Studies On The Mechanism Of Arabidopsis Endophytes Bacillus Sp. LZR216 Affectting Arabidopsis Growth And Development

Endophytes are important microbial resources.Endophytes do not interfere with normal growth of host plants and do not cause diseases in hosts.Sometimes,endophytic microbes can promote plant growth.In this project,we used the model plant Arabidosis as the experimental material.We isolated and identified endophytes from Arabidosis and studied the mechanism of endophytes in affecting Arabidopsis growth and development.The results are as follows:1.Isolation and identification of endophytes in Arabidopsis thaliana and their promotion on Arabidopsis growth.We isolated five endophytes from different tissues?roots,stems and leaves?of Arabidopsis:one from roots?named Bacillus sp.LZR216?,two from stems?named Bacillus sp.LZS1-216 and Bacillus sp.LZS2-216?,and two from leaves?named Bacillus sp.LZL1-216 and Bacillus sp.LZL2-216?.Furthermore,we found that all five endophytes can promote the growth of shoots?including an increase in leaf area and fresh weight?and roots.No interference on growth promotion among different endophytes.Mixed endophytes can not only promote the growth of shoots and roots,but alos promote the accumulation of soluble sugars in Arabidopsis seedlings.To study the mechanism of growth-promoting effect of endophytes,we chose endophyte LZR216 which shows significant growth-promoting effects.Microbes produce active substances to affect plant growth and development.We used liquid chromatography-mass spectrometry to detect whether metabolites of LZR216 contain hormones auxin?IAA?,gibberellin?GA₃?,zeatin,and abscisic acid?ABA?.Results showed that the metabolites of LZR216 do not contain the four kinds of plant hormones,suggesting LZR216 may produce other regulatory substances to promote plant growth.Previous studies have shown that 3-hydroxyl-2-butanone can promote the growth of Arabidopsis thaliana.We used gas chromatography-mass spectroscopy to examine LZR216 metabolites and detected 3-hydroxy-2-butanone.Furthermore,exogenously applied 3-hydroxy-2-butanone?5?M?promoted the growth of Arabidopsis.2.LZR216-regulated root growth and development is dependent on auxin signaling and polar auxin transport in Arabidopsis seedlings.LZR216 not only promotes the shoot growth,but also alters the root system structure,including inhibition of primary root elongation,increased lateral root numbers,and increased root hair length and density.LZR216 suppressed primary root elongation by reducing CYCB1;1::GUS expression in root tips and shortening the length of the meristem region of root tips.It has been shown that auxin plays important roles in the development of roots.Using the auxin responsive reporter gene DR5::GUS and DR5::GFP,we examined the roles of auxin in the process of LZR216-promoted root growth.Results showed that LZR216 enhanced the DR5::GUS and DR5::GFP expression in root tips.Furthermore,LZR216 notably induced the transcriptional levels of NIT1,TAA1,and YUCCA1?auxin biosynthesis-related genes?compared to the control.These data implied that LZR216strengthens f auxin response through,at least partially,enhancing the transcription of auxin biosynthesis genes.In addition,we used a series of auxin signaling and polar auxin transport mutants to testify the roles of auxin in the process of LZR216-altered root system architecture.Compared to the wild type,LZR216 did not inhibit the primary root growth of both auxin signaling mutants?axr2 and arf10,16?and polar auxin transport mutants?aux1-7 and pin2?.and did not increase lateral root numbers of polar auxin transport mutant aux1-7 and auxin signaling mutant axr4-1.Using qRT-PCR,we found that LZR216 down-regulated AUX/IAA transcription inhibition factor genes?IAA12,IAA13 and AUX1?,but up-regulated auxin response factor genes?ARF5,ARF19?and auxin transport genes?PIN1,PIN2 and PIN3?in roots.Using transgenic lines PIN1-GFP,PIN2-GFP,PIN3-GFP,and AUX1-YFP,we detected the protein abundance of PIN1,PIN2,PIN3,and AUX1.Results indicated that LZR216down-regulates abundance of PIN1,PIN2,PIN3,and AUX1 protein.These results showed that LZR216 uses different ways to regulate transcription levels and protein levels of PINs and to achieve the regulation of root system architecture.In addition,we found that auxin transport inhibitor NPA eliminated the roles of LZR216 in primary root elongation and lateral root numbers.Similarly,NPA relieved the down-regulation of abundance of PIN1/PIN2/PIN3/AUX1 proteins by LZR216.In summary,an intact auxin signaling pathway in Arabidopsis is necessary for LZR216-regulated root development.3.LZR216-promoted Arabidopsis growth and development involves cytokinin signaling.Cytokinins play important roles in microorganism-regulated growth and development of the host.We used cytokinin receptor,Arabidopsis histidine phosphotransfer proteins?AHP?family and Arabidopsis response regulator?ARR?family mutants to investigate the roles of cytokinin signaling in LZR216-promoted growth and root development.The results showed that LZR216 did not promote shoot growth nor altered the root system architecture in ahp2,3,5 mutant seedling.Then we used transgenic plants ARR5::GUS and qRT-PCR method and found that LZR216enhanced the ARR5::GUS expression in roots,but decreased the ARR5::GUS expression in shoots.Furthermore,LZR216 up-regulated the transcript abundance of AHK3/AHK4,AHP1/AHP3 and ARR4/5/7/10/12/15 genes in roots,but down-regulated the transcript abundance of AHK3/AHK4,AHP1 and ARR4/5/7/10/12/15 genes in shoots.In summary,LZR216 strengthened the cytokinin signaling in roots but weakened the cytokinin signaling in shoots to achieve the promotion of shoot growth and alteration of root development.An intact cytokinin signaling pathway is necessary for LZR216-promoted plant growth and altered root system architecture in Arabidopsis.4.Plant growth promotion and root system architecture alteration by LZR216involves ethylene signaling.Ethylene plays important roles in microbe-plant interactions.First,we added ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid?ACC?to examine the effects of ethylene.Results showed that ACC eliminated the effects of LZR216-increased leaf areas.When the concentration of ACC was 10?M,LZR216did not inhibit the primary root elongation.When the concentration of ACC was 1?M,lateral root numbers of LZR216-treated seedlings were almost the same as the control.Then,we used the ethylene signaling mutants and transgenic lines EIN3-OX to test the roles of the ethylene signaling pathway in the process of LZR216-regulated growth.The results showed that LZR216 promoted the shoot growth and affected root development of ethylene receptor mutants?etr1-3,etr1-7,etr1-6/etr2-3,and etr2-3/ein4-4?and ethylene signaling mutants?ein2-1,ein3-1,and ein3-1/eil1-1?,but did not affect the root development of transgenic line EIN3-OX.LZR216 increased the transcript level of ACC synthase ACS2,ACS4,ACS6,ACS7,and ACS8 genes in roots.We also found that LZR216 increased the transcript level of EIN3,ERF1 and ERF2 genes in roots.Collectively,we conclude that plant growth promotion and root system architecture alteration by LZR216 involves ethylene signaling.5.LZR216 interplay with auxin and ethylene controls the primary root growth in Arabidopsis seedlingsEthylene biosynthesis precursor ACC can inhibit Arabidopsis primary root elongation,but ethylene synthesis inhibitor aminooxyacetic acid?AOA?has little effects on primary root growth.ACC affects the growth of the primary root mainly through the influence of auxin biosynthesis and redistribution.So,we used DR5::GUS as the research material to examine the roles of ethylene-auxin interaction in LZR216-altered root system architecture.ACC induced the DR5::GUS expression in the elongation zone of Arabidopsis root tips,but LZR216 eliminated the DR5::GUS expression of the elongation zone.LZR216 also eliminated the ACC-increased expression of PIN1,PIN2,PIN3,and AUX1 proteins.These data implied that LZR216 acts downstream of the ethylene signaling.Furthermore,we examined the roles of the ethylene signaling and auxin signaling in LZR216-regulated primary root growth.The results showed that LZR216 did not suppress the primary root growth of pin2 mutant,whether ACC,or 1-Naphthalene acetic acid?NAA?was added.However,LZR216 was able to inhibit the primary root growth of ethylene signaling mutants etr1-3 and ein3-1.Collectively,polar auxin transport plays a very important role in LZR216-regulated primary root growth and LZR216 acts downstream of ethylene to affect the growth of primary roots.