H₂S Signal Regulates The Growth Of Arabidopsis Hypocotyls In Response To Blue Light

Light is one of the basic environmental factors for plant growth and development.Photoreceptors play an important role in regulating growth and development of plants,which is in the way of mediating light signal transduction pathway.The blue light receptors cryptochromes are involved in physiological processes such as hypocotyl inhibition,photoperiodic regulation of floral initiation,development of stomatal and chloroplast,regulation of circadian clock,and tropism growth.Hydrogen sulfide has been identified as a gasotransmitter and proved to participate in various physiological and biochemical processes and also response to biotic and abiotic stresses.It was reported that one of the mechanisms of hydrogen sulfide is post-translational modification of proteins?S-sulfhydrylation?on the cysteine residues.In this paper,wild-type Col,blue light receptor mutants cry1,cry2,and cry1/2 of Arabidopsis were used as experimental materials.The seedings were exposed to blue light for 5,10,or 15 minutes to detect H₂S content,H₂S production rate,and transcription changes of H₂S producing enzymes encoding genes.The effects of H₂S on seed germination and hypocotyl elongation under dark or blue light were detected also.An improved biotin switch method was also provided for detecting the level of protein sulfhydrylation.The experimental results were as follows:?1?Biotin switch assay.According to Mustafa's manuscript,two extra steps of precipitating with acetone and resuspending with HEN buffer need be performed,while in this paper using the improved method could reduce the repeated steps of precipitation and resuspension and then reduce the loss and content error of protein samples.Besides,this method is suitable for the detection of total plant protein or purified recombinant protein from E·coli.?2?Effect of blue light on H₂S content in Arabidopsis.The H₂S content of wild-type Col and mutant cry1 increased significantly after 5 minutes of blue light irradiation but it continued to increase in Col at 5-15 minutes.The rising rate in cry1 was faster than Col and the H₂S content of cry1 was stable at 5-10 minutes.The H₂S content of cry2 increased in 5-15 minutes,but the content and the rising rate were lower than those in Col and cry1.The H₂S content did not increase significantly from dark to blue light exposure for 15 minutes in cry1/2 mutant.?3?Effect of blue light on H₂S production rate in Arabidopsis.During the first 5 minutes of light irradiation,the H₂S production rate in Col increased,and gradually decreased to the level in dark,and then remained stable.It was not increased significantly in cry1 within the first 10 minutes of blue light irradiation.There was no significant change in cry2 and cry1/2 mutants during the 15 mins of blue light treatment.?4?At-LCD phosphorylation detecting.After blue light treatment,the total protein of Arabidopsis reacted with the purified His-LCD protein,and there were weak phosphorylation bands on Western blotting after 40 min and 60 min reaction.?5?Effects of blue light on transcription of endogenous H₂S-producing enzyme genes in Arabidopsis.After blue light treatment,the expression levels of At-LCD and At-DES1 in Col,cry1,and cry2 were increased,while not changed in cry1/2.?6?Effects of exogenous H₂S on Arabidopsis germination under blue light.10 ?mol/L H₂S inhibited germination under dark conditions,while showed no significant effectiveness under blue light.?7?Effects of exogenous H₂S on the hypocotyl elongation of Arabidopsis.There was no difference in the length of the hypocotyl of each plant material under the same H₂S treatment concentration.40,60 ?mol/L H₂S inhibited the hypocotyl under dark conditions,and showed more obvious effect under blue light conditions.In addition,when treated with blue light,the higher the concentration of H₂S,the shorter the hypocotyl length.