Molecular Mechanisms Of DEFECTIVE IN ANTHER DEHISCENCE1(DAD1) In Regulating Silique Length In Brassica Napus

Rapeseed?Brassica napus L.?is widely planted all over the world,which is an important oil crop.In addition,rapeseed is also a major protein feed and industrial raw material crop.In China,rapeseed oil is the largest source of edible vegetable oil,accounts for about 55%of total consumption.In previous study,we identified a DEFECTIVE IN ANTHER DEHISCENCE1?Bna.A05DAD1?gene that positively related to silique length based on the rapeseed Full-length c DNA over-expression gene hunting?FOX-hunting?library.The Bna.A05DAD1 gene encodes a chloroplast phospholipase A₁?PLA₁?protein,which participates in the first catalytic reaction in the jasmonic acid?JA?biosynthesis pathway,and can convert phospholipids into linolenic acid.At present,several studies have shown that DAD1 gene is involved in JA biosynthesis pathway,and associated with anther cracking and wounding induction,but it has not been reported that DAD1 is involved in plant silique development and seed weight increased.Therefore,we compared the gene structures,protein characteristics,evolutionary characteristics,and gene expression patterns of Bna.A05DAD1 and its homologous proteins,and detected the expression pattern of Bna.A05DAD1 in rapeseed by quantitative Real-time PCR?q RT-PCR?and GUS histochemical staining.Then,Bna.A05DAD1 recombinant protein was obtained by prokaryotic expression and purification.The protein activity assay showed that the protein had conservative catalytic activity and could promote the production of linoleic acid and linolenic acid.The silique length,seed number per silique and thousand seed weight of Arabidopsis and rapeseed overexpressing Bna.A05DAD1 transgenic lines were significantly higher than those of wild-type?WT?plants.In addition,we also analyzed the changes of fatty acid composition in transgenic plant seeds by Near infrared reflectance spectroscopy?NIRS?,Gas chromatography?GC?and q RT-PCR.The change of bound water content in transgenic seeds and its effect on seed vigor was revealed based on water content measurement and seed germination test.Finally,based on the comparative results of transcriptome,marker gene expression patterns induced by wound and JA,and JA and auxin content between WT and Bna.A05DAD1 overexpressing lines,the regulation mechanism of silique length increase due to the overexpression of Bna.A05DAD1 was proposed.The main results are as follows:1.Bioinformatic analysis of Bna.A05DAD1 and its homologous genes in rapeseedUsing 12 At PLA₁ proteins as query,a total of 32 Bna.PLA₁ members were identified from rapeseed based on the results of BLASTP alignment and domain analysis,including 2 DAD1 proteins,12 DAD1-LIKE LIPASE?DALL?proteins,2DONGLE?DGL?proteins,13 DAD1-LIKE ACYLHYDROLASE?DSEL?proteins and3 DAD1-LIKE SEEDING ESTABLISHMENT-RELATED LIPASE?DLAH?proteins.The phylogenetic tree constructed by 32 Bna.PLA₁ protein sequences and the subcellular localization prediction results both suggested that Bna.PLA₁ proteins were divided into three classes.The members in class?,?and?were located in chloroplast,mitochondria and cytoplasm,respectively.Gene structures analysis showed that each class members have similar gene structure and exon number,except for the class?members.All the Bna.PLA₁ genes were unevenly distributed in the chromosomes of rapeseed except A02,C02 and C09.Analysis of physical and chemical properties showed that the polypeptide chain length was between 368-523 aa,molecular weight?Mw?was 42.41-59.46 k Da;protein isoelectric?p I?was 5.28-9.83,among which23 members?more than 70%?were basic proteins with p I greater than 7.0.Among the five DAD1 homologous proteins of Arabidopsis and Brassica,At DAD1 lacks the functional unknown motif 7,Bna.A05DAD1 lacks the functional unknown motif 13.Results of the three-dimensional structure comparison showed that Bna.A05DAD1 had one more?-sheet than At DAD1,but it was located outside the protein core functional region and had no significant effect on the protein structure,so it was speculated that it would not affect the function of the protein.2.Tissue-speficific expression pattern of Bna.A05DAD1 in rapeseedTranscriptional data of Bna.PLA₁ genes those retrieved from the Brassica EDB database showed that the Bna.PLA₁ genes displayed diverse expression patterns in different classes.Majority of Bna.PLA₁ genes were expressed in specific stages and organs,while partial genes were silent in all investigated organs.It is worth noting that Bna.A05DAD1 and Bna.C04DAD1,two copies of Bna.DAD1 in rapeseed,were only specifically expressed in seeds and seed coats,especially those at 40 days after pollination?DAP?.q RT-PCR results showed that Bna.A05DAD1 gene was specifically expressed in seeds and seed coats at 40 DAP.In order to further identify the expression pattern of Bna.A05DAD1 gene,we cloned 1500 bp upstream of Bna.A05DAD1 coding region sequence?the putative promoter?,and constructed the plant expression vector of GUS reporter gene driven by this promoter and transformed into Arabidopsis.Histochemical staining of GUS activity in T₃ transgenic plants showed that the gene was specifically expressed in anthers and seeds.Combined with the results of transcriptome,we found that Bna.A05DAD1 was mainly expressed in seeds,seed coats and anthers in rapeseed.3.Overexpression of Bna.A05DAD1 significantly increase silique length and thousand seed weight in rapeseedThe full coding sequence?CDS?of Bna.A05DAD1 driven by the Cauliflower mosaic virus 35S promoter was inserted into the binary vector p Early Gate101 to generate the recombinant p35S::DAD1-YFP plasmid.Transformed into Arabidopsis and rapeseed by Agrobacterium tumefaciens,and the positive transgenic lines of T₃generation were obtained.In Arabidopsis,the average silique length?1.55 cm?,the number of seeds per silique?67?and the thousand seed weight?21.15 mg?of overexpressed transgenic lines were increased?16.8%,9.80%,11.26%,respectively?,compared with WT plants?1.33 cm,61,19.01 mg?.In rapeseed,the average silique length?11.51 cm?,the number of seeds per silique?23?and thousand seed weight?3.76g?of overexpressed transgenic lines,were increased?42.59%,43.75%,25.65%,respectively?,compared with those of WT?8.07 cm,16,2.87 g?.Our results showed that overexpression of Bna.A05DAD1 can significantly increase several yield traits of plants,which is of great significance in promoting the yield of rapeseed and other crops.4.Overexpression of Bna.A05DAD1 participates in fatty acid elongation to change fatty acid compositionGC analysis results showed that compared with the WT?72.89%?,the average oleic acid concentration of the three lines overexpressing Bna.A05DAD1 in rapeseed significantly decreased 67.21%,while the average content of linoleic acid and linolenic acid increased significantly,and positively correlated with the expression level of Bna.A05DAD1?r²=0.99,P=0.007?.In addition,the differentially expressed genes?DEGs?analysis between transgenic plants and WT showed that the expression of multiple copies of FATTY ACID DESATURASE-2/3/6/7/8 genes were up-regulated in the process of fatty acid biosynthetic process?GO:0006633?,fatty acid metabolic process?GO:0006631?.q RT-PCR results further showed that the expression of linoleic acid biosynthesis key gene Bna FAD2 and linolenic acid biosynthesis key gene Bna FAD3in the seeds of overexpressing Bna.A05DAD1 transgenic rapeseed plants were significantly up-regulated compared with the WT,and their expression trend was consistent with the expression pattern variation of Bna.A05DAD1 genes in the transgenic rapeseed plants.The coding sequences?CDS?of Bna.A05DAD1 was inserted into the prokaryotic expression vector p GEX-4T1 to generate the construct for Bna.A05DAD1 recombinant protein expression.After purification of Bna.A05DAD1,the catalytic activity was assayed with anchovies oil as the substrate,and fatty acid components in the samples were compared usging GC.The content of oleic acid decreased significantly from2.65%to 2.14%,and the contents of linoleic acid and linolenic acid increased from0.074%to 0.212%and from 0.177%to 0.314%,respectively.These results showed that Bna.A05DAD1 can hydrolyze fatty acids to produce linoleic acid and linolenic acid,which is similar to the reported of PLA₁ family members.The aforementioned results confirmed that Bna.A05DAD1 was involved in the elongation of fatty acids in seeds to change the composition of fatty acids in overexpressed transgenic rapeseed.5.Overexpression of Bna.A05DAD1 participates in the regulation of bound water content in rapeseed seedsThe changes of seed bound water content and its effect on seed vigor were analyzed by measuring seed water content,seed germination rate,absolute germination rate and the length of radicle and hypocotyl of seedlings after germination in WT and overexpressing Bna.A05DAD1 transgenic plants of rapeseed.The results showed that the content of bound water in seeds of transgenic plants was significantly higher than WT,and the trend was in agreement with the expression variation of Bna.A05DAD1.The change of bound water content in seeds had no obviously effect on the absolute germination rate of seeds.It is worth noting that,without swelling,the overexpressed Bna.A05DAD1 transgenic rapeseed seeds germinated 6 h earlier.The average radicle length in three transgenic lines increased 7.2%,34%,23%,16%,and 30%,respectively,compared with wild type?1.26,2.95,5.50,6.98,and 7.61 cm?at 36,48,60,72,96,and 108 h after germination.And the average hypocotyl length increased by 26%,29%and35%respectively compared with WT?1.27,1.40,and 1.50 cm?at 72,96,and 108 h after germination,indicating that Bna.A05DAD1 has a positive regulatory effect on seed vigor.In addition,the results of DEGs analysis between transgenic rapeseed plants and wild type transcriptome showed that 245 differential genes related to seed water content were enriched,among which the up-regulated genes were mainly enriched in regulation of body fluid levels?GO:0050878?,multicellular organismal water homeostasis?GO:0050891?and other metabolic pathways,while the down-regulated genes were mainly enriched in response to water deprivation?GO:0009414?and other metabolic pathways.These results suggested that Bna.A05DAD1 gene may be involved in the regulation of seed water content in rapeseed and has a certain effect on seed germination rate.6.Overexpressionof Bna.A05DAD1 activates Bna.A09ARF18-mediated auxin synthesis pathway to increases silique lengthCompared with the WT,the silique length of overexpression of Bna.A05DAD1transgenic rapeseed plants increased significantly at 10 and 35 DAP.Observation of silique peel cells by scanning electron microscope showed that the increase of silique length of transgenic plants was mainly related to the increase of cell size but not cell differentiation.In addition,the results of DEGs analysis between transgenic rapeseed plants and wild type transcriptome showed that the up-regulated genes were enriched in auxin mediated signaling pathway?GO:0009734?and other metabolic pathways,including Indole-3-Acetic Acid Inducible 9?Bna.C01IAA9,Bna.C02IAA9?and Yucca 8?Bna.C01YUC8?,as well as PIN-formed 1?Bna.PIN1?and covalently modified jasmonic acid Gretchen hagen 3?Bna.GH3?related to auxin output.According to the q RT-PCR results,the expression level of Bna.A09ARF18,which was negatively related to silique length,was significantly down-regulated,while the Bna.A02PIN1 and Bna.A03GH3 related to cell size was increased.The changes in the expression of these genes was consistent with the change trend of JA and IAA content in wild type and transgenic plants.In addition,JA induction could lead to the inhibition of Bna.A09ARF18 gene expression in rapeseed leaves,indicating that Bna.A09ARF18 may be the interaction point of JA and IAA,and plays an important role in the crosstalk between the two phytohormones.According to the abovementioned results,we speculated that when ectopic overexpression of Bna.A05DAD1 in the silique,the key genes in JA synthesis pathway could significantly increase the JA content in the silique of transgenic plants.The increase of JA content inhibits the activation of IAA synthesis pathway gene by Bna.A09ARF18,which might be the key gene of signal interaction between two phytohormones JA and IAA.Resulting in the increase of IAA content in silique,the enlargement of cell size during silique development and the phenotype of increased silique length.In general,our study confirmed that overexpression Bna.A05DAD1 could change the fatty acid composition of rapeseed seeds,increases the silique length,and increase the thousand seed weight by increasing the content of bound water in seeds.At the same time,the improvement of seed vigor might improv the seed quality.Furthermore,this study clarified the important role of Bna.A05DAD1 in promoting rapeseed yield,and preliminarily revealed the mechanism of JA and IAA crosstalk in crop yield formation,which provided a novel insight into of high yield breeding of rapeseed.