The Function Of Hydrogen Sulfide In Soybean Nudule Senescence

Hydrogen sulfide?H₂S?,known as the third type of gas signal molecule after nitric oxide?NO?and carbon monoxide?CO?,plays an important role in plants growth as well as the process of plants responding to a variety of biotic and abiotic stresses.However,the effect of H₂S involved in regulating the nitrogen-fixing symbiotic system of soybean plants and on their nitrogen fixation and senescence process remained unknown.Therefore,this study intends to explore the regulation mechanism of H₂S in the senescence of soybean root nodules and nitrogen fixation efficiency.In order to get a clear view of H₂S's effect on nodules senescence,we use soybean?Glycine max cv.Zhonghuang 13?as plant material.Then we analysed plant growth phenotype,nitrogen fixation and nitrogenase activity,protein and gene expression.The main results are as follows:1.Plant physiology assaySoybean?Zhonghuang 13?was grown in a greenhouse with inoculation of Sinorhizobium fredii Q8 stain.Three weeks later,H₂S was added in the treatment group.Samples were collected at six periods?3-8 weeks,at increments of 1 week unit?to estimate the physiological indexes of root and shoot dry weight,root nodule number and senescence ratio,root and shoot dry weight,chlorophyll content,root nodule nitrogenase activity,plant ammonium nitrogen,nitrate nitrogen and total nitrogen content.The results showed that with treatment of H₂S,the dry weight of soybean plants increased and the decrease of chlorophyll content was slower.Simultaneously,the number of nodules increased but the proportion of nodules senescence decreased.What's more,nitrogenase activity began to decrease at 5weeks.And 6wi witnessed a sharp decrease in nitrogenase activity as well as nitrogen nutrient fast transfered to legumes.Our data illustrated that H₂S could influence the nodules'nitrogenase activity,and thus prevent nodule senescence in soybean plants.2.Histological assay of nodulesParaffin sections were carried out to observe the infection of Q8 in the nodules and the arrangement of the symbionts under different treatment conditions?observed at 6 periods?.The sections indicated that there was no significant difference between the distribution of the symbionts in the nodules and the infection of Q8 during 3 to 5 week under the treatment of H₂S.However,from the 6th week,the symbionts in the nodules began to disappear which contribute to an empty cavity and then formed a central senescence zone in the control group.The senescence zone of nodules only appeared at 7wi as a response to the H₂S treatment,whereas the senescence zone of the control group was enlarged and some nodules structure partially destroyed.The ultrastructure of nodules was analyzed using transmission electron microscopy to observe the morphology of the bacteroids in individual infected cells.It was observed that at 3wi,there are few bacteriods in both groups.But,H₂S increased the number of PHB.At 5wi,the arrangement of the bacteriods in the two treatment groups became tight,and there was no significant difference.However,the bacteroids of Q8 began to degenerate,and the vesicles showed voids after bacteroid degradation.At that time,Q8+NaHS had no obvious aging characteristics.At 7wi,the edge of the Q8 bacteroid was observed to shrink,and the number of rod shape bacteroids was transformed to spherical shape due to senescence,and the voids of the vesicles became larger.At the same time,Q8+NaHS also began to show the symptom of degeneration.H₂S treatment delayed the senescence of nodules and prolonged the activity period.3.Western-blot assayThe antibody of the nitrogenase iron-protein?NifH?and glutamate-ammonia ligase-like protein?NodGS?were prepared.The total protein of six stages of soybean root and nodule was extracted and subjected to Western-blot.It indicated that after treatment with H₂S,the protein NifH in the nodule is highly accumulated in the 3-6 weeks when the nitrogenase activity is high,and gradually decreases since the 7th week.In the control group,NifH was less in 3-4wi,and peaked at 5-6wi and a decrease appeared at 7wi.And the quantitative analysis showed that the NifH content was also lower than that in H₂S treatment group.NodGS was expressed more after treatment with H₂S treatment,and it was more expressed in the early stage and then decreased gradually.The most significant difference of NodGS expression between Q8 and Q8+NaHS was at 4 and 5wi.The quantitative analysis showed that both proteins the control group expressed were lower than that the H₂S treatment group.It indicates that exogenous application of H₂S may enhance the role of nodulin protein in the development and signal transduction of primary root and nodule,but it can not prevent the declined expression during senescence,but it can keep nitrogenase activity for a long time and prolong the nitrogen fixation cycle.Plant gene expression analysis4.Plant gene expression analysisThe soybean roots and nodules RNA of the above periods were extracted,and the relevant gene-specific primers were designed.Then the genes related to plant senescence and nitrogen fixation were analyzed by qRT-PCR.It was found that the application of exogenous H₂S interferes with the expression of nucleoporin genes associated with rhizobia infection and increase the expression of related symbiotic genes such as Ca²⁺signaling.Simultaneously,it was found that the expression of some cysteine proteases decreased and superoxide dismutase genes in the roots and nodules of the plants increased after the addition of exogenous H₂S.That is to say,H₂S delayed the ageing process of plants by affecting the genes expression involved in the establishment of the symbiotic system and the senescence process of the nodules.