| Low temperature is the main stress problem affecting the growth of sorghum seedlings in northeast China.It is also an important factor affecting the development of plants and leading to the reduction of sorghum quality and yield.Enhancing cold resistance of sorghum is an effective method to improve yield and quality of sorghum.Therefore,It is important to clarify the cold response mechanism of sorghum to create cold tolerant sorghum varieties.In this study,cold-sensitive sorghum material’Huangkezi’（H21）and cold-tolerant variety’Pingdingxiang’（P61）,which were obtained by screening low-temperature resistant materials of about 80sorghum germplasm in the laboratory,were selected as research materials.The seedlings were treated at 4℃at 10 days of seedling age,and the growth biomass,photosynthetic fluorescence and physiological indexes were measured.At the same time,combined with RNA-Seq sequencing and Lipidomics analysis,the root growth,physiological and biochemical changes,membrane lipid metabolism and gene expression patterns of sorghum seedlings under low temperature were determined,providing a certain reference for further understanding of cold tolerance mechanism of sorghum.The results of this study are as follows:1.Effects of low temperature on physiology and related genes of sorghum seedlings with different cold toleranceAt 4℃,the leaves of H21 curled and wilted,and the root development was delayed,while P61 had little change.The chlorophyll content and photosynthetic fluorescence parameters of H21 were lower than those of P61 after low temperature treatment.Under low temperature,the antioxidant enzyme system and osmotic regulation system of P61R were stronger than those of H21R（R represents root）,and the relative electrical conductivity,malondialdehyde,hydrogen peroxide and superoxide anion contents of root were lower.At the same time,the expression data of genes related to the above physiological indicators were screened from the RNA-Seq data,and it was found that SbSODs,SbPODs,SbCATs,SbLOXs and SbP5CSs showed up-regulated expression in different time points of H21R and P61R response to cold.2.Analysis of RNA-Seq in roots of sorghum seedlings with different cold tolerance under low temperatureIn this study,the RNA-Seq analysis of sorghum seedling roots of H21R and P61R at 12,24and 72 h of low temperature treatment found that the screening condition was|Log2FC|≥2,and there were 1244 common differentially expressed genes（DEGs）for H21R and 882 common DEGs for P61R at low temperature.The number of up-regulated and down-regulated DEGs in the roots of the two varieties at three time points was 354 and 188,respectively.The above DEGs analysis showed that there were differences in the enrichment pathways of GO and KEGG pathway.Transcription factors involved in cold response of sorghum roots were detected in both H21R and P61R,and MYB,NAC,bHLH,WRKY and ERF were the main transcription factors responding to low temperature.Log2FC values of Sobic.006G192100（MYB-related family gene）and Sobic.008G021800（NAC family gene）were higher than 4 at 12,24 and 72 h.3.Membrane lipid remodeling of roots of sorghum seedlings with different cold tolerance under low temperatureThe determination of H21R and P61R glycolipids,phospholipids,lysolipids and their molecular species at 0,3,7 and 14 days under low temperature showed that the content of PC was the highest in sorghum root membrane lipid components.At 4℃,the PC content of H21R and P61R decreased,while the PA content increased.The phospholipid PG content in plastids increased,especially the PG（C34:4）level.The contents of MGDG,DGDG and SQDG increased,while the molecular species of C36 MGDG and C36 DGDG were observed to dominate galactose,which contained a higher level of 18:3 fatty acids,which proved that sorghum was an18:3 plant.4.Construction of the interaction network between lipid-related genes and metabolites in sorghum roots under low temperatureCold resistance of two different sorghum seedling root RNA-Seq data involved in lipid metabolism in DEGs（|Log2FC|≥1）analysis found that most DEGs involved in lipid oxidation,fat fat fat signal transduction and galactose,sulfur and phospholipid synthesis process;Moreover,lipid components in root membrane of sorghum seedlings also changed significantly under low temperature,indicating that low temperature promoted lipid metabolism to improve the adaptability of roots to cold.Lipid data were combined with RNA-Seq data to establish lipid metabolism molecular regulatory network in response to cold in sorghum seedling roots.The results showed that low temperature activated the de novo synthesis of PC in the Kennedy pathway of the endoplasmic reticulum,enhanced the PG and glycolipid synthesis pathway in the eukaryotic pathway and plastid of sorghum roots,and most of the genes that catalyzed these reactions showed up-regulated expression.In this study,a variety of omics techniques such as RNA-Seq,combined with the measurement of growth,photosynthesis and physiological and biochemical indexes,were used to study the physiological metabolism,transcription regulation and membrane lipid remodeling of roots of sorghum seedlings with different cold tolerance under low temperature to a certain extent,and the response of roots to low temperature was systematically elaborated.At 4℃,the photosynthetic damage degree of cold-resistant material P61 was lower than that of H21,and the level of root reactive oxygen scavenging and osmotic regulation system was higher than that of H21R.The root cell membrane adapts to low temperature by lipid remodeling,which is manifested as the degradation of main phospholipids PC and PE and the generation of PA,leading to the increase of the level of PA.The mole percentage of plastolipid glycolipids MGDG,DGDG,SQDG and plastidic phospholipid PG increased,and the high grain seedlings were protected against cold injury by these spontaneous protection mechanisms.Compared with the sensitive variety H21,the resistant variety P61 had a stronger protection mechanism. |
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