Identification Of The NADPH Oxidase Complex Encoding Genes In Volvariella Volvacea And The Mechanism Of The Abiotic Stress Response

The Straw mushroom Volvariella volvacea is an important(sub)tropical mushroom which has a very high nutritional value and its massive cultivation(the third largest produced mushroom in the world)represents a large economic value.Abiotic stress conditions such as low temperature inhibit mushroom formation and can even cause autolysis,which is a big obstacle for the production and preservation of Volvariella volvacea.Better understanding of abiotic stress will help to improve cultivation and post harvest preservation.NADPH oxidases,originally found in animal neutrophils and macrophages,are a major oxidizing enzyme complex in animals,plants and most microbial organisms.It produces reactive oxide species(ROS)which in turn are important for adverse stress.However,the mechanism of how fungi respond to abiotic stress remains unclear.Single spore strains PYd15,PYd21 and heterokaryotic strain H1521 of the edible fungus Volvariella volvacea were used in this study and we identified six NADPH oxidase complex genes based on the recently completed genome sequence.Abiotic stress induction in hyphae from H1521 was performed in order to study the NADPH oxidase stress response mechanism.The results of this study provide a new direction for studying abiotic stress responses in edible fungi.Following are the main results of this study:(1)Six NADPH oxidase complex components were identified of which the gene sequences and structure were analyzed.The names and lengths of these genes are:VvNoxA(2836bp),VvNoxB(3122bp),VvNoxR(3199bp),VvBemA(2515bp),VvRacl(1406bp)and VvCdc24(4277bp).(2)Subsequent analysis showed that all of the oxidase subunits possess conserved domains but no signal peptides.Five out of six have at least one transmembrane domain(VvBemA has no transmembrane domain)and VvNoxA and VvNoxB each contain seven transmembrane domains which is characteristic for membrane proteins.(3)In order to determine appropriate abiotic stress conditions,we performed survivability tests of H1521 hyphae at different pH,temperature and H2O2 and NaCl concentrations.The results showed that pH=8 is optimal for H1521,while the limits for strain H1521 lie at pH=4 and pH=ll,0.3mol/L NaCl and 10mmol/L H2O2.Incubation of hyphae at 4? from 0 to 4 hours followed by continued cultivation at 35? showed that hyphae could restore growth,yet at decreased speed.1h low temperature incubation showed a significant difference with control conditions and was determined to be a good stress time in the later study.(4)qRT-PCR was used to test gene expression levels of the six genes after treatment with different abiotic stresses for 1h(pH=4,pH=11,0.3mol/L NaCl,10mmol/L H2O2and 4?).Only Cdc24 showed a large increase in expression(about 7-17 times).It suggests that VvCdc24 may have some other roles in responding to the abiotic stress.In order to better understand the role of Cdc24,we analyzed the gene expression patterns during different growth periods and different periods of low temperature treatment.Gene expression of Cdc24 was found to change during different hyphal growth stages.Expression was high during the first two days of cultivation in PDA,down-regulated at the third day and up-regulated from the fourth to twelfth day.Different periods of low temperature(0 to 60 min at ?)revealed expression patterns of Cdc24 similar to the Gst01 gene,and both genes show changed expression in relation to the concentration of H2O2.These results suggest that Cdc24 and Gst01 could participate in the same signal pathway controlled by H2O2.(6)Using DPI,DDC and Vc to change the ROS concentration of hyphal cells we found that DPI(50mmol/L)not only inhibits the concentration of O2-produced by NADPH oxidase,but also effectively restrains the up-regulation effect caused by abiotic stresses pH=4,pH=11,0.3mol/L NaCl and 4?.However,it only partly restrains up-regulation induced by 10mmol/L H2O2.We hypothesize that H2O2 can directly enter the cell and acts as a signal molecule that induces Cdc24 gene expression.Both DDC(1mmol/L)and Vc(2mmol/L)effectively reduce the H2O2 content of hyphae,but show different abilities in restraining the up-regulation effect caused by low temperature(4?,1h).Vc absolutely inhibits up-regulation caused by low temperature,but DDC does not.According to the function of DDC and Vc,we suggest that both O2-and H2O2 can induce Cdc24 up-regulation.Taken together,the results indicated that the NADPH oxidase can generate reactive oxide species such as 02-and H2O2,and these ROS can act as signaling molecules that induce expression of NADPH Oxidase genes.In this way,the cell can make a series of adaptive changes to suit new environmental conditions.During this process,Cdc24 can act as a subunit of NADPH oxidase,triggering the formation of ROS while on the other hand,ROS up-regulate Cdc24 gene expression.