| Plant cell division is an important life feature of multi-cellular plants,since it is the basis of growth and tissue renewal.Variety of factors are involved in the regulation of plant cell division,such as plant hormones-cytokinin,auxin,transcriptional regulators of cell division and cellular machineries for cell division such as microtubules and their regulators.Although many reports have explained the regulatory mechanism of plant cell division,mechanisms to coordinate activation and inactivation of cell division with organ growth are not well understood yet.Arabidopsis thaliana leaf epidermis contains several differentiated cells,such as stomatal guard cells,trichomes and pavement cells(PC)for gas exchange.The PC in Arabidopsis thaliana have different concave and convex shapes,and they fit into each other to form a stable structure to maintain the homeostasis of Arabidopsis epidermis.The shape of the PC in the abaxial and adaxial axes of Arabidopsis thaliana leaves is different,and they are separated by several layers of marginal cells.Recent studies have shown that marginal cells in cotyledons or leaves have meristem activity,especially at the early stage of development.However,how margin cell meristematic activity is activated or repressed along with development of cotyledons or leaves is unknown.CLE(CLAVATA3/Embryo surrounding region-related)is a kind of important plant polypeptide hormone,which participates in the signal transmission between cells during plant growth and development,especially plays a key role in the balance between stem cell proliferation and differentiation in SAM(Shoot Apical Meristem),RAM(Root Apical Meristem),and vascular(Pro)cambium.In the SAM of Arabidopsis thaliana,CLV3 acts as a ligand for receptor kinases such as CLV1 or BAM1.This ligand binding leads to inhibit the transcriptional level of WOX gene in the SAM and thus inhibit the number of stem cells.Interestingly,we have found that BAM1 and WOX1 are contributing to the regulation of margin cell division.However,the mechanism of CLE and its receptor system regulating the development of marginal cells is not clear.Therefore,we aim to explore whether there are similar signal regulatory pathways in the marginal cells of leaves to regulate the development and growth of leaves.Many methods have been found to observe cell division.Edu staining has the characteristics of simple,rapid and better protection of cell morphology,and can accurately detect the replication activity of DNA.However,this method has no cell specificity and thus unsuitable to observe the mitotic activity of specific cells.Another method to observe cell division is to use the cell cycle marker protein-GUS/GFP to label specific cells,the expression of related genes can be quickly known by staining or microscopic observation.And the mitotic activity can be known through the expression of specific genes in specific cells.However,recent studies revealed that marker expression and cell division is not always coincide well.These prompted us to establish novel method for accurately detecting cell division in a cell type specific manner.We found that PIN2 is specifically expressed along margin cells in a transient manner.Previous studies have found that Tub protein shows dynamic localization change upon cell division.Based on this,we constructed a genetic system for real-time observation of margin cell division,using these two marker genes: PIN2-GFP and Tub6-mcherry to observe the proliferation and division of marginal cells in real time.This method only needs to construct transgenic plants through vectors and observe the proliferation and division of marginal cells in real time with the help of confocal microscope,which provides another method for understanding the development of marginal cells.By utilizing physiological experiments of synthetic peptides combined with our new methods,we found that CLE19 and CLE25 may act as positive regulators of marginal cell development.At the same time,we found that exogenous CLE19 and CLE25 peptides enhanced the expression of PIN2,indicating that CLE19 and CLE25 could promote the transport of auxin and promote the division of marginal cells.The numbers of marginal cells increased significantly in bam1 mutant and wox1 mutants,indicating that BAM1 and WOX1 is a negative regulator of marginal cell development.CLE peptides can regulate the growth and development of marginal cells,and BAM1 and WOX1 are also involved in this process.Our results suggest that there is a CLE19/25-BAM1-WOX1 regulatory network in cotyledon marginal cells,and there is another auxin pathway to regulate the development of cotyledon marginal cells.Our findings provide a new mechanism for understanding the development of marginal cells.At the same time,they proved utility of our novel method to detect cell division regulatory mechanism in specific tissues. |
PDF Full Text Request