Functions Of TCH1-3Genes In Root System Mechanoresponses In Arabidopsis Thaliana

The functions and morphogenesis of plant root systems are directly affect plantsgrowth and development. In a variety of soil environmental factors, such as, rockobstacles, soil resistance, gravity and the contact between roots and soil stimulation,represented by mechanical stimulation is one of the abiotic stress factors that focused bybotanist. Therefore, the correctly responses to mechanical stimulation of plant roots arethe premise and basis of plants survival and development.Previous studies have reported that there are four TCH genes (touch induciblegenes) involving in Arabidopsis thaliana mechanical touch responses. Among the genes,the Calmodulin (CaM) and Calmodulin-like protein (CML) are encoded by TCH1-3genes, which have major roles in pathway of mechanoresponses. However, thefunctions of TCH1-3in Arabidopsis thaliana roots mechanoresponses remain unclear.Therefore, in the present study, we based on the reverse genetic theory and comparedthe root systems mechanoresponses behaviors among tch1（cam2-1、cam2-3）、tch2（cml24-2、cml24-4）、tch3（cml12-2）mutants and wild type (WT), to explore thepotential functions of TCH1-3in Arabidopsis thaliana mechanical touch responses andpenetration abilities.Firstly, the wild-type Arabidopsis thaliana were cultured on the inclining agarsurfaces, and the inclining angles were0,15,30,45and60from vertical.According to the length and growth direction of primary root, and the developmentsituation of lateral root, we got the best angle of culture and built a loading method oftouch stimulation. The results provided evidences that altered mechanical environmentcould significantly influence WT Arabidopsis thaliana roots growth behaviors. Besides,we also found that when the angle was30from vertical, roots growth and developmentwere significantly better than those of others. Therefore, we selected the30as the bestloading method of touch stimulation for subsequent experiments.Furthermore, we cultured tch1-3mutants and WT Arabidopsis thaliana on theinclining (30) hard agar surfaces and compared roots growth and development betweenthe mutants and WT to explore the potential roles of TCH1-3in Arabidopsis thalianaroots touch mechanoresponses. Results showed that two tch2mutants, cml24-2andcml24-4, exhibited significantly reduced root length and decreased density of lateralroot (DLR) phenotypes. In addition, primary root length and DLR of tch3(cml12-2) were also significantly decreased on inclining agar surfaces. Observation of growthdirections found that the cml24-2and cml24-4exhibited significant left and rightskewing, respectively. These results indicates that the normal functions of TCH2andTCH3are necessary for the mechanical response of Arabidopsis thaliana root, and theCML24-4, CML24-4, and CML12-2protein have the major roles in keepingArabidopsis thaliana primary roots and lateral roots growth and development.The gravitropism of plant roots is one of the most important physiologicalcharacteristics. The situations of synthesis and distribution of starch grain in the root capcells directly affect the directions and behaviors of plant roots. Therefore, in order tobetter understanding the skewing growth behaviors of tch1-3primary roots under touchmechanical stimulation, we stained and observed the starch grain in root cap cells of tch1-3and WT. Results showed that the synthesis and aggregation extent of starch grains incml24-2and cml24-4were less than that of WT. Therefore, these results indicates thatthe functions of CML24-2and CML24-4can regulate the directions of primary rootspossibly through having roles in the synthesis of starch grains in root cap cells.The penetration growth of Arabidopsis thaliana primary roots in different hardnessagar medium can mimic the primary roots growth in the soil. In order to investigate thephysiology functions of TCH1-3in primary roots penetration growth, we cultured thetch1-3and WT Arabidopsis thaliana in stacking agar medium layers with differentconcentrations of agar. Firstly, we tested the elastic modulus of agar medium, and gotthe constitutive relationship between concentration of agar medium and elastic modulusof agar medium. Besides, after testing the penetration ratio (PR) of primary roots, theresults showed that when the concentration of upper/lower layer is1.0%/0.5%(2times), the PR of WT Arabidopsis thaliana primary root was highest (96%6.255%).Finally, comparing with the PR of tch1-3mutants and WT Arabidopsis thaliana, thePRs of cml24-2and cml24-4were lower than that of WT, indicating that tch2mutantprimary roots were more sensitive in response to mechanical properties of growthcondition than wild type Arabidopsis thaliana. Therefore, we believed that the TCH2gene could have the important roles in penetration growth of primary roots.