFP and AUX1-YFP in the plasma membrane were rapid internalized and formed intracellular agglomerations that we termed as "NCS bodies". The observation of fluorescent images showed that NCS promoted general endocytosis traced by styryl dye FM4-64staining labeled vesicles. Results further indicated that NCS-induced intracellular compartments of PIN2-GFP partially co-localized by the staining to PIN2-GFP seedlings in NCS treatment with the membrane-selective endocytic tracer FM4-64, suggesting that the internalization of auxin carrier proteins and formation intracellular agglomerations induced by NCS was dependent on endocytosis pathway. The treatments of CHX (an inhibitor of protein biosynthesis) and NCS indicated that the effect of NCS on endocytosis was independent of protein synthesis and the delivery of newly synthesized proteins to the PM. Furthermore, the membrane localization and the intracellular transfor of the three membrane fusion proteins BRI1-GFP, PIP2-GFP and LTI6B-GFP was not affected by NCS treatment, demonstrating that the effect of NCS on the stability of membrane proteins has pronounced proteins specificity. The analysisi of subcellular localization by using endomembrane markers ARA7-GFP, NAG-GFP, SYP22-YFP and SYP61-CFP showed that NCS also exhibited the specificity. In addition, the results by using the transgenic plant ABD2-GFP of actin cytoskeleton demonstrated that NCS could disrupt the structure of actin cytoskeletal organization and lead to decrease in the protein filaments. Using real-time live-cell microscopy, we also found that NCS influences actin-based vesicle motility through endosomal markers protein ARA7-GFP.4. NCS modulates the development of root system in rice seedlings.NCS inhibits rice root growth and diminishes the number of lateral roots, but it promotes adventitious root formation in rice seedlings. NCS also inhibits the promotion of exogenous auxin on rice root architecture, which is similar to the observations in Arabidopsis, suggesting that NCS may also interferred with auxin signaling or auxin transport in rice seedlings. Histochemical staining showed that auxin-induced DR5gene expression was markedly decreased in rice root tips after NCS treatment, impling that the inhibitory effect of NCS on auxin-responsive gene expression is also achieved by directly affecting the auxin signaling. Semiquantitative RT-PCR analysis demonstrated that NCS inhibited the expression of putative OsPIN genes in rice roots. The assay of in nanoscale [3H]-IAA radiotracer transport in rice roots showed that NCS reduced the acropetal or basipetal [3H]-IAA transport, respectively. These results revealed that NCS also affects auxin transport in rice roots. Very interesting, the phenotype of spiral roots was observed in0.5μM NCS treatment, which maybe due to the alteration in auxin distribution by disruption of auxin transport in NCS treatment.