| Wheat(Triticum aestivum L.)is one of the important food crops in the world,and the development and quantity of its floral organs are directly related to the yield and reproduction coefficient of wheat.The floret of common wheat has a fixed number of floral organs,but in some mutants,the number of floral organs also increases.Multi-pistil wheat MA is a naturally mutated wheat material with 2-3 pistils,while the number of other floral organs is unchanged.Most of the added pistils have normal functions,and after pollination,they can develop into grains,which makes multi-pistil wheat have a clear advantage in the grain number per spike.In production,if multi-pistil wheat can be applied to hybrid wheat seed production,it may effectively increase the reproduction coefficient of hybrid wheat,reduce the cost of seed production,effectively promote the application and development of hybrid wheat,and eventually make hybrid wheat widely used in production.At the same time,if the gene associated with multi-pistil trait can be locked and the formation mechanism of multi-pistil wheat can be revealed at the molecular level,it will greatly enrich the relevant theories of wheat floral development.For these reasons,we used multiple rounds of backcrossing MA and normal wheat 77(2)to create a set of near-isogenic lines Mu77(2)and 77(2).We had revealed the potential formation mechanism of multi-pistil wheat at the molecular level via a series of analyses such as phenotype,proteome,transcriptome,hormone content and function of related genes.The main conclusions were as follows:1.It was determined that secondary pistils are derived from the extra stem cells between the carpel and lodicule primordia,by using scanning electron microscopy(SEM)and analyzing the expression level of Ta KNOX1.Compared with the flower meristem(FM)of normal wheat,the size of the FM of multi-pistil wheat did not increase significantly,and at the initial stage of the main-pistil carpel,there was still accumulation of extra stem cells in the FM of multi-pistil wheat.In multi-pistil what,a floret contains 2-3 pistils with no change of other floral organs,which is different from the pistillody in wheat.Because it lags behind the development of the main pistil,in the order of development,the secondary pistils are the fifth round of floral organs.Most of the secondary pistils have normal functions,and they can develop into normal grains after pollination,which makes the multi-pistil wheat have obvious advantages in the grain number per spike.2.By comparing the proteome profiles of the young spikes of Mu77(2)and 77(2),334 DAPs that may be related to the formation of multi-pistil wheat were identified.The DAPs,located primarily in the cell,were involved in the translation and the metabolisms of carbohydrate,nucleotide,and amino acid.Differential expression analysis showed that Ta HUA2,Ta RF2 a,Ta CHR12 and Ta HEN2 may play vital roles in the regulation of stem cell activity in wheat floral meristem.The low expression level of Ta HUA2 and Ta HEN2 may result in the low expression of wheat AG homologous genes,which in turn leads to the accumulation of extra stem cells.This also suggests that similar AG pathways may also be involved in the termination of the floral meristems in wheat.In general,proteomics analysis supports the results of phenotypic analysis to a certain extent,and also provides possible candidate genes for further study on the regulation of wheat floral organs.3.By comparing the transcriptional profiles of the young spikes of Mu77(2)and 77(2),204 differentially expressed genes(DEGs)that may be related to the formation and development of multi-pistil wheat were identified.According to the KEGG enrichment analysis,the DEGs were mainly involved in the carbon metabolism,glyoxylate and dicarboxylate metabolism,peroxisome,plant hormone signal transduction,m RNA surveillance pathway,and plant-pathogen interaction.Further analysis showed that in the plant hormone signaling pathway,the DEGs were mainly concentrated in the auxin signaling pathway.Ta ARF11 was a DEGs in auxin signal pathway,and its FPKM value was significantly higher in normal wheat than in multiple-pistil wheat based on RNA-seq.In addition,a series of q PCR results also showed that Ta ARF11 has abnormally low expression levels in multi-pistil wheat,and its expression level was correlated with multi-pistil trait.Further analysis shows that Ta ARF11-D was also abnormally low in multi-pistil wheat,which was the main reason for the low total expression of Ta ARF11.Combining the results of previous QTL mapping,we identified Ta ARF11-D as an important candidate gene associated with multi-pistil trait.The results of hormone content analysis showed that IAA content accumulated differentially in the young spikes of MA,Mu77(2)and 77(2).These results indicated that auxin and auxin signaling are necessary to maintain the stable number of floral organs in wheat.4.Our research also confirmed that Ta ARF11 is a nuclear-localized transcriptional activator.Overexpression of Ta ARF11 in Arabidopsis(Ta ARF11-OE)could promote the development of organs such as leaves,roots,stems,flower buds and branches.However,overexpression of Ta ARF11 did not directly inhibit the development of floral organs,nor did it reduce the number of floral organs.The expression level of Ta KNOX1 ortholog gene STM in the leaves and inflorescences of Ta ARF11-OE was significantly reduced.The promoter of Ta KNOX1 contains a conserved auxin response element.Our research also showed that Ta KNOX1 could negatively respond to the corresponding auxin signal.The expression trend of Ta KNOX1 in the young spike of multi-pistil wheat was opposite to that of Ta ARF11,and our research also confirmed that Ta ARF11 cannot directly bind to the promoter of Ta KNOX1 gene.These results indicated that Ta ARF11 might be indirectly inhibits the expression of Ta KNOX1 to regulate stem cell activity. |
