| Banana(Musa acuminata)is one of the important staple food crop in the world.Originally,banana is tropical crops,and the optimal cultivation temperature condition was between 24 ~ 35 ?.Because of climate change,banana plants have to endure many abiotic stresses including high-temperature stress which limits the growth and development when the temperature is higher than 35 ?.Piriformospora indica(P.indica),an endophytic root fungus,similar to that of Abuscular Mycorrhizal Fungi(AMF),sustains the growth,development and enhanced tolerance of plants to biotic and abiotic stresses.However,little is known about the physiological and molecular mechanism of high temperature stress response in banana leaves when inoculated with endophytic fungus.In this study,the cultivated variety “Tianbao” banana of Fujian is used as the material.The changes of physiological and biochemical,transcriptome analysis and differential expression analysis of small RNA in banana under high temperature stress after colonization by the endophyte fungus P.indica were studied.The purpose of this study was to explore physiological,biochemical and molecular mechanisms of P.indica to improve the heat tolerance of banana,and to provide scientific basis for further using P.indica a beneficial endophyte fungus.and to find a feasible method in the form of comprehensive management and chemical control of high temperature,which is helpful for plant growth and development and can resist high temperature stress.At the first time,this research,determinated and comparised the changes of antioxidant activity or antioxidant molecules' contents and phytohormone contents in leaves of “Tianbao” banana colonized and non-colonized by P.indica before and after of high temperature(45 ?)treatment for 0 h,3 h,6 h,12 h,24 h,and 3 d.At the second time,the BGISEQ-500 sequencing platform was used to perform transcriptome sequencing and small RNA sequencing analysis on banana leaves under normal temperature(28 ?)and high-temperature stress(45 ?,3 d).It is planned to explore the response pathway and heat tolerance mechanism of banana high temperature stress.Provide the scientific basis for digging banana high temperature-resistant genes and breeding high temperature-resistant varieties.The main results are as follows:1.Physiological and biochemical mechanism of the leaves of “Tianbao” banana colonized with Piriformospora indica under high temperature stressEvaluations involving plant height,leaf number,leaf area magnitude,and pseudostem circumference showed to be affected by the presence of the symbiotic fungi under both conditions.Our findings showed banana growth and development to be significantly higher under P.indica treatment.Mean leaf number,leaf area magnitude,pseudostem circumference,and plant height of potted banana colonized with P.indica were significantly higher than the non-colonized ones.Also,the application of the fungi proved to improve the antioxidant capacity of the plant which is crucial for plant tolerance.High activity for many enzymatic procedures tested was observed before and after high temperature treatments.The H2O2 contents in leaves of banana colonized and non-colonized by P.indica both trend to increased first and then decreased after HT treatment.The content of proline(Pro)and MDA in banana leaves decreased after HT treatment.Moreover,the contents of H2O2,Pro and MDA in leaves of banana colonized by P.indica were higher than those of the non-colonized controls.After 3 d,Pro and H2O2 in leaves colonized were both significantly lower than non-colonized,and the MDA content of colonized was similar to non-colonized.The activities of SOD,CAT,POD and PAL in banana leaves increased gradually under HT treatment,and these indexes in leaves of banana colonized by P.indica were all lower than those in noncolonized group.The PPO activity in leaves of P.indica colonized banana showed no significant change after HT treatment in the banana colonized by P.indica,but the PPO activity in the non-colonized group increased significantly at 3 h after HT treatment.Before and after HT treatment,the IAA and GA contents in leaves of P.indica colonized banana seedlings were lower than those of the non-colonized group,while the ABA contents were significantly higher than those of the noncolonized group,and the JA and SA contents showed no significant difference.After 3 d of high temperature,JA,GA and IAA illustrated an increase in colonized compared to non-colonized,while ABA and SA showed opposite change patterns.It was noted that SA content was lower in colonized than the non-colonized after 3 d of high temperature.The fundings obtained in this study indicated that P.indica could improve the heat resistance of banana by increasing the content of H2O2,Pro,MDA and ABA,and by inhibiting the content of IAA and GA.2.Transcriptome Analysis of banana leaves under high-temperature stressBased on transcriptomics data,we identified the related genes and transcription factor regulatory networks involved in the growth promotion and high temperature tolerance conferred by P.indica.For this purpose,leaf samples were harvested at control(28 ?)and severe high temperature(45 ?,3 d)in P.indicacolonized and non-colonized “Tianbao” banana,then extracted the total RNA from banana leaves cultured at 28 °C(control with / without P.indica)and 45 °C(with/without P.indica,treated by high temperature)(3 replicates for each)and constructing a c DNA library,then used the BGISEQ-500 sequencing platform for the transcriptome sequencing analysis.The results showed that the average length of the transcripts assembled from each sample is between 90 bp and 100 bp;the length of assembled transcript sequences number were between 252 to 2912 nt in size,Q20 and Q30 of each library were respectively higher than 97 % and 88 %,and the uncertain base ratios were 0.12 %,0.13 %,0.15 % and 0.16 %;the content of GC is over 47 %.The analysis between the six comparisons showed at least 24019 differentially expressed genes;4386 DEGs with 1877 up-regulated and 2509 down-regulated in C28-vs-P28 comparison,802 DEGs with 653 upregulated and 149 down-regulated in C45-vs-P45 comparison,19996 DEGs with 7560 up-regulated and 12436 down-regulated in C28-vs-C45 comparison,21027 DEGs with 8715 up-regulated and 12312 down-regulated in P28-vs-P45 comparison,20472 DEGs with 8402 up-regulated and 12070 down-regulated in C28-vs-P45 comparison,and 20157 DEGs with 7534 up-regulated and 12628 down-regulated in P28-vs-C45 comparison.GO enrichment of DEGs found that the photosynthetic system,cell and membrane system of banana leaves were significantly affected under high temperature stress.KEGG database results indicated that the number of differential genes rich in metabolic pathways was the largest.Compared with non-colonized seedlings,the pathways that are significantly enriched under high temperature treatment are secondary metabolite,biosynthesis,photosynthesis,glycosaminoglycan degradation,metabolic pathways,glyoxylic acid,dicarboxylic acid,and the pathways including plant hormone signal transduction,MAPK signaling pathway,plant-pathogen interaction,phenylpropanoid biosynthesis,photosynthesis,anthocyanin biosynthesis,starch and sucrose biosynthesis,lipid metabolism,photosynthesis-antenna protein,and monoterpenoid biosynthesis.Quantitative results show that the sequencing results have high accuracy,and HSFs,HSPs genes play an important role in the high temperature stress response of banana.3.Small RNA analysis of banana leaves in response to high temperature stressSmall RNA sequencing of the same sample used for transcriptome sequencing using the BGISEQ-500 sequencing platform.The sequencing results showed that there were 185 known mi RNAs,93 novel mi RNAs and 5,039 si RNAs in response to P.indica and high temperature stress.Analysis of differentially expressed mi RNAs shows that some mi RNA families respond specifically under P.indica colonization after high temperature stress,and different members of the same family have different expression patterns under different treatments of P.indica and high temperature stress.For example novel_mi R85,novel_mi R11,novel_mir71,novel_mir35,mi R156,mi R171,mi R172 a,mi R164e-5p,mi R396 b,thus indicates that these mi RNAs may play an important role in the high temperature stress process of bananas.GO and KEGG analysis revealed that 1411 predicted target genes of DE mi RNAs,most of which were encoded either by genes or transcription factors involved in nutrient sources uptake,secondary metabolism,growth regulators,auxin-responsive factor,plant hormones,proline metabolism and so on.Many of these are known to be stress responsive genes.Interestingly,P.indica affected the expression level of differential abundance mi RNAs regulated genes,and transcription factors linked to high temperature stress tolerance.Moreover,q RT-PCR verification results showed that the accuracy of mi RNAs sequencing data is high,and novel_mir11,mi R396,mi R159,mi R156,mi R172,mi R164,mi R171 may play a role in heat stress of bananas. |
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