| The Stay-green mutation of plants is a phenomenon that chlorophyll is not degraded or degraded slowly at the late breeding period,etiolation does not occur after plants maturation,and plants still stay green.Currently,a number of stay-green mutants have been discovered or obtained by artificial mutation in many crops.It is believed that differences exist among formation causes and mechanism of various stay-green mutants.Most of the researches on stay-green mutants focus on mechanism of formation of mutants and analysis of structure and function of mutant genes.Besides,in the minority of crops such as wheat and rice,some researches on carbon-nitrogen metabolism,antioxidant physiology,and stability of photosynthetic system were conducted.However,reports on physiological and biochemical characters and relevant molecular mechanism of soybean stay-green mutants in the process of aging remain few.By work of our team for years,a new stay-green soybean cultivar named Jinda Zhilu 1 was bred with the female parent of Jinda74 which is super-high-yield and the male parent of Z-Lurenshuangqing which is a natural stay-green mutant.Jinda Zhilu 1 combines excellent traits of the two parents,has obvious stay-green characteristics in leaves and performs better than the parents in yield and quality.In this study,from the perspectives of antioxidation,glycometabolism,and photosynthetic physiology,effect of the stay-green mutant on leaf aging and relevant physiological and molecular mechanism was studied at the key stage of formation of seed yield in Jindazhilu1 and its parents after blooming.Also,research was conducted on germination characters of different genotypes of soybeans under stress of PEG.The results are as following.1.PEG stress obviously inhibited the traits of germination percentage,germinative force,length of radicel and hypocotyl of seeds of different cultivars(lines)of soybeans.Weight of radicel and transformation rate of storage materials of seeds declined.Compared with non-stay-green parents,the stay-green one performed better under the stress of osmosis,relative germinative force,relative germination percentage,relative length of radicel,radicel/ hypocotyl index were significantly higher than those of the non-stay-green ones.By comprehensive evaluation of drought resistance of different cultivars(lines)of soybeans at budding stage using subordinate function,Jindazhilu1(Z1)was a strong drought resistant cultivar,and its parents were medium drought resistant cultivars.2.Due to stay-green mutation in leaves,completeness and stability of proteins in reaction center of photosystems and proteins combining light-harvesting pigment were enhanced in the process of leaf aging,dissociation of chlorophyll from pigment combining proteins was reduced,damage of photosynthetic structures was declined,utility rate of luminous energy and efficiency of photosynthetic electron transfer were increased.All of the differences and changes performed in the chlorophyll fluorescence parameters.Compared with non-stay-green cultivars,chlorophyll degradation of stay-green cultivar was inhibited resulting in green staying in mature leaves instead of etiolation.On the other hand,the stay-green cultivar reserved higher light-harvesting and light-delivering ability ensuring it to maintain high level of photosynthetic rate for a long period,especially at the seed-filling stage,higher photosynthetic capacity means better yield performance.3.In the key period of yield formation of soybeans,the levels of antioxidant protective enzymes in the stay-green cultivar was high which declined accumulative degree and velocity of active oxidant material such as H2O2、O2-,lessened oxidant damages,and prolonged aging of leaves.Molecularly,transcription of SOD isogenes such as Mn-SOD,chloroplast Cu/Zn-SOD was higher than that of the non-stay-green cultivar JD74.The change of APX isogenes of cultivars was similar,but APX isogene of Z1 expressed more at the late stage.MDHAR and DHAR isogenes had a same co-expression pattern,but two genes of JD74,MDHAR2 and DHAR4,were inhibited from 29 th to 42 nd day after blooming,so did CAT1,which was because contents of H2O2 of JD74 increased rapidly at the period.Additionally,under the darkness treatments,activities of antioxidant enzymes declined,the balance of production and clearance of POS was broken,soluble protein and chlorophyll were degraded,leaves turned yellow.The stay-green cultivar Z1 was not affected by darkness and could endure longer of darkness.4.The stay-green mutation had obvious effect on expression of genes of glycometabolism,focusing on the stage of seed-filling.At this stage,the expression level of sucrose phosphate synthase SPS4 in the stay-green cultivar was higher that in the no-stay-green cultivar JD74,indicating the stay-green cultivar had higher sucrose synthesis ability.SPS3 had low expression,assuming it had little effect on sucrose.Among invertase Inv participating sucrose splitting and isogenes of sucrose synthase SS,CInv,CWInv1,SS2-1,and SS2-2 had same expression pattern.The stay-green cultivar had higher expression amount and sucrose splitting ability.Hxk and Frk,the isogenes of Hexokinase and Fructose kinase had higher expression amount and provided supplement hexose phosphorylation substrate for glycolysis,providing more energy and medium mebabolism products for plants’ growth.In the non-stay-green cultivar JD74,Hxk and Frk and had low expression resulting in accumulation of hexose and leaf aging.In the reproductive growth stage,especially the maturation period,the stay-green cultivar had higher expression of SUTs,sucrose transporter isogene,which help transport sucrose into seed rapidly,ensuring soluble sugar content in leaves decreased and lower than that in the non-stay-green cultivar JD74.5.The results indicate that the stay-green mutant stg is the SGR1/SGR2 double mutant.Deletion mutation was occurred in SGR1 gene,and shortened the second exons sequence,encoded the SGR1 protein without function or weaker function.Insertion mutation in SGR2 result in the variation of function of SGR2 protein.In sum of the theory above,the stay-green mutation of soybeans could prolong leaf aging.Especially in the key period of yield information such as seed-filling stage,stay-green mutation had positive effect on antioxidant protective enzymes,photosynthetic structures and capacity,glycometabolism.The cultivar Jindazhilu1 bred by cultivar improvement performed better than parents in aspects.The outcome of the study had theoretical and substantial significance on germplasm innovation and high-yield breeding strategy of soybeans. |
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