Molecular Mechanism Of BnMAPK1 Regulating Drought Response And Growth And Development Process In Brassica Napus L.

Rapeseed（B.napus）has high yield and nutritional value,which is widely planted in the Yangtze River Basin in China.With a relatively complex genome and many mutations in domestication,rapeseed,which is an allopolyploid species,has important scientific value in evolutionary researches.The soil environment and water supply directly affect the accumulation of dry matter and oil in rapeseed.During the long growth period,rapeseed has a great demand for water.China has the widest arid and semi-arid areas in the world,and rapeseed losses caused by drought have been severe.It has become a research hotspot to make the best use of genes that have excellent resistance to drought through genetic engineering to improve the drought resistance in plants.Therefore,digging drought-related pathways and genes,and exploring these molecular network in rapeseed become one of the important ways to improve drought resistance,yield and quality in rapeseed.MAPKs（Mitogen-activated protein kinases）cascade is a common pathway for multiple signal transmembrane transmission.After receiving signals,plants amplify signals to cells through phosphorylation of the MAPKs cascade to respond to stresses,with MAPKKKs（MAPK kinase kinases）transmitting signals to MKKs（MAPK kinases）and further to MAPKs.Our previous results indicated that the growth preoid of overexpression BnMAPK1 transgenic rapeseed was significantly advanced,and the drought resistance was significantly improved.In this study,many molecular biological techniques were performed to identify BnMAPK1-interacting drought-/growth-related proteins and upstream regulatory factors of BnMAPK1.In addition,the interaction relationships between the BnMKKs and the C-group BnMAPK1/2/7 were clarified.Our work revealed the molecular network of BnMAPK1 in drought responses and growth and development processes.The main results are as follows:1.The transcription level of BnMAPK1 was positively correlated with the resistance of drought in rapeseedWith dehydration treatment to drought-sensitive and drought-resistant rapeseed,the water loss rates of drought-sensitive materials which with more serious wilting were higher than drought-resistant materials.And the phenotypes of these materials under PEG treatment were basically the same as that of dehydration treatment.The transcription levels of BnMAPK1 in the roots and leaves of drought-sensitive materials were lower than those of drought-tolerant materials.These results showed that the expression level of BnMAPK1 was positively correlated with the resistance of drought in rapeseed.2.Enrichment analysis of drought response-related pathways in RNA-Seq of BnMAPK1transgenic rapeseedRNA-Seq analysis was performed on overexpression-BnMAPK1（OE-BnMAPK1）,interference-BnMAPK1（RNAi-BnMAPK1）,and wild-type Zhongyou821（WT）plants.33 differential expression genes（DEGs）in water deprivation/water/ABA,water transport,and H₂O₂/ROS metabolism process were obtained both in OE-WT group and RNAi-WT group.These data indicated that BnMAPK1 may regulate the transcription of some water-related genes,then changing the expression of H₂O₂-/ROS-/polysaccharide metabolism-related genes.3.Overexpressing BnMAPK1 in Arabidopsis increased drought resistance in plantsThe overexpression-BnMAPK1 recombinant vector was transformed in Arabidopsis to perform dehydrated treatment.Our data showed that the water loss rate of AtOE-BnMAPK1 Arabidopsis was significantly lower than WT（Col-0）.The natural soil drought treatment data showed that the wilting degree of AtOE-BnMAPK1 plants was significantly lower than that of WT.After rewatering,the wilting of AtOE-BnMAPK1plants was almost disappeared,indicating the overexpressing BnMAPK1 in Arabidopsis can increase the drought resistance of plants.4.The spatial and temporal expression of BnMAPK1 was ubiquitousThe qPCR data in Zhongyou821 and the RNA-Seq data in Zhongshuang11 showed that BnMAPK1 expressed in different growth stages and tissues such as roots,stems,leaves,seeds,and silique pericarps.After transforming the promoter of BnMAPK1（ProBnMAPK1:GUS）into Arabidopsis,GUS signals were detected in different growth stages and tissues,indicating that ProBnMAPK1 was non-specific.In addition,the GUS signals were enhanced in mannitol-induced ProBnMAPK1:GUS Arabidopsis.These data showed that BnMAPK1 may participate in mannitol response to involve in drought stress.5.BnMAPK1 regulated drought resistance of rapeseed through ABA pathway and redox process,and participated in growth and development of rapeseed through cytokininCombining Y2H and Split-LUC assays with IP-MASS analysis,we found that BnMAPK1 may interact with CYP20-3（Cyclophilin 20-3）by ABA-dependent signaling pathways to response ABA in drought stress,and may interact with ADH2（Alcohol dehydrogenase 2）by ABA-independent signaling pathways to regulate redox process in drought stress,to improve the drought resistance of plants.In addition,BnMAPK1 may also interact with CYP20-3 and TUF（Vacuolar ATP synthase subunit E1）by cytokinin signaling pathways to regulate cell expansion,to advance the growth period of rapeseed.6.The transcription of BnMAPK1 was regulated by RHON1 and PIG3 to response to drought,and regulated by SnRK1-PV42a and FRL3 to regulate growth and development in rapeseedY1H and Y2H assays showed that RHON1（RHO-N domain-containing protein 1）,PIG3（Quinone oxidoreductase PIG3-like）,SnRK1-PV42a（SNF1-related protein kinase regulatory subunit gamma-like PV42a）,and FRL3（FRIGIDA-like protein 3）regulated the transcription of BnMAPK1 by binding to ProBnMAPK1-PF motif,but did not participate in translation and modification of BnMAPK1.Y1H and transcriptional activation assays indicated that RHON1 can bind to ProBnMAPK1 by ABA-dependent signaling pathways to response to drought,in which Rho N domain was important,and PIG3 can regulate BnMAPK1 by ABA-independent signaling pathways to regulate redox process in drought stress,in which all domains of PIG3 worked.Furthermore,plant-specific SnRK1-PV42a may regulate BnMAPK1 to participate in the development process though CBS domains binding to ProBnMAPK1-PF,and the Frigida domain of FRL3 may regulte BnMAPK1 to function in cell differentiation and flowering by cytokinin signaling pathways.7.Directly regulation to BnMAPK1 by BnMKK3/9 dependent on MAPKs cascadeThe BnMKKs genes of rapeseed were cloned,and the results showed that there were two copies of BnMKK1/2/3/5/8,one copy of BnMKK4/6/9/10,and BnMKK7 may not exist in rapeseed.Based on Y2H assays,both BnMKK3 and BnMKK9 could interact with C group BnMAPK1/2/7,indicating that BnMKK3 and BnMKK9 may directly regulate BnMAPK1 to participate in drought response and growth and development process through the phosphorylation of MAPKs cascades.