Effect And Mechanism Of Combined Application Of Indole Acetic Acid And Kinetin On Cadmium Hyperaccumulation In Solanum Nigrum L.

Cadmium（Cd）is a common element among many heavy metals that cause soil pollution.Application of exogenous plant hormones can effectively alleviate the damage caused by heavy metals to plants.Many achievements have been made in the effects of single plant growth regulator（PGRs）on the growth and soil remediation efficiency of hyperaccumulators.However,there are few studies on the application of two PGRs to a certain hyperaccumulator,as well as on the influence of culture time and spraying method of PGRs on one hyperaccumulator.Therefore,in order to study the effect and mechanism of the application of indole acetic acid（IAA）and kinetin（KT）on Cd hyperaccumulator of Solanum nigrum L.,firstly,an orthogonal experiment with 2 factors and 4 level in a pot trial was designed.Different concentrations of IAA(0,15,25,35mg·L^-1)and KT(0,10,20,30mg·L^-1)were applied to S.nigrum L.which grown in soil containing 3.9 mg·kg^-1Cd,and the best ratio of two PGRs with the highest extraction efficiency of Cd by S.nigrum was selected.Then,based on the best ratio of two PGRs,IAA and KT were sprayed to S.nigrum in different periods（the fifth and seventh leaf germination）and in different ways（only spraying soil,only spraying leaves,spraying both soil and leaves）,the best time and method of PGRs spraying for the best Cd extraction efficiency of Solanum nigrum were determined,and the changes of physiological and biochemical indexes,including photosynthetic pigment contents,root activity,antioxidative enzyme activities and malonaldehyde（MDA）content,in Solanum nigrum were also measured.At the same time,the physiological and biochemical changes in Solanum nigrum were also investigated at CK（no PGR application）,single PGR（IAA or KT）and combined PGR treatment.Finally,under the condition of the best ratio of PGRs and the best time and method of PGRs spraying,the changes of subcellular distribution and chemical forms of Cd in plant were analyzed.The results showed as follows.（1）The combination of IAA and KT could promote the rapid growth of S.nigrum grown in soil containing 3.9 mg·kg^-1Cd compared to that of the control,and the plant height,Cd concentration,Cd extraction efficiency and bioconcentration factor were significantly increased at 35 mg·kg^-1IAA combined with 10 mg·kg^-1KT treatment.Although the extraction efficiency of Cd was only 0.49%,it was about 2.3times higher than that of the control group.Therefore,35 mg·kg^-1IAA combined with10 mg·kg^-1KT was the best ratio to improve the extraction efficiency of Cd in S.nigrum.（2）At the optimal ratio of 35 mg·kg^-1IAA combined with 10 mg·kg^-1KT and the fifth leaf germination,the plant height,dry weight of each part,Cd concentration in the shoots and the extraction efficiency of Cd were significantly increased when the soil and leaf were sprayed at the same time.Under this optimal conditions,the biomass,photosynthetic pigment content,leaf CAT and POD activities of S.nigrum were the highest.The result from stepwise regression analysis showed that Cd extraction efficiency was positively correlated with dry weight of shoot and chlorophyll b content of S.nigrum.At the treatment of IAA combined with KT,the root activity,photosynthetic pigment content and activities of antioxidative enzymes in S.nigrum were significantly increased compared to those of single application of IAA or KT,and the MDA content was decreased more significantly at combined treatment of two PGRs.（3）Combined application of IAA with KT could distribute more Cd in the cell wall and vacuoles of S.nigrum,especially in vacuoles,which strengthened the compartmentation of Cd in the cells of S.nigrum.In vacuoles,Cd ions could combine with metal ligands and the toxicity of Cd to plant cells was alleviated.When IAA and KT were used in combination,the proportions of deionized water-extracted Cd（mainly water soluble organic acid-Cd and phosphate-Cd）and Na Cl-extracted Cd（mainly pectate-,protein-bound and adsorbed-Cd）in leaves of S.nigrum were increased significantly,and Na Cl-extracted Cd in plant roots was also increased significantly.Cadmium may bind with organic acids to form chelates,which reduces the toxicity of Cd.It also combines with proteins to form a stable metal organic complex,which effectively reduces the free Cd in leaves and avoids the damage caused by oxygen free radicals to cells.In conclusion,35 mg·kg^-1IAA combined with 10 mg·kg^-1KT was the best ratio to improve the extraction efficiency of Cd in S.nigrum.At this best ratio of PGRs,the best spraying method was the fifth leaf germination and the simultaneous spraying of soil and plant leaves.By doing so,the Cd extraction efficiency was the highest and it was positively correlated with dry weight of shoot and chlorophyll b content of S.nigrum.Combined application of IAA with KT could distribute more Cd in the cell wall and vacuoles of S.nigrum,especially in vacuoles,which strengthened the compartmentation of Cd in the cells of S.nigrum.