Genome-wide Characterization Of Phospholipase Families In Brassica Napus

Brassica napus is an important crop mainly grown for oil purpose around the globe.Its production is severely affected due to the prevalence of environmental stresses such as drought salinity,extreme temperature and so on.However,the mechanism by which Brassica napus respond to these stresses remains largely unknown.Phospholipase families including phospholipase As(PLAs),phospholipase Cs(PLCs),and phospholipase Ds(PLDs)have vital functions in plant growth and development and enhance plants acclimation to abiotic stresses.The aim of this research was to identify the phospholipase gene families,number of genes in each family,monitor their expression and corresponding lipid changes in response to different abiotic stresses such as ABA,salt,dehydration and cold stress(4℃) at two different time points.Then we selected PLDα1 gene to further perform functional characterization by generating its overexpression and mutant lines in Brassica napus.We classified each family into different subfamilies based on protein structures,sequence similarities,conserved domains,subcellular localization,and substrate preferences.In this study,29 BnapPLA genes were identified and classified into three main subgroups,pPLAI,pPLAII(α,β,γ) and pPLAIII(α,β,γ,and δ).Several differences were found among these gene subfamilies,such as protein size,motifs and their number with in domains and other physiochemical properties.BnapPLAs shared high similarity with their orthologs in AtpPLAs,but the number of genes in each pPLA subfamily in Brassica napus was around three-fold of Arabidopsis.The gene expression analysis showed that BnapPLAIIIs were highly expressed in most tested tissues compared to other subfamilies.BnapPLAIII β,BnapPLAIIIα,and BnapPLAICnn were significantly induced by different abiotic stresses including dehydration,salt,ABA,and cold stress,except BnapPLAIIIδ whose RNA transcripts significantly accumulated under ABA stress.Lipidomic analysis revealed that lysolipids including LPC accumulated significantly under dehydration stress and ABA 4H after stress.LPE levels rose significantly in response to dehydration stress,while accumulated medially under other abiotic stresses at different time points.The level of LPG decreased gradually in response to all the stresses.The molecular species such as16:0,18:3 and 18:2 of LPC,LPE,and LPG significantly increased while LPG18:1 decreased under the applied stresses.Moreover,40 BnaPLC genes were identified and classified into two subfamilies:phosphoinositide phospholipase C(BnaPI-PLCs) and non-specific phospholipase C(BnaNPCs).High similarity existed between BnaPLCs and AtPLCs genes and their protein structures,whereas BnaPI-PLCs and BnaNPCs genes were 3 and 2 times more than Arabidopsis respectively.Gene expression analysis showed that BnaPI-PLC2 and BnaNPC1 had much higher expression than other PLCs in different tissues.In response to dehydration and salt stress,BnaNPC1 and BnaNPC2 genes were significantly induced while,BnaNPC1,2,3,BnaPI-PLC1,2 and BnaPI-PLC4 exhibited significant expression in response to ABA stress.Cold stress mostly induced BnaPI-PLCs than BnaNPCs with the time course of the stress.Lipidomic analysis showed that DAG accumulated with the extended duration of dehydration and cold stress while,its physiological levels altered in a non-significant manner in response to salt and ABA stresses.The DAG molecular species such as 34:6 and 36:6 accumulated under dehydration,while 34:3 accumulated in response to all stress treatments.DAG 36:5 and 36:4 accumulated at higher levels only under cold stress.Furthermore,we identified 32 BnaPLD genes and classified them into 6 subgroups(α,β,γ,δ,ε,and ζ) depending on the conserved domain present in their protein structures.High similarity in genes and protein structures was found between BnaPLDs and AtPLDs.Apart from this,the number of genes in each subfamily in Brassica napus were found more than that of Arabidopsis.The gene expression analysis showed that BnaPLDα1s and BnaPLDδs had much higher expression than other PLDs in different tissues.BnaPLDα1 and BnaPLDδ were also significantly induced by the different abiotic stresses applied.Lipidomic analysis revealed that the content of main membrane phospholipids including PC,PE,and PG decreased gradually under the stresses,except PG which increased under ABA stress and PE which accumulated in response to cold stress.Correspondingly,their product of PA increased with a mode of oscillation.Based on the gene expression and lipidomic data in response to the aforementioned abiotic stresses,we selected BnaPLDα1and generated its overexpression and mutant lines.The CRISPR/Cas9 mutants of PLDα1had dwarf phenotype compared to wild-type and overexpression lines.Agronomic traits such as yield,seed number per silique,silique number per inflorescence and branches per plant etc.were significantly impaired in pldα1 mutants compared to the wild-type.BnaPLDα1-overexpression lines produced seeds with higher levels of desirable fatty acids than the mutant lines suggesting the role of PLDα1 in improving oil nutritionalquality.Taken together,our study suggested the significant roles of phospholipase families in membrane lipids remodeling,maintaining the growth and abiotic stress tolerance in Brassica napus.Moreover,PLDα1 gene can be further characterized in improving the growth,development as well as yield and oil contents in Brassica napus.