| Maize(Zea may L.)is an important crop for food,forage and economy,originating from tropical and subtropical regions,which is sensitive to low temperature.The northeast of China is one of the main areas for maize production.Due to its geographical location,maize severely suffers from low temperature stress in spring,resulting in devastating yield loss.Therefore,it is particularly important to study the mechanism of maize chilling tolerance.In nature,it is not unusual for plants to experience temperature differences between shoots and roots.However,little is known about how these temperature differences affect maize growth and development.The crown tissues,at the intersection between the shoots and roots,are critical for maize survival from stress as it contains meristems.Whether and how crown tissues contribute to chilling tolerance in maize is unknown.Based on these research background,three chilling treatments were firstly set up,including whole-plant,shoot-only and root-only chilling treatments.Through comparing their effects on maize seedlings,it revealed the contribution of shoots and roots to maize lethality and the survival mechanism of crown tissues under chilling stress.Secondly,transcriptomic analysis of the different chilling responses of different tissues and maize inbred lines with different chilling tolerances were done to reveal the chilling tolerance mechanism and explore the genes associated with maize chilling tolerance.Finally,the distal chilling responses in shoots and roots of maize were preliminarily explored to analyze the possible chilling signals for long-distance transduction.This study lays foundation for the future research on the mechanism of chilling tolerance and long-distance signaling in response to chilling in maize.The main research contents and conclusions are as follows:1.To explore the contribution from shoot and root in maize chilling tolerance,three chilling treatments including chilling treatment on whole plant(4S4R: 4℃ shoot + 4℃ root),shoot-only(4S26R: 4℃ shoot + 26℃ root)and root-only(26S0R: 26℃ shoot + 0℃ root)were set up.Three-leaf stage seedlings of maize B73 were treated by the above three chilling treatments for 3-7 days and the survival rates were counted after recovery.We found that the whole-plant chilling led to the lowest survival rate compared with shoot-only or root-only chilling.The surviving plants all generated new leaves and roots from the crown tissue during recovery,suggesting that the crown tissue is very important for maize survival.Further analysis of the survival mechanism of maize crown tissues by RNA-seq and q RT-PCR showed that the crown maintained survival by inhibiting its cell cycle progression under chilling stress and the degree of inhibition was proportional to the degree of chilling stress and possibly inversely proportional to the survival rate.2.The Principal Component Analysis(PCA)of the transcriptome data with 48 samples revealed a tissue specificity of chilling responses in maize.Differentially Expressed Genes(DEGs)and Biological Processes(BPs)enrichment analyses of maize leaves,roots,top-crown and bottom-crown tissues suggested that chilling responses in top crown and bottom crown were more similar to each other and the transcriptional regulation was activated and the heat response was repressed.In leaves,the abscisic acid(ABA)signaling pathway was upregulated.In roots,the ROS scavenging was activated and the protein degradation was inhibited.In addition,the ABA biosynthesis and response induced by chilling stress were also different in maize different tissues.The q RT-PCR analyses revealed that the Aldehyde Oxidase 3(AAO3)gene for ABA biosynthesis was induced by chilling stress in maize leaves,roots and crown tissues,but more highly induced in leaves and roots,indicating the main sites for ABA biosynthesis.However,the BPs of ABA responses were enriched in leaves and crowns,but not in roots,in response to chilling.This suggests that ABA may act as a long-distance signaling molecule in response to chilling in maize.Besides,the maize,rice and Arabidopsis had the species specificity of the chilling induced BPs enrichments by comparative analysis of transcriptome data.The 76 of total 90 enriched BPs were specifically enriched in one of the species.This indicated that the chilling responses of maize were different from other model plants,which needs to be further studied.3.To identify the association of chilling response BPs in different tissues with maize chilling tolerance,two relatively tolerant lines,B73 and B104,and two susceptible lines,CML333 and M37 W,were selected from a collection of 11 maize inbred lines based on phenotypic observations and relative electrolyte leakage following chilling treatment.Further analysis of RNA-seq and q RT-PCR showed that the upregulation of biological processes related to ‘transcriptional regulation’,‘ABA biosynthesis and response to ABA’,‘response to environmental stimulus’,‘photosynthesis’,‘growth and cell communication’,‘nitrogen compound metabolism’ and ‘response to water’,and the downregulation of ‘translation’ were associated with chilling tolerance in leaves,while the upregulation of ‘transcriptional regulation’ and ‘response to ABA’,and the downregulation of ‘response to heat’ were associated with chilling tolerance in crowns.Moreover,we selected 32 genes associated with maize chilling tolerance through transcriptomic analyses and protein-protein interaction network analyses.4.The functional identification of ZmERF4 from 32 genes indicated that the candidate genes related to chilling tolerance were feasible.We found the upregulation of AP2/ERF4 family genes were associated with maize chilling tolerance.Among them,ZmERF4 is one of the most significantly upregulated AP2/ERF4 family genes in all four tissues in response to chilling treatments.Bioinformatics analysis and transient expression in maize protoplast revealed the ZmERF4 protein was localized in the nucleus.Furthermore,overexpression of ZmERF4 shortened the petioles and enhanced chilling and freezing tolerance of Arabidopsis with reduced the electrolyte leakage in leaves under chilling treatments.In addition,the q RTPCR analyses indicated the overexpression of ZmERF4 gene activated the expression of CBF pathway genes including ICE1,CBF1 and CBF2,in Arabidopsis.5.In addition to the identification of BPs and genes associated with maize chilling tolerance,the distal chilling responses of maize leaves and roots were further analyzed.The comparative analysis of the effects of whole-plant,shoot-only and root-only chilling on maize leaves and roots indicated that the distal chilling response was distinct from both local and whole-plant chilling responses for both leaves and roots.ABA signaling and nutrition deficiency signaling pathways may involve in distal chilling signaling transduction in leaves.In contrast,distal chilling upregulated genes involved in photosynthesis in roots,and the specific functions and signaling mechanisms need further analysis.In sum,comprehensive transcriptomic analyses in this study have revealed potential tissue specific chilling survival and tolerance mechanisms and distal chilling responses in maize.This lays a theoretical foundation for future research on the chilling tolerance mechanism and the long-distance transduction of chilling signals. |
