Photosynthetic And Physiological Response To Shade And Genetic Screening Of Glycine Soja

In recent years,the maize-soybean intercropping system has been rapidly spreaded in China.The planting mode guarantees the production of soybeans,improves the utilization rate of solar energy and the land equivalent ratio,and increases farmers’income and agricultural production in the case of no reduction or reduced production of corn.However,under the maize-soybean belt-like intercropping mode,the soybeans are shaded by the corn plants from the seedling stage,leading to excessive elongation of the internodes,increased plant height,thinning of the stems and easy lodging,etc.Soybean growth and production are seriously threatened.Therefore,the mechanism of soybean response to shade and the cultivation of shade-tolerant soybean need further work.Glycine soja is a closely relative species of Glycine max,because it grows in an environment where plants shaded,which has the characteristics of low requirements for light,and it is not artificially domesticated,and it contains abundant stress-resistant gene resources,which can be used as a material for excavating genes about shade-tolerant.Therefore,in this study,Glycine soja,Glycine max Nandou 12 and Nan032-4 were used as experimental materials to study their morphological and photosynthetic characteristics under shade.Meanwhile,gibberellin GA₃ and its inhibitor paclobutrazol PAC,auxin IAA and its inhibitor3-iodobenzoic acid（TIBA）,brassinolide（24-epiBL）and its inhibitor brassinazole（BRZ）were discussed about their effects of pretreatment on the phenotype and photosynthetic and physiological characteristics of Glycine soja seedlings in a shaded environment.The aim is to preliminarily determine the response of wild soybeans to the light environment,and provide theoretical support for the subsequent mutant screening work of using wild soybeans as mutagenic materials.And then,this study used EMS and ⁶⁰Co-γ-ray irradiation to mutagenize Glycine soja,and initially selected some mutants with variant phenotypes,which provided potential experimental materials for excavating new stress-resistant related genes.It also established a theoretical basis for cultivating shade-tolerant soybean varieties and the mechanism of soybean response to shaded environments.The main results are as follows:1.Morphological characteristics and photosynthetic physiological characteristics of Glycine soja and Glycine max under shading stressCompared with white light,the plant height of the three soybean plants grown in the shade increased significantly,but the increase of the Glycine soja was greater than the other two Glycine max.It is indicated that Glycine soja are more sensitive to shaded environments than Glycine max in plant height changes.Under the shading treatment,the area of the two cultivated soybean leaves decreased to different degrees,while the area of the two wild soybean leaves didn’t decrease significantly.The reason may be may be due to the area of wild soybean leaves itself small,resulting in no significant difference from that of the wild soybean leaves growing under normal light.After shade treatment,the maximum photochemical efficiency,electron transport efficiency,actual photochemical quantum efficiency of PSII and non-photochemical quenching coefficient of Glycine soja were consistent with the change of shade-tolerant variety Nan032-4.both photosynthetic organs are greatly affected by the shade stress.However,the electron transfer efficiency,actual photochemical quantum efficiency of PSII and non-photochemical quenching coefficient of the shade tolerant variety Nandou12 hardly changed.And the maximum photochenical efficiency increased,indicating that the photosynthetic mechanism of Nandou12 was least affected by shade stress.Compared with Glycine max,the content of Glycine soja‘s photosynthetic pigment was most affected by shade,and the content of all three photosynthetic pigments was largely inhibited by shading.2.Effects of hormone and inhibitor pretreatment on shade avoidance in Glycine soja（1）Effects of IAA and TIBA on Glycine soja seedlings’phenotype and photosynthetic characteristics under shadeIn white light environment,IAA treatment can significantly increase the Glycine soja seedlings’height;in the shade environment,IAA treatment has no effect on plant height.Compared with the control,TIBA-treated Glycine soja showed no significant change in plant height under white light,and plant height in the shade was significantly reduced.IAA pretreatment can improve the photosynthetic efficiency and heat dissipation ability of Glycine soja leaves in white light or shaded environment,but has no significant effect on chlorophyll content,actual photochemical quantum efficiency of PSII and photosynthetic electron transport rate.This indicates that IAA pretreatment has no significant influence on the shade-avoiding response of wild soybeans in terms of plant height variation,and its corresponding transport inhibitor TIBA pretreatment weaken the shading response of wild soybeans;from the change of photosynthetic physiological indicators,IAA Pretreatment can improve the photosynthetic capacity of wild soybeans under shading and protect the photosynthetic apparatus from damage.（2）Effects of 24-epiBL and BRZ on the phenotype and photosynthetic characteristics of Glycine soja seedlings under shadeCompared with the control,24-epiBL significantly promoted the plant height in both light environments;while BRZ treatment had no effect on the plant height in the white light environment,and significantly inhibited the plant height under shade environment.24-epiBL pretreatment can enhance the capacity of photosynthetic and heat dissipation of Glycine soja leaves in both white and shaded environments.In the shade environment,24-epiBL pretreatment can enhance the photosynthetic efficiency and light energy utilization of Glycine soja leaves.After 24-epiBL pretreatment on Glycine soja seedlings,the chlorophyll a content increased significantly in shaded environment,but didn’t change significantly under white light;chlorophyll b content increased significantly under white light,and there was no significant change in shading environment;carotenoid content was in significantly decreased under white light,there was no significant change in the shade;the Chla/b ratio decreased significantly in the white light environment,and there was no significant change in the shaded environment.This indicates that 24-epiBL pretreatment can significantly enhance the sensitivity of wild soybean in the shaded environment from the aspect of plant height.The corresponding synthetic inhibitor BRZ pretreatment can weaken the sensitivity of wild soybean in the shaded environment.From the change of photosynthetic physiological indicators,24-epiBL pretreatment has a certain promoting effect on the photosynthetic capacity of wild soybean under shading.（3）The effects of GA₃ and PAC on the phenotype and photosynthetic characteristics of Glycine soja seedlings under shadeCompared with the control,GA₃ significantly promoted the plant height in the two light environments;while the PAC treatment did not have an influence on the plant height under the white light environment.In the shaded environment has a significant inhibitory effect on plant height.In the white light environment,GA₃ pretreatment improves the photosynthetic electron transport,heat dissipation,photosynthetic efficiency and actual photochemical quantum efficiency of PSII;in the shaded environment,GA₃ pretreatment destroys the photosynthetic electron transfer process and reduces its heat dissipation.And also reduced heat dissipation capability,photosynthetic efficiency and electron transfer rate.Under white light,GA₃ treatment had a great influence on the photosynthetic pigment content of plants.SPAD,chlorophyll a content,chlorophyll b content and carotenoids were significantly reduced.Under the shade,PAC treatment had a greater effect on the photosynthetic pigment content of plants.Among them,SPAD,chlorophyll a content,chlorophyll b content and carotenoids all increased significantly.This indicates that GA₃ pretreatment can enhance the sensitivity of wild soybean in the shaded environment from the changes of plant height and photosynthetic physiology.The corresponding synthetic inhibitor PAC pretreatment can weaken the sensitivity of wild soybean in the shaded environment.Gene expression analysis showed that PAC treatment could up-regulate the expression of photosynthetic pigment synthesis and photosynthetic electron transport related genes GsCAO,GsPSAE1 and GsPSAG in wild soybean under shading.This indicates that the PAC pretreatment can deepen the leaf color and photosynthetic pigment content of wild soybean under the shaded environment,which is mainly achieved by the up-regulated expression of genes related to photosynthetic pigment biosynthesis.3.Screening of Glycine soja mutantsGlycine soja was induced by ⁶⁰Co-γ-ray irradiation mutagenesis at a dose of 200 Gy for 5 h.The emergence rate and survival rate of M1 generation were 63.4%and 43.8%respectively.194 mutations with variant phenotype were screened in M2 generation.The EMS mutagenesis experiment determined that 1%EMS（ethyl methane sulfonate）treatment for20 h was the best EMS mutagenesis condition for Glycine soja,and the M1 generation emergence rate and survival rate were 64.2%and 30%respectively.A total of 71 mutants with a variant phenotype were screened in the M1 generation.In summary,this paper compares the sensitivity of wild soybeans and cultivated soybeans to the shaded environment.It is known that wild soybeans are more sensitive to shading than cultivated soybeans;at the same time,the effects of pretreatment of hormones and inhibitors on the shading response of wild soybeans were further confirmed,and it was further verified that wild soybeans were more sensitive to the shading environment.The above research provides theoretical support for the selection of mutagenic materials in the subsequent screening of soybean mutants.Finally,the appropriate EMS mutagenesis treatment combination was screened out and some mutants with trait mutations were obtained.Among them,the mutant strains M2-31-1 and M2-2-1,as wild soybean materials with weak sensitivity to shading environment,provide the basic materials for excavating soybean new genes and changing the response of plants to light environment.