Study Of Trichoderma Asperellum T6 On Arabidopsis Thaliana Growth Promotion Under Calcium Stress

Calcium is involved in regulating signal transduction,growth and development in the organism,and it is one of the essential nutrients in plant life activities.The content of calcium in the lithosphere is rich,but most of calcium exists in the form of insoluble calcium,such as calcium carbonate(CaCO₃),which can not be directly absorbed and used by plants.The crop yield and quality were declined because of the calcium deficiency.Therefore,it is urgent to explore effective ways to solve the calcium deficiency and insoluble calcium stress.Trichoderma spp.can promote plant growth and increase plant resistances of stress,it is a rhizosphere probiotic fungus which has attracted much attention and great application potential.Our laboratory has found that T.asperellum Q1 can produce siderophore under Fe-deficiency stress,which not only transforming insoluble iron in the environment for absorpting and utilizating by plants,but also increasing the content of auxin in roots of plant and promoting plant growth.However,there is no research on whether T.asperellum can promote the growth of plants and the mechanism of growth under calcium stress.In this study,on the basis of results that calcium can affect the growth of Arabidopsis thaliana,it was found that inoculation of T.asperellum T6 can promote the growth of Arabidopsis thaliana under calcium stress.Then the effects of different concentrations of calcium on the content of siderophore and IAA in T.asperellum T6 were measured,and the effects of CaCl₂,NaCl and pH on the expression of the key gene for the synthesis of siderophore?SidA?and the catalyzed subunit gene of calcineurin?CnA?gene in T6 also were researched.The results preliminarily identified the growth-promoting mechanism of T.asperellum on Arabidopsis thaliana under calcium stress and the regulatory relationship between siderophore synthesis and calcineurin pathway.The specific results are as follows:?1?The effects of different concentration of soluble and insoluble calcium on the germination of Arabidopsis thaliana seeds,the root length and endogenous IAA content of transgenic Arabidopsis thaliana were determined.The results showed that it is beneficial to Arabidopsis thaliana seed germination,root growth,and endogenous IAA production,when the calcium ion concentration is between 1 mmol/L and 3 mmol/L.All of too high or too low calcium ion concentrations and the insoluble calcium?CaCO₃?can inhibit plant growth.?2?To further study the effect of calcium on the expression of IAA in the roots of Arabidopsis thaliana,the paper were analyzed the effects of Ca²⁺and CaCO₃ on the expression of auxin gene family?YUCs?by qRT-PCR for the first time.The results indicates that calcium stress can reduce the relative expression of YUCs gene family in the root of Arabidopsis thaliana,which was consistent with the reduction of endogenous IAA fluorescence intensity obtained by using the DR5::GFP transgenic type.?3?The growth-promoting effect of T6 on Arabidopsis thaliana DR5::GFP seedling under low calcium stress and CaCO₃ stress were studied.Results show that it can promote the root length and endogenous IAA production of Arabidopsis thaliana after indicating T.asperellum T6.Compared with the control,Arabidopsis thaliana root length increased by 75.31%(0.5 mmol/L Ca²⁺),55.91%?0.5 g CaCO₃?,88.94%?1 g CaCO₃?,and the endogenous IAA content in the roots also increased significantly.it means that T.asperellum T6 can promote the expression of IAA in Arabidopsis thaliana roots,and T6 can relieve the damage of calcium stress on plants and promote plant growth.?4?We have determined the effects of different Ca²⁺and CaCO₃ concentrations on the yield of T6 siderophore and IAA,the results shown that both of low calcium stress and CaCO₃ stress could accelerate the siderophore content of T6,the final content was increased by 9.40%(0.5mmol/L Ca²⁺),2.95%?0.5 g CaCO₃?and 3.2%?1 g CaCO₃?,but there was no significant effect on IAA yield.These results preliminarily presumed that one of the mechanism of T.asperellum T6 promoting plant growth under calcium stress maybe by its siderophore.?5?Since the expression of calcineurin gene is related to the resistance of microorganism,this paper uses semi-quantitative PCR technology to study the relationship between SidA and CnA in T.asperellum T6 under abiotic stresses such as NaCl,CaCl₂,and pH.The results show that high concentration of sodium chloride and high pH can inhibit the expression of SidA gene and increase the expression of CnA gene,and the expression of SidA gene is up-regulated and the expression of CnA gene is down-regulated under low calcium stress.It suggested that there is a negative regulatory relationship between SidA gene and CnA gene.This finding initially indicates that abiotic stress factors may affect the siderophore biosynthesis through the calcineurin pathway.It provide a reference for future research the resistance mechanismnd and the siderophore synthesis mechanism in T.asperellum T6.