Effects Of Ethylene Regulation On The Vegetative And Reproductive Growth Of Soybean (Glycine Max (L.) Merr.)

Plant growth and development have been affected by a wide range of environmental conditions.In order to meet the complex and ever-changing external conditions,the plant in the long-term evolution has developed a variety of gene expression system to regulate and adopt to cope with the challenge from environment.Modulation of ethylene biosynthesis of the plant is a fundamental physiological response to the fluctuations in the internal or external cues.The soybean cultivar Tiefeng-31 was used to study the effects of ethylene on the growth and development of soybean.Ethylene emission and the relative gene expression of 1-aminocyclopropane- 1-carboxylate synthase gene(ACS) were detected during the soybean growth and development;on the other hand,RNAi technology as well as ethylene regulator aminoethoxyvinylglycine(AVG) and 1-aminocyclopropane-1-carboxylic acid(ACC) were used to study the physiological function of ethylene during soybean seed germination and plant growth.(1) The ethylene production and the ACS gene expression were detected at different development stages and multiple soybean tissues,including vegetative and reproductive abscission zones and flanking tissues during full flowering stage of soybean.There is a significantly differences of ACS gene expression and ethylene emission at the different stages and different organs of soybean.The results showed that ethylene emission was at the late stage of the plant life cycle,and mainly produced by the aging organ.There is a maximum emission of ethylene at the soybean ripening period,especially before soybean harvest.The abundance of expression of ACS showed similar rules.Different concentrations of ethylene inhibitor AVG and promoter ACC were sprayed to soybean cultivar Tiefeng 31,and their effects on plant photosynthesis and CO₂ response curve were measured.The results showed that through the treatments of AVG,net photosynthetic rate,transpiration rate and stomatal conductance were significantly increased;intercellular CO₂ concentration were markedly decreased;light saturation point,photosynthetic rate at light saturation point,apparent quantum efficiency,CO₂ saturation point,carboxylation efficiency and maximum RuBP regeneration rate were markedly increased;light compensation point,CO₂ compensation point were significantly decreased.ACC,the precursor of ethylene showed an opposite effects. In addition,the chlorophyll contents were significantly enhanced.The results above indicated that AVG treatment could improve photosynthesis characteristics of soybean seedlings.And with the improvement of photosythesis characteristics,the plant height and the dry weight of seedlings were significantly enhanced by AVG treatment.The ACC treatment showed the opposite effects.(2) The relationship between ethylene production and metal mobilization and transport was investigated.Soybean seeds were incubated with or without inhibitors of ethylene synthesis AVG or in the presence of the natural ethylene precursor ACC,the ddH₂O as control. The kinetics of ethylene emissions,the ethylene key enzyme gene ACS expression,the ACS enzyme activity and the concentration and extractability of metal in axis tissues were measured.The results showed that the ACS gene relative expression,the ACS enzyme activity and ethylene evolution reached the peak value during the visible stage of germination (pericarp splitting) and the prolonging stage of the hypocotyls.Correspondingly,the Zn²⁺ concentration and extractability in axis tissues significantly increased.The concentration of Fe²⁺ and Mn²⁺ showed no obvious change before prolonging stage but declined at the 7^th day,and their extractability showed no rule change.The ethylene inhibitors inhibit the ethylene emission;promote the axis prolonging and Zn²⁺ concentration and HCl-extractability. The ACC showed the contrary effect.These findings suggested that,the triple response in soybean affected by ethylene may be regulated the concentration of Zn²⁺ into axis tissues from cotyledon or HCl-extractability in axis tissues.(3) Different concentrations of ethylene inhibitor AVG and promoter ACC were sprayed to soybean,and their effects on soyben flower traits,pollen morphology and yield were studied.Results showed that total number of flower,flower length,petal length, stamina column length,pollen quantity,pollen tube length,straw weight,pod number per plant and yield increased along with higher AVG concentration,while abortion ratio of pollen decreased.ACC,the precursor of ethylene showed an opposite effect.Both AVG and ACC treatments had no significant effects on pollen germination ratio.Scanning electron microscope(SEM) observation results showed that AVG treatments promoted pollen length,while distorted deformation occurred after ACC treatments.(4) A linear gene cassettes direct transformation method was developed and used in transient expression of a GUS reporter gene in onion epidermal cells and soybean cotyledon nod system,respectively.Linear gene cassette was labeled by 5'-fluorescent dye(fluorescein isothiocyanate,FITC) and its delivery pathway was traced.The basic transformation solution used was Murashige and Skoog basal salt mixture(MS) liquid medium.Hypertonic pretreatment of explants and transformation cofactors,including Ca²⁺,surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated.Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h,the onion low epidermal explants were pre-cultured in darkness at 27℃for 48h.After transformation for 3h,the explants were transferred to MS solid media for 72h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei.By GUS staining and flow-cytometry-mediated fluorescent detection,a significant increase of the ratios of fluorescent nuclei as well as expression of the GUS reporter gene was observed by each designed transformation solution.This potent and feasible method showed prospective applications in plant transgenic research.(5) RNAi vector of conservative region from ethylene biosynthesis limiting enzyme ACS gene was constructed.The liner DNA cassettes of RNAi-ACS acquired by PCR action was used as the transformation elements and the soybean cotyledon nod was transformed as the explants to study the effect of ethylene on flower bud differentiation of soybean.The results of PCR and RT-PCR showed that the linear RNAi-ACS gene cassettes have entered into the cotyledons node of soybean.Ethylene emission and ACS gene expression abundance was significantly inhibited after the cotyledon node was transformed.Compared with the control,more flower bud was differentiated at the cotyledon section.The above results showed that the ethylene affects soybean growth and development through its lifecycle.The typical ethylene triple response was relative with the Zn²⁺ concentration and extractability in axis tissues regulated by ethylene.And inhibition of ethylene in soybean increased soybean yields by improving photosynthesis characteristics, promoting the vegetable growth,and enhancing the forage quality and quantity.The results presented above provide a general theoretical basis for the soybean cultivation and breeding.