| Rice is a kind of important cereal crop that provides staple food for most of the world’s population.Improving the yield and quality of rice remains a concern for researchers.Because the development of floral organs can directly produce seeds,which finally determines grain yield and quality,it is of great practical significance to understand the regulatory mechanism of floral organ development.In this study,we identified a mutant named afg1(abnormal flower and grain 1)with abnormal palea development in the EMS mutant line of Yunjing 17(YJ17).First of all,we compared the phenotypes of the afg1 mutant and YJ17 under the field planting conditions,and observed the morphological characteristics of floral organs using stereomicroscope and scanning electron microscope(SEM);then we observed the structural characteristics of each whorl of floral organs by paraffin sectioning;the gene was precisely located and cloned by map-based cloning;the transcriptional activity of the protein was detected by yeast assay and dual luciferase reporter method;and the quality of afg1 and YJ17 grains were also examined.The main results are as follows:In the field,we identified a mutant afg1 with abnormally enlarged palea,elongated lodicules and reduced stamen number,but no obvious abnormality was found in other floral organs.Further observation by paraffin sectioning showed that the palea structure of afg1 had indeed changed,it lost the smooth marginal structure and showed the cell morphology similar to lemma,resulting in the failure to form the interlocked structure at the junction of palea and lemma and the appearance of glume splitting.SEM was used to observe the early floral primordia.It was found that the development of inner floral organs in afg1 was significantly delayed compared to that of the wild type.The statistical results showed that the grain length of afg1 was decreased,the grain width was increased,and the 1000-grain weight was also significantly decreased.We further detected the expression levels of genes related to grain size and cell proliferation and expansion,and found that the expression of these genes changed to varying degrees.The results of the physicochemical properties of the grains showed that the contents of total starch and amylose in afg1 were notably decreased,while the contents of protein and soluble sugar were increased.In addition,the gelatinization temperature was significantly increased,and the starch solubility in urea solutions was significantly different from those of the wild type.The results of q RT-PCR showed that the expressions of some rice quality-related genes were affected.Using a large number of mutant individuals,the gene was finally located in a76 kb region on chromosome 2 by map-based cloning.We found that there was a previously reported gene related to floral organ development in this region.Sequencing analysis showed that the gene leading to the phenotype of afg1 mutant was indeed Os MADS6.q RT-PCR showed that AFG1 was only expressed in spikelets and seeds at different developmental stages,but not in other organs such as root,culm,leaf or leaf sheath.In addition,we constructed CRISPR-Cas9 knockout lines and confirmed that it was LOC＿Os02g45770 that caused the abnormal phenotype of afg1.The subcellular localization showed that AFG1 protein was located in the nucleus,and we speculated that it was a transcription factor.Further more,the results of yeast experiment and dual luciferase assay showed that AFG1 protein had transcriptional activation activity.In conclusion,the above results showed that AFG1,as a transcriptional activator,not only interacts with some floral organ development related genes,but may also regulates the expression levels of the downstream cell proliferation and expansion,and grain quality related genes,thereby affecting grain yield and quality. |
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