Study On The Mechanism Of Calcium Nanoparticles Improving Drought Resistance In Brassica Napus

Climate change,food insecurity,water scarcity,and population growth are some of the world’s frightening problems.Drought stress exerts a constant threat to field crops and often considered as a serious constraint to global agricultural productivity and its strength and frequency will increase in near future.Because of similarities in some characteristics with drought tolerant species,rapeseed（Brassica napus L.）known as one of the best choice crop to grow in water deficit areas.In spite of the fact that this is a requirement for making this crop species drought-tolerant,the methods by which rapeseed uses nanotechnology to withstand drought at both the vegetative and reproductive phases have not yet been fully understood.Investigations carried out in this thesis aimed to decipher the genetic variability responses and the role of calcium nanoparticles（Ca-NPs）imparting drought tolerance abilities in rapeseed genotypes,with emphasis on integrated transcriptome and metabolome profiling.Analyses on plant reproductive fitness in terms of seed quality were expanded in order to improve the commercial viability of drought tolerance.Therefore,both short-term（hydroponics）and season-long（soil-based）experiments were carried out.To exploit the natural variations of drought tolerance,a screening of 79 B.napus genotypes with different genetic background were carried out.After two runs of screening,9 genotypes selected and tested to understand the drought tolerance response.Notably,variability of drought tolerance was observed,and four genotypes（ZD622,ZD630,ZD619 and ZS11）and one genotype（GY605）were identified as drought tolerant and sensitive,respectively.The fact that only four out of 79 tested genotypes were identified as showing drought tolerant behavior suggested that this trait might be rare in B.napus;hence,these genotypes（drought tolerant）focused in subsequent investigations.The drought tolerance of B.napus further examined in relation to the relative contribution of Ca-NPs responses.To allow a comparative analysis,genotypes with distinct phenotype i.e.tolerant genotype（ZD622）integrated in this study.The Ca-NPs had spherical and uniform shape with an average size of 86 nm.Foliar application of 100 mg L^-1 Ca-NPs enhanced biomass of rapeseed plants and considered as optimal dose.Ca-NPs at 100 mg L^-1 had a favorable impact on mesophyll ultrastructure,PSⅠ and PSⅡ efficacy,gas exchange parameters,chlorophyll content,and mineral absorption.The Ca-NPs treatment increased NPQ and Y（NPQ）under drought condition,indicating a higher PSⅡ protective response to stressed conditions with better heat dissipation as a photo protective component of NPQ.Ca-NPs application also reduced oxidative stress damage as measured by a reduction in reactive oxygen species（ROS）generation in terms of hydrogen peroxide and malondialdehyde（H₂O₂ and MDA）and increased antioxidative defense enzymes（SOD,POD,CAT,APX）,seed quality and potential genes expression PsbB,CP29,CP24,PCaA,CP26 and cytochrome f,CAS,CDPK5,POD,MYC2 and MPK6.Ca-NPs may play a vital function in sustaining photosynthesis and stress tolerance in rapeseed.Taken together,the findings of this work provide new understanding into the physiological and molecular processes that regulate B.napus responds to Ca-NPs exposure.In order to explore the drought tolerance mechanism in rapeseed following Ca-NPs treatment under drought stress examined at transcriptome levels.According to results,the enhanced expression of Ca-NPs responsive genes related to osmotic regulation such as IDH,ProDH1,ProDH2,APs;hormones metabolism IAA9,IAA10,ATPase,PYL4,AOC3,GA20ox2,GA20ox3,GA20ox4,GA3ox2,and GA30ox6;proteins regulation KRP1,Ca BP25,BBx19,BBx20,PIP1-1,PIP1-2,HSPs1,HSPs2,SAG21,and KIN2.In addition,Ca-NPs response the marked increase in genes like BnLhcb1.3,BnLhcb1.5,BnLhcb1.10,BnLhcb2.1,BnLhcb2.4,BnLhcb2.5,BnLhcb2.8,BnLhcb5.3,Ps Bs28 and PSA3 reveal their key role in controlling the photosynthetic stability and stress tolerance.Moreover,stress tolerance related genes such as Ces A,PE,DHN1,and HSPs;and transcription factors such as HSFs3,HSFs4a,HSFs8,HSFs C-1,VOZ1,VOZ2,HY5,MYB14exhibited marked increase in expression pattern of drought tolerant ZD622 onset to Ca-NPs treatment under drought stress.These results demonstrated the feasibility to rescue B.napus plants under drought stressed conditions through supplemented of exogenous Ca-NPs,actions of which shown to be in relation with better preservation of endogenous hormones regulation,stable cellular structure and photosynthesis performance by triggering the up-regulation of these genes for controlling stomatal movement in rapeseed plants.For a holistic view of Ca-NPs mediated drought tolerance in drought genotype,investigations extended at whole metabolome level,using liquid chromatography/mass spectrometry.Metabolomics analysis revealed that metabolites differentially expressed in leaves under drought stress.These metabolites significantly enriched in the biosynthesis of flavonoid,terpenoids,tyrosine metabolism,purine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,oxidative phosphorylation,iso-flavonoid and flavonoid biosynthesis.Interestingly,Ca-NPs recovered the 38%of deferentially expressed leaf metabolites to normal level,thereby facilitating the plant growth under drought stress.However,the correlation analysis further suggested that these metabolites including the tryptophan,glycine,alanine,phenylalanine,glutamate,proline,thrionine and 4-aminobutyric acid exhibit positive correlation with the plant growth.Thus,these findings indicate that Ca-NPs can impart in plant growth regulation and reveal the involved mechanism of drought stress.The main conclusions from the joint analysis of DEGs and DEMs in response to Ca-NPs treatment included:the accumulation of metabolites related to carbohydrates metabolism because of BnGAPCP1,BnGAPDH3 and BnLhcb5.3 like genes expression and synthesis of secondary metabolites including the carboxylic acids and derivatives（amino acids）,flavonoids,prenol lipids（terpenoids）,benzene and derivatives,indoles and derivatives,and phenols（phenylproponoids）.Therefore,the carbohydrates metabolism may transcriptionally regulated by Ca-NPs responsive BnGAPCP1,BnGAPDH3 and BnLhcb5.3 genes,which may play a critical role in stress tolerance mechanism in rapeseed.