Theoretical And Experimental Study Of Regulation Of Arabidopsis Root Development And Growth By Mechanical Environment

Growth,development,and morphogenesis of plant regulated by mechanical environment is a significant and theoretical and practical topic.However,there are few researches related to quantitative analysis of root growth behavior and its controlled mechanical environment.This study focuses on establishment of experimental model with controllable mechanical environment,and investigation of the adaptability of root organ,tissues and cells during root development in medium resistances through this experimental model,improving the understanding of relationship between plant growth and mechanical environment.This study has important theoretical significance and potential application value for plant development and production.The main research contents and results are as follows:The establishment of a experimental model of controllable mechanical environment for root development regulation was based on the change of gel concentration in growth media.Mechanical properties of media with gradient concentrations were systematically measured for model evaluation.The rheological properties of agar and Phytagel were explored by uniaxial compression testing,together with rotational rheometer.For 0.5-2.0% agar and Phytagel media,the variation of gel concentrations resulted in different mechanical strength.There was a good linear relationship between the observed data points of breaking load and gel concentrations,indicating that gel media with gradient concentration could provide gradient mechanical strength in root growth environment.The spline regression model fits to the Young's modulus for agar and Phytagel media data quite well,indicating that significant differences of stiffness exist among the media with various concentrations,thus quantitative mechanical environment for root growth can be provided through changing gel concentrations.0.5-1.2% agar and Phytagel media were viscoelastic structures based on results that elastic modulus > viscous modulus.Increasing agar concentrations resulted in a stiffer structure at 0.5-1.2%.So these agar media could used for the design of gradient resistance during root helical growth.Therefore,the experimental model of controllable mechanical environment was verified to be rational and feasible.The relationship between root growth and mechanical environment was evaluated by growing Arabidopsis in 1.0-2.0% agar and Phytagel.Correlation between phenotypes of 4-6 days old roots and mechanical parameters was not found.In agar media,7-day-old root length reached the maximum value at 1.3% agar concentration.Negative correlations between root length and fracture stress,between straightness and Young's Modulus were found in 1.0-2.0% agar.In Phytagel media,7-day-old root length decreased with Phytagel concentration increasing from 1.0% to 2.0%.Root length reached the maximum value in 1.0% Phytagel concentration.There were negative correlations between root length and fracture stress,between root length and Young's Modulus,and between straightness and Young's Modulus in 1.0-2.0% Phytagel.Measurement of one-month-old plant electrolyte leakage rate reflects plant growth.Resutls displayed that electrolyte leakage rate were <45% in both media,thus 1.0-2.0% agar and Phytagel media were suitable for the primary root penetration growth.Therefore,plant development was affected by the response of root to penetration resistance.Varying amounts of agar and Phytagel have been used successfully to regulate root elongation after 7 days growth.The effects of media rheology on root elongation enriched the root expansive growth theory.Controllable mechanical environment were designed with different concentration agar(0.6,0.8,1.2,1.4,1.6,1.8%)and Phytagel(0.6,0.8,1.0,1.2,1.4,1.6,1.8%)to simulate the gradient penetration resistance during root growth.Under continued,quantitative mechanical stress,root tip shape,fluorescence intensity,and cortical microtubules(CMTs)orientation of epidermal cells of elongation zone(EZ)for GFP-MBD seedlings were characterized in vivo microtubule visualization.Microtubule visualization revealed mechanical environment-dependent difference in root tip that fluorescence intensity and cell division in EZ of roots in agar media are greater than those in Phytagel media.Root diameter of 7-day-old Arabidopsis in different media exhibited significant difference,and roots in Phytagel even showed significant difference in root tip length.Growing in media with gradient mechanical strength CMTs orientation of epidermal cells in EZ of 3-day-old roots were clearly different.In order of low to high strength of media,the patterns of CMTs of 3-day-old root were consisted with the morphology of 7-day-old root during root penetration.For quantitative study the role of auxin in the process of root responsing to mechanical stimulation from media,Arabidopsis wild-type Ws,DR5-GUS,and pin1 were planted in 1.0-2.0% Phytagel.Auxin expression and distribution in the root tip of each medium were founded by GUS staining method.Increasing medium resistance,the scope of auxin distribution in root tip narrowed to the root cap area.Growing in the gradient strength phytagel for 7 days,the average root length of Ws was much longer than that of pin1,and average root length of both genotype decreased remarkably with the media strength.This difference was not absent in both genotype,which consisted with a hypothesis that PIN1 may have a role in thigmotropism of Arabidopsis primary roots via regulation of cell elongation.To explore the mechanisms of root circumnutation,Arabidopsis root waving and helix were investigated by loading roots quantitative mechanical stimulation from agar media.Roots were cultivated on inclined hard agar surface and inside agar.Root waving and helix observed,and the shape of root were quantified.(1)To examine the role of forces in root waving,we used a physical approach,to analyze forces acting on the root apex.Root waving occurs mostly through the contribution of growth force,and resistance could promote and inhibit the root waving.(2)For root helix observation,Arabidopsis roots were cultivated in 0.5-1.2% agar media with gradient stiffness which lead to gradient resistance during root penetration.Roots were preferred to develop a helical pattern in agar media with concentration from 0.5% to 1.0%.As stiffness of agar increased,the percentage of helical roots and helix diameters in each agar medium declined;root lengths and auxin distributions varied.We demonstrate that the size of the helical deformation decreases with agar stiffness as expected by the analysis based on a combination of growth-induced mechanical buckling.Therefore,helical roots growth is driven by growth force,and the resistance from agar media impacts the properties of root helix.Growth force and external mechanical forces modulate root phenotypes in Arabidopsis.It is an equilibrium between the intrinsic rolling and the restoring resistance force.In conclusion,how mechanical environments impact on root growth and development have been investigated successfully through this model.Under quantitative mechanical stress,varying patterns of root tip shape,microtubules orientation of epidermal cells and auxin distribution consisted with the change of root morphology.Furthermore,the important role of mechanical resistance played in mediating roots waving and circumnutation was proved by experimantal and theoretial analysis.