Study On The Physiological And Biochemical Response Mechanism Of Carthamus Tinctorius L. To Drought Stress

C.tinctorius was one of the oldest cultivated crops,which was a species of the genus Carthamus in Compositae family,and its flower was used as medicine.Its oil was mainly for edible purposes.Safflower was grown for flowers used for coloring,flavoring foods,dyes,medicinal properties,and livestock feed.Drought stress was one of the most serious environmental stresses in global agricultural production,which seriously affects the growth,development and yield of crops.Therefore,it was of great significance to search for drought-adapted plants or study the drought-resistant mechanism of plants response to drought stress.Future climate changes and environmental changes（high temperatures,droughts,etc.）might lead to changes in the appropriate distribution of safflower.The current and future distribution of safflowers were modelled by using a maximum entropy model（Max Ent）and geographical information system（GIS）in China.Meanwhile,the comprehensive evaluation of 14 safflowers was carried out by statistical analysis method under drought stress during germination period.Drought-tolerant and drought-sensitive materials were taken as experimental material in response to drought stress by pot experiment.The physiological and biochemical response mechanisms of drought tolerance in safflower were systematic researched by agronomic traits,physiological,RNA-Seq,metabonomics.The main results are as follows:（1）99 safflower distribution records and 11 Worldclim environmental factors were used in this paper,and the current and future distribution of safflowers were modelled by using a maximum entropy model（Max Ent）and geographical information system（GIS）in China,based on three representative concentration pathways（RCP2.6,RCP4.5 and RCP8.5）for the year 2050s and 2070s.The results showed that the area under the receiver operating characteristic（ROC）curve（AUC）was used to evaluate model performance.All of the AUCs were greater than 0.970,thereby placing these models in the―Excellent‖category.The results also showed that maximum temperature in March（tmax3）,maximum temperature in February（tmax2）,maximum temperature in November（tmax11）,precipitation in July（prec7）and precipitation in August（prec8）were the main variables.From the area of current distribution,the total area of suitable areas accounts for 65.36%of China’s total areas,,27.28%area was highly suitable for safflower in China,and the highly suitable area（with suitability between 66 and 100）was mainly concentrated in Sichuan（2.74%）,Yunnan（2.09%）,Shaanxi（1.82%）,Hubei（1.76%）,Guizhou（1.66%）,Henan（1.66%）and Hunan（1.64%）.Xinjiang was the largest suitable area,which accounts for 9.70%.Compared to the current distribution,the total area of the poorly suitable regions and moderately suitable regions for safflower would increase in 2050s and 2070s.However,the total area of the highly suitable regions would decrease in 2050s and2070s..（2）The seeds of PI305192 and PI401472 were used as the tested materials under the simulated drought conditions with 6 different PEG concentrations.The results showed that the concentration of 20%PEG–6000 could be the optimum condition for the simulation of drought stress.The germination rate,germination potential,germination index,root length,bud length,total length,germination fresh weight,root fresh weight and total fresh weight decreased with the increase in osmotic potential within the scope of the PEG concentration 5%～25%,whereas proline content and soluble proteins increased.（3）The germination characteristics and physiological and biochemical indexes of14 safflowers were studied by simulated soil drought with 20%PEG-6000 solution.Principal component analysis and fuzzy subordinate function analysis were used for comprehensive evaluation of drought resistance in germination period.Meanwhile,multiple indexes of drought resistance of safflower were identified and screened.The results showed that there were significant differences among all the 14 genotypes and their interaction（P﹤0.05）for all tested parameters.The relative values of germination rate（GR）,germination potential（GP）,germination index（GI）,germination drought resistance index（GDRI）and superoxide dismutase（SOD）activities were significantly positively correlated with drought resistance of safflower,which could be used as the screening index of drought resistance identification.Principal component analysis was conducted on GR,GI,GP,GDRI,contents of osmotic-adjustment products,malondialdehyde（MDA）and antioxidant enzymes activities,and 5 principal components were obtained whose accumulated contribution rate is 92.75%.The analysis of principal component and comprehensive subordinate function showed that 14 accessions of safflower were divided into different drought tolerant levels.PI401470（No.12）,PI401477（No.16）,PI470942（No.23）and PI544021（No.28）were identified as highly drought resistant genotypes.（4）In this syudy,the influences of drought stress on photosynthetic characteristics,contents of osmoregulation substance,protective enzyme activities and the other physiological characteristics of two drought-tolerant and two drought-sensitive materials were studied at safflower seedling stage by drought stress.The results showed that the net photosynthetic rate,stomatal conductance,intercellular CO₂concentration and transpiration of 4 safflower materials declined under the drought stress,and the difference among the different materials was significant（P﹤0.05）.The decrease of photosynthetic characteristics of drought-tolerant materials was lower than that of the drought-sensitive materials.The contents of permeability regulation substance of leaves such as soluble sugar,soluble protein,proline and the protective enzyme activities of SOD,POD and CAT are all increased in 4 materials under drought stress.Moreover,the increase rate of drought-tolerant materials was higher than that of drought-sensitive.The increase rate of MDA content in drought-tolerant materials was also lower than that in drought-sensitive materials.Two principal components were obtained by principal component analysis of 11 indexes,and the cumulative contribution rate is 89.50%.The net photosynthetic rate,stomatal conductance,intercellular CO₂ concentration,transpiration rate,SOD and CAT activities were used as the first principal component of drought resistance index,while soluble protein and proline were used as the secondary principal component of drought resistance identification index.（5）Transcriptional responses under drought stress were studied by using two safflower materials（the drought-intolerant material PI560169 and the drought-tolerant material PI401477）.A total of 3280（including 1,862 up-regulated and 1,418down-regulated）and 2260（including 1,161 up-regulated and 1,099 down-regulated）differentially expressed genes（DEGs）were identified in PI401477 and PI560169between the drought and well-watered conditions,respectively.Among these differentially expressed genes,only 46 common genes were differentially expressed among all four group treatments.KEGG enrichment analysis results showed that there are 31 and 33 significantly pathways in PI401477 and PI560169（P-value≤0.05）,respectively.For PI40147,there were nine significantly enriched pathways with a large number of DEGs included plant hormone signal transduction,ribosome,metabolism of xenobiotics by cytochrome P450,drug metabolism-cytochrome P450,galactose metabolism,steroid hormone biosynthesis,tryptophan metabolism,sesquiterpenoid and triterpenoid biosynthesis and carotenoid biosynthesis.A total of561（304 up-regulated and 257 down-regulated genes）and 335（159 up-regulated and176 down-regulated genes）significant genes encoding putative TF genes were identified from PI401477 and PI560169 leaves,respectively.These TF genes were classified into MYB,b HLH,NAC,WRKY,TRAF,bZIP,C2H2,MYB-related,FAR1,MADS,AP2-EREBP,PHD,ABI3VP1,C3H,orphans and other transcription factor families and subfamilies.（6）In this study,a total of 359 and 209 differential metabolites（DMs）were identified between the two treatments in PI401477 and PI560169 by liquid chromatography mass spectrometry（LC-MS）,respectively.Among these differential metabolites,13 differential metabolites are shared between these two safflower materials.Moreover,we also detected several main metabolites such as N-gluconyl ethanolamine,7-beta-D-glucopyranosyloxybutylidenephthalide,elenaic acid,galactitol,arbutin,glycerol alpha-monochlorohydrin oleacein and N-gluconyl ethanolamine in PI401477.（7）Several key candidate genes,such as MYB62,MYB2,NECD4,ABA2,CYP707A4,ZDS,Gol S1,P5CS1,GST23,GST3,GSTL1,Cu-ZnSOD1,OEE2 and ALDH3F1 were identified by integrated analysis of the transcriptome and metabolomics,which were more likely to determine tolerance to drought stress in PI401477.In addition,it was found that three key metabolites（galactitol,neoxanthin and arbutin）might be correlated with the drought-tolerance and differently regulated in PI401477.These key candidate genes and metabolites were found to belong to the galactose metabolism,carotenoid biosynthesis,glycolysis/gluconeogenesis,biosynthesis of amino acids pathway,glutathione metabolism,peroxisome,flavonoid biosynthesis and photosynthesis pathway,which suggested the importance of these metabolic pathways during drought stress.These results provided a new perspective on the complex mechanisms underlying drought stress responses to safflower.