Functional Analysis Of Agaricus Bisporus Para-aminobenzoic Acid Synthase Gene In Arabidopsis Thaliana

Agaricus bisporus is a kind of edible mushroom.It is widely cultivated as it has high yield property and is consumed largely.Temperature is the main restricted factor for its growth.It thus has great economic and ecological values to study its thermotolerance mechanism and to breed a novel thermotolerant strain.Comparing the differentially expressed genes between thermal-insensitive A.bisporus 02 and conventional A.bisporus 8213 by integrative transcriptomic,para-aminobenzoic acid synthase gene(Pabs)was screened in our laboratory.It encodes a protein regulating the biosynthesis of para-aminobenzoic acid(PABA)in mushroom.PABA is a precursor of folate synthesis to maintain the fungi growth.Significantly,it also can enhance heat tolerance of mushroom.PABA as a mediator of environmental stress israrely reported.We obtained Pabs transgenic Arabidopsis by floral dip protocols.We further investigated the morphology changes and the tolerance-improving mechanisms of mushroom-derived Pabs on the perfect platform of Arabidopsis.After being introduced into Col-1 mediated by Agrobacterium tumefaciens,Pabs can be integrated into the Arabidopsis genome DNA and stably inherited to the offspring.It can express the correspondingly active protein(PABS)which promotes the synthesis of PABA in plants.Pabs overexpressing plants displayed at best 3 times PABA increase compared to wild type.PABA can regulate the growth and development of Arabidopsis thaliana.Aminodeoxychorismate synthase gene(Adcs)is a key gene of PABA biosynthesis in Arabidopsis.Homozygous Adcs mutants lead to defective embryos.In this study,overexpression of Pabs did not result in any obvious phenotypic alteration in mature Arabidopsis,except its slight inhibition on primary root growth.It suggested that PABA may affect the growth of Arabidopsis roots.Exogenous PABA(100 ?mol/L PABA)significantly decreases the length of Arabidopsis roots,and promotes the development of root hairs.In addition,PABA can lead to an accumulation of auxin in root apical.Our further research found that overexpression of Pabs induced ROS generation in root apical,especially in lateral root cap and columella(COL).We proposed that Pabs might act as a novel regulater of the Arabidopsis roots growth and development via ROS.ROS is a central messenger in a variety of signal transduction networks.The alteration of ROS homeostasis not only regulated the growth and development of organisms,but also improved the plants tolerance to biotic stress and abiotic stress.In Arabidopsis,we found that over expression of Pabs can induce the expression of Pathogenesis-related-1 gene(PR-1),while SA content was not changed.Both overexpression of Pabs and exogenous PABA reinforced the thermotolerance of mushroom.However,there was no significant morphologic difference between Pabs overexpressing lines and wild type in heat stress.Interestingly,under normal conditions,overexpression of Pabs slightly induced the expression of HSP101 and HSP70.After heat shock,the relative increase of HSP70 expression in Pabs plants was significantly higher than that in wild type.Our experiments indicated that the improvement of HSPs expression induced by Pabs did not likely alter the phenotype of Arabidopsis in heat stress.Interestingly,we also found that Pabs transgenic Arabidopsis was hyposensitive to UV-C irradiation.Pabs overexpression lines exhibited reduced root growth inhibition and cotyledon bleach upon UV-C stress.UV-C radiation could lead to the accumulation of PABA,which indicated that PABA was involved in response of UV-C stress.Reduced UV-C-induced DNA damage was observed in Pabs lines suggesting overexpression of Pabs may protect DNA against UV-C exposure.We assumed that PABA could enhanced plants resistance to UV-C in more than one aspect.PABA can directly absorb UV-C reducing the damage of UV-C radiation on cell.It also promotes ROS generation which in turn acts a signal to increase antioxidant enzymes SOD and APX activity in plants.Higher activity of SOD and APX prevented the accumulation of ROS induced by UV-C.Finally,PABA induced the DDM1 expression,maintaining the stability of the genomic DNA.In summary,this thesis showed that the PABA synthase gene of Agaricus bisporus could be stably and inherited expressed in Arabidopsis thaliana,increasing the PABA level of Arabidopsis.Besides its role in folate synthesis,PABA may act as an active plant hormone to participate in the growth,development and environmental resistance of Arabidopsis by regulating the balance of ROS.Details of the underlying molecular mechanisms are needed to facilitate genetic improvements of crops.