Cytological Observation And Epigenetic Variation During Cell Differentiation In Root Of Maize

Maize(Zea mays L.),its genome size is about 2.5 x 103 Mb.Maize is not only an important economic crop but also commonly used as a model organism for genetic research.In recent years,the differentiation and development mechanism of embryonic stem cells has been a research hotspot in animal studies.However,there are few researches on tissue differentiation in plant.In this paper,the differences between the meristem zone and differentiation zone of maize root tip during apical cell differentiation were investigated and the related studies were employed based on cytology and epigenetic inheritance.1.Differences on cytological level between the meristem zone and differentiation zone of maize root tipFirstly,the root tip meristem zone and differentiation zone were distinguished by the Feulgen staining method.Then,the differences in nucleus of the two areas were observed by the DAPI staining method.The results show that the areas of nuclei in the meristem zone are smaller and more uniform than those of nuclei in the differentiation zone.Moreover,the nuclei containing chromocenter in the two zones is detected.The chromocenter is less and smaller in the meristem zone with relatively looser chromatin,while the chromocenter is more and larger in the differentiation zone with more condensed chromatin.In addition,there are significantly differences in the nucleolar area of the two zones.The nucleolus in the meristem zone is larger,while in the differentiation zone the nucleolus is smaller.Moreover,the ratio of nucleoli to nuclei is relatively larger in the meristem zone than that of the differentiation zone.2.Differences on chromatin configuration and whole-genome DNA methylation levels in nuclei between the meristem zone and differentiation zone of maize root tipThe nucleus chromatin configuration and genome-wide DNA methylation level differences between the meristem zone and differentiation zone in maize root tips were detected by the micrococcus nuclease sensitivity experiment and HPLC method.The results indicate that the chromatin of meristem zone is more sensitive,looser,and easily be cut into short pieces with lower genome-wide DNA methylation level,while the chromatin of the differentiation zone is more condensable with higher genome-wide DNA methylation level.3.Differences on epigenetic level of Knob and 45S rDNA sequence in the nuclei between the meristem zone and differentiation zone of maize root tipAccording to the above results,the two apparent differences are the chromocenter and nucleolus between the meristem zone and differentiation zone in maize root tips.The chromocenters are mostly composed of repeats in which mostly are knob repeats.The 45S rDNA repeats sequence is an important component of nucleolus and participates in the formation of ribosome.Therefore,we mainly study the epigenetic differences of these repeates sequences in the nuclei of meristems and mature nuclei.(1)Differences on epigenetic level of Knob repeats in the nuclei between the meristem zone and differentiation zone of maize root tipKnob mainly includes 180-bp and 350-bp repeats sequences.The chromatin configurations of the two repeats were tested by means of CHART-PCR(Chromatin Accessibility by Real-Time PCR).The results show that the chromatin configurations of the two repeats is relatively looser in the meristem zone,while that in the differentiation zone is more condensed,which is verified by the results of fluorescence in situ hybridization(FISH)experiments.Then the DNA methylation levels of Knob repeats were analyzed by the bisulfite sequencing technology.There are three types of DNA methylation(CG,CHG and CHH)in plants.CHG in Knob-180-bp repeat,CHH and total DNA methylation levels in Knob 350-bp repeat are significantly higher in differentithan zone than those of meristem zone.(2)Differences on nucleoli activity and epigenetic level of 45 S rDNA sequence in the nuclei between the meristem zone and differentiation zone of maize root tipNucleoli are involved in the synthesis of ribosomes,processing and cutting of pre-rrna and other life activities.Silver staining showed that the meristem zone had more silver staining granules than the differentiation zone,indicating higher levels of nucleolus activity.Next,the signal distribution of fibrillan in the meristem zone and differentiation zone of maize root tips using fibrillan as the antibody was analyzed by the fluorescence immunostaining technology.The result showed that the nucleoli are large and round,and the fibrillan signal is bright in the meristem zone.However,the signal in the differentiation zone is weak only scattered around the nucleolusm,which is consistent with that of DAPI staining.These results verify that the meristeme nucleoli activity is much higher than that of the differentiation zone.The result of CHART-PCR demonstrate that the chromatin configuration of 45 S rDNA is relatively looser in the meristem zone,while that of the differentiation zone is more condensed,which is also verified by FISH experiment.The methylation level of CG in 45S rDNA sequence of the meristem zone is lower than that of the differentiation zone with significant differences in some special sites.However,the methylation level of CHG and CHH is very low and has no significant difference in the two zones.Above all,chromatin remoded during cell differentiation in plant root tips,which resulted looser chromatin and lower level DNA methylation in the meristeme zone,condend chromatin and higher DNA methylation level in the differentiation zone.