| Plant development contains two phases:embryogenesis and post-embryonic development. During embryogenesis, the basal body plan is established, and later in post-embryonic development, mature plant is formed through reiterated organogenesis and organ growth. Lots of researches carried out with auxin related mutants or by toxicological treatment to inhibit polar auxin transport, showed that such polar auxin transport and auxin gradient play a key role in regulating both embryogenesis and post-embryonic development. However, most of these mutants had their adult defects early from embryogenesis, i.e., the phenotype observed might be accumulative results or indirect effects, thus they may not precisely represent the real influence of auxin dynamics. Whereas, blocking polar auxin transport by inhibitor always brought in large-scale change in auxin distribution or other side-effects, consequently the results may not stand for real natural responses of plant to auxin. Therefore, we need more delicate experimental design to observe the role of auxin dynamics in specific tissue and in specific development stage, meanwhile try to avoid both accumulative effects and dramatic changes in auxin transport. Using inducible expression system to manipulate the expression of appropriate gene that regulating auxin transport may provide us such a tool for transient control of auxin dynamics, which allow us to examine the response and self-adjustment of plant tissues, as well as the dynamic change in differentiation and developmental fate of specific cells to auxin.In Arabidopsis, PIN family proteins play an essential role in polar auxin transport. These kinds of proteins are actively recycled between cytoplasm and plasma membrane through vesicle trafficking, resulting in fine-tuning of auxin transport. It was shown that GNOM protein was participated in regulating the recycling process. GNOM gene encodes an ARF-GEF (guanine nucleotide exchange factor of ADP-ribosylation factor), which is required for basal localization of auxin efflux carrier protein PIN1, by mediating endosome to plasma membrane recycling and GNOM dependent PIN1 transcytosis to basal cell membrane. The activity of GNOM protein has an obvious dosage effect on the regulation of polar auxin transport, as well as the modulation of primary root growth and lateral root formation. Therefore, in the present study we constructed an inducible anti-sense GNOM expression system in Arabidopsis, which allow us to adjust the transport and accumulation of auxin in relatively small scale by regulating the expression level of GNOM protein, focusing on the response of embryogenesis, primary root growth and lateral root formation to transient manipulation of auxin distribution through above system. The main results are as follows:1. We have constructed a reliable anti-sense GNOM expression system that effectively manipulate the expression of GNOM protein. We generated transgenic arabidopsis that contained antisense GNOM (against partial sequence of 5'coding sequence) construct in pTA211 vector that allow induction of the antisense mRNA by applying dexamethasone (DEX) to the plants. After antibiotic resistance screening and phenotype observation, we chose InAGN9 line, which showed the strongest phenotype, for further experiments. Expression of the anti-sense GNOM transcripts reached about 110 times of endogenous GNOM expression level in the second day of DEX induction. InAGN9 seedlings, which were germinated on DEX medium, displayed similar phenotype with gnom mutants, and also exhibited a decreased expression of GNOM protein. However, the anti-sense transcripts could not be detected 1 day after the induced InAGN9 seedlings were transferred from DEX medium to MS medium. These results showed that our established system could suppress expression of GNOM protein or restore to normal by DEX treatment or retrieving it, thus allow us to transiently adjust auxin dynamics.2. The inducible anti-sense GNOM expression system was capable of regulating auxin dynamics. Primary root elongation in DEX induced InAGN9 seedlings became more sensitive to auxin than in uninduced controls. The induced InAGN9 seedlings displayed an abnormal auxin distribution pattern as revealed by the auxin response marker DR5-GUS, which had an additional expression in cells of lateral root cap, as well as a weakened expression in stele cells of root tip. This abnormal distribution pattern could be observed in part of the induced root tips 10 hours after DEX application, and appeared in all the treated root tips after 24 hours. Once the induced roots were transferred to plain medium, a gradual recovery of the abnormal DR5-GUS distribution pattern to normal could be observed. The recovery could be observed in part of the roots 10 hours after the transfer and in all roots 24 hours later. It's worth noting from the experiments that auxin transport in plant tissues can respond in 10 hours after DEX application, and restore to normal in one day when DEX induction is removed, thus plant possesses a machinery of self-adjustment to maintain normal pattern of development when confronts with harsh environment.We further examined the expression and localization of several auxin efflux carrier protein, and found that PIN1 and PIN7 proteins played an active role in regulating auxin dynamics in root tips. After DEX induction, the expression of PIN7 protein had extended to basal part of lateral root cap, however, they remained normal polar distribution within cells, which may result in the abnormal DR5-GUS signal in lateral root cap by transporting extra auxin into lateral root cap. We did not observe any changes in either expression region or polar distribution of other PIN family proteins (PIN1, PIN2, and PIN3). However, the expression level of PIN1 and PIN7 protein in stele cells within the root tip was obviously lower than in the control roots, which could account for the decreased DR5-GUS signal in this region because of the reduced capacity of acropetal auxin transport.3. After DEX induction to transiently disrupt polar auxin transport during embryogenesis, we observed a general lag of embryo development compared with the controls, and a slight expansion in radial axis of hypocotyls and cotyledons as well. Some embryos had uncoordinated cell division and even defected in establishing apical or basal patterns. These suggested that apical-basal pattern formation of embryos relys on proper polar auxin transport within them, and is quite sensitive to changes of auxin dynamics in both apical basal differentiation and directional elongation.4. During primary root growth, transient interruption of auxin transport and distribution initially caused inhibition of primary root elongation; whereas this effect restored normal shortly after the auxin transport and distribution in root tip went back to normal. Further analysis showed that although the division and differentiation of meristem cells remained unaffected, the elongation of root cells in root elongation zone was severely inhibited. The abnormal accumulation of auxin in lateral root cap may played an important role in inhibiting the elongation of cells underneath the lateral root cap. The inhibition caused by transient disruption of auxin dynamics did not persist, as the elongation of both root cells and primary root recovered to the same level as in the controls after the re-establishing of normal polar auxin transport in root tips. These results referred that morphogenesis during primary root growth responds rapidly to transient manipulation of auxin dynamics, meanwhile the determination of cell fate was unchanged, therefore the effects are reversible.5. Transient impairing auxin transport and accumulation severely inhibited lateral root formation, due to inhibition of both initiation and development of lateral root primordia. Moreover, the inhibition persisted even after DEX induction was removed. Decreased auxin level in the cells of meristem zone may account for this inhibition, since addition of auxin during DEX induction could efficiently restore both initiation and development of lateral root primordia. Those part of primary root, which had no lateral root formation, never grew new lateral roots again even after the seedlings had recovered on a medium without DEX for 2 weeks. It had already been shown that establishment of proper auxin distribution pattern was very important for organogenesis. In addition, our results demonstrated that it was also critical for organogenesis to establish this auxin distribution pattern at proper time. Once the cells have missed the moment for fate determination, they will never catch up the development program. It seems that there is a significant difference between organogenesis (such as lateral root formation) and organ growth (eg. primary root growth) in the reliance on auxin dynamics.6. Further observation found that lateral root formation was more sensitive to changes in auxin dynamics than primary root growth was. By controlling concentration of DEX in the induction experiments, we disrupted auxin transport and accumulation at different level. It was observed that low concentration of DEX could already severely inhibit the formation of lateral roots, however, the inhibition of primary root elongation happened at higher DEX concentration. Besides, transient interruption of auxin dynamics had a permanent inhibiting effect on lateral root formation, but not on primary root elongation. These results emphasize the importance of observing natural responses of different plant tissues in aspects of cell fate determination and cell differentiation by means of adjusting auxin dynamics in relative small scale.7. The phenotypes of seedlings after suppression of GNOM protein expression by DEX induction were not always consistent with gnom mutants. It can be interpreted from that the defects in gnom mutants are produced by accumulative effects, but the phenotype after transient suppression of GNOM may be a natural response of plant to change of auxin dynamics.In summary, by transiently interrupting auxin dynamics through an inducible anti-sense GNOM expression system, we find that organogenesis and organ growth has different reliance on auxin dynamics, and they differently respond to changes of auxin dynamics. Besides, the sensitivity of plant cells to auxin is variable, which always changes with their development program, thus indicates a temporal effect and non-repeatability of the role of auxin dynamics in specifying cell fates. |
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