Transcriptome Analyses Of Maize (Zea Mays L.) Under Three Different Stresses In Southwest China And Functional Characterization Of The Candidate Genes

Plants are persistently and simultaneously confronted with a range of biotic and abiotic stresses.To cope with and adapt to different stresses,plants have evolved sophisticated defense network in response to external stimulus.Emerging evidence suggests that phytohormones,transcription factors,kinase cascades,transporter,pathogenesis-related proteins（PRs）play key roles in plant defense responses.Recent evidence highlights the complex crosstalk between biotic and abiotic stress responses,which may be synergistic and/or antagonistic.A comparative study of plant response mechanisms under a variety of stress conditions will enrich our understanding of the interaction between these response mechanisms,as well as provide useful information for genetic improvement of crops.Fusarium graminearum infection,nitrogen starvation,and Cd exposure were important stress factors in southwest China,which severely restricted the development of the corn industry.Based on previous research work,this study used different maize varieties to investigate the molecular mechanism of maize defense responses to nitrogen starvation,Cd exposure,and Fusarium graminearum infection using comparative RNA-Seq analysis.The results obtained from this research were as follows:1.To elaborate the molecular basis underlying maize resistance against Fusarium graminearum,the resistance（IBM₈1）and susceptible（IBM₈5）maize ear rot DH lines were selected from IBM DH population on the basis of the previous research.Differentially expressed genes from disease-resistant and disease-susceptible materials were detected at 48 h,72 h,and 96 h after inoculation.A total of 5760 DEGs were found in six comparative groups after F.graminearum inoculation.Six co-modulated DEGs related to defense response were identified in all comparative groups.Interestingly,one long intergenic non-coding RNA（lincRNA）was strongly induced by F.graminearum.Functional analysis revealed that DEGs were enriched in defense responses,plant hormone signal transduction,plant-pathogen interaction,and phenylpropanoid biosynthesis.In IBM₈5,DEGs involved in photosynthesis were repressed at 72 h,whereas inhibition of photosynthesis appeared in IBM₈5 at 96 h.Several ZmJAZs had higher expression abundance in IBM₈5.As a negative regulator,the low expression levels of ZmJAZs were beneficial to activating JA responses.In addition,ZmOPR1,ZmOPR2,and ZmOPR5 also participated in the stress responses to F.graminearum.2.Pathogenesis-related protein 1（PR1）play diverse roles in mitogen-activated protein kinase（MAPK）signaling pathway,plant hormone signal transduction,and plant-pathogen interaction.In this study,12 PR1 genes were identified in maize with typical structure domain.Transcriptome profiling revealed that four genes encoding PR1 were responsive to F.graminearum.Among them,ZmPR1-11 predominantly expressed in bracts and showed higher expression abundance in IBM₈5,which may have broad-spectrum disease resistance to fungi.3.To investigate the difference of root morphological and transcriptomic responses to low-N stress between the maize mutant rtcs and its wild-type,we explored the changes of root morphology and identified genotype-and/or treatment-related differentially expressed genes（DEGs）.The differences between the two genotypes were determined at 12 h,24 h,48 h and 96 h after two N treatments.The inhibitory effect of low-N stress on total protein content and glutamine synthetase（GS）activity was observed in both genotypes.The common feature that length of primary root（PRL）and total lateral root on primary root（LRL）increased after low-N treatment was observed in both genotypes,while larger changes of LRL were detected in rtcs.This result suggested that the primary roots elongation and lateral root compensatory growth are main response strategies to offset the deficiency of seminal and shoot-borne roots in rtcs under N-deficiency.4.Subsequently,we performed a comparative RNA-Seq analysis to compare gene expression profiles between mutant rtcs roots and wild-type roots under different N conditions.We identified 786 co-modulated differentially expressed genes related to root development.These genes participated in various metabolic processes.Among them,transcription factor ZmJAZ7 and bHLH103 showed higher abundance in wild-type root and dramatic up-regulation expression profiles under low-N stress.The co-expression cluster analysis and cis-regulatory motifs analysis revealed the importance of the AP2-EREBP transcription factor family in the rtcs-dependent regulatory network.Further analysis of the differences in gene transcript levels between rtcs and wild-type under different N conditions revealed 403 co-modulated DEGs with distinct functions.A comparative analysis revealed that the regulatory network controlling root development also controlled gene expression in response to N-deficiency.The modules of AUX/IAA-ARF-LBD-bHLH-AP2-EREBP and JAZ-dependent pathway may play important roles in maintaining the balance between root development and N-deficiency response.5.In order to elucidate the gene networks of the plant respond to cadmium（Cd）stress,transcriptomic changes during B73 and Mo17 seedlings development responsive to Cd pollution were investigated using comparative RNA-Seq-based approach.115 DEGs with significant alteration in expression were found co-modulated in both genotypes during the maize seedling development.Of those,most of these DGEs were found comprised of stress and defense responses proteins,transporters,as well as transcription factors,such as ZmNAC70,ZmRAN1,ZmJAZs and ZmOPRs.Moreover,12 co-expression modules associated with specific biological processes or pathways were identified by consensus co-expression network.Remarkably,several Cd-responsive genes were involved in different biotic and abiotic stresses,such as ZmJAZs and ZmOPRs.6.Based on the results of RNA-Seq,we further focused on the function of 12-oxophytodienoic acid reductases（OPRs）in response to Cd stress.Eight ZmOPR genes were identified in maize at whole-genome scale.Of those,ZmOPR5 encoding OPR I protein was identified to be highly inducible by Cd stress.Functional disruption of AtOPR2 homologous to ZmOPR5 showed obviously inhibited growth of roots and Cd-sensitive phenotype under Cd stress,compared with wild type.ZmOPR2 appeared to function in defense responses to pathogens infection and Cd stress.The results suggested that OPRs play diverse roles in response to Cd in maize.7.Comparative analysis revealed that some important functional genes were involved in a variety of stress responses.ZmJAZ7 participated in root development and stress responses of low-N stress,Cd exposure,and F.graminearum infection,which may regulate the balance between plant growth and responses to biotic and abiotic stresses by the regulation of JAinduced responses.Transcription factors also play an important role in regulating plant growth and stress responses.In this study,multiple members of AP2-EREBP family in maize were involved in root development and stress responses.ZmOPR2 and ZmOPR5 had distinct roles in response to Cd stress and F.graminearum infection by JA-response pathway,which may have broad-spectrum disease resistance to fungi.ZmPR1-2 and ZmPR1-11 were involved in defense response of F.graminearum infection in maize bracts and silk,respectively.These results indicate that the above genes are involved in crosstalk between abiotic and biotic stress responses.It has significance to dissect the functions of candidate genes in response to diverse stresses,which will expand our understanding of the crosstalk between biotic and abiotic stress signaling pathways and provide useful information for genetic improvement of crops.