Morphological Characteristics And Expressional Analysis Of A Dl/Osjag Double Mutant In Rice

The floral formation plays an important part in plant reproductive development. Many genes are involved in the regulation of floral organ development. Two mutants, dl and Osjag, related to floral organ development were found in rice. Now, it is known that DL(DROPPING LEAF) and OsJAG belong to Zinc-finger genes. Loss of DL function causes drooping leaf phenotypes and complete transformation of carpels into stamens, loss of OsJAG function results in significant change of four floral organs, causing complete transformation of stamens into the pistils. Based on the above results, we constructed dl/Osjag double mutant. The morphological characteristics and gene expression of the dl/Osjag double mutant were investigated in this study. The main results are as follows:1. Morphological Characteristics of the dl/Osjag Double MutantThe dl/Osjag mutant exhibited obvious abnormalities in plant morphology and floral organ development. The dl/Osjag plant morphology is similar to that of dl mutant. Because of lack of midrib, its leaves are drooping. The dl/Osjag mutant does not change the number of floral organ primordia, but seriously affect the morphology of the four whorls of floral organs. The lemma and palea in whorl 1 look like hooks and can not be closed. The lodicule primordia in whorl 2 do not show significant change but occasionally swelling elongation. Both stamen primordia in whorl 3 and pistil primordia in whorl 4 are completely transformed into stamen-like organs. The floral organ development in dl/Osjag mutant flowers shows some similarities to that of the dl and Osjag mutant. The development of lemma and palea primordia and lodicules primordia is similar to Osjag mutant. The development of pistil and stamen is similar to the process of pistil dissimilation in dl mutant. It is thus speculated that DL and OsJAG genes may act in a chronological order in the regulation of floral organ development.2. Gene expression analysisComparison of the expression levels of various types of flower organ identity genes by Quantitative PCR in single/double mutant. The results show that(1) OsMADS14/15/2/4/16/58/1/7/19/8/6/17were upregulated in the early develo- pmental stages of floral organ primordia, which OsMADS6 significantly upregulated, OsMADS5 slightly downregulated, other genes basically unchanged in double mutant dl/Osjag. However, OsMADS6/19 significantly upregulated and OsMADS14/2/4/16/8 basically unchanged as well as other genes were varying degrees of downregulated, Which OsMADS3/5 was significantly reduced in the pistil and stamen formation. This implies that DL/OsJAG might regulate the floral development by affecting the expression of floral identity genes.(2) Compared with the expression levels of different types of genes in the dl, Osjag and dl/Osjag mutants. This showed that the expression levels of some genes are similar to Osjag mutant at the dl/Osjag mutant flower primordia early stages. the expression level is similar to dl mutant at the process of the stamen-like organ formation in the double mutant. This suggests that the DL and OsJAG genes have chronological order in the regulation of floral organ development.(3) The DL gene plays a decisive role in pistil development. The expression level significantly upregulated in Osjag mutant. While the expression level of OsJAG gene, which plays a decisive role in the development of stamen significantly reduced in dl mutant.This indicates that the DL gene may be negative regulation of OsJAG gene.3. in situ hybridization analysisThe situ hybridization results of OsMADS16, which belongs to Class B genes, further validates the spatial and temporal expression characteristics of the DL and OsJAG gene. This shows that (1) the ectopic expression of OsMADS16 gene was found in the whorl 4 of the dl mutant. While, the expression levels of other whorls were similar to wild-type. (2) With the formation of pistil-like organs which changed from both stamens primordia in whorl 3 and pistil primordia in whorl 4, the expression levels of OsMADS16 gene significantly decreased in the Osjag mutant. (3) In contrast, the expression level of OsMADS16 gene is similar to Osjag mutant in the early floral primordia development of dl/Osjag mutant. And, the expression area of OsMADS16 was significantly increased in the formation of stamen-like organs. This results confirmed that OsMADS16 inhibition DL expression and promote OsJAG expression. Also, the DL and OsJAG gene have chronological order in the floral organ development of dl/Osjag double mutant:The development of lemma and palea primordia in whorl 1 and lodicules primordial in whorl 2 and stamen primordia in whorl 3 was regulated by OsJAG gene. While, the development of stamen primordia was regulated by DL gene. So, the expression pattern of stamens and pistil characteristics of development is proposed in this study:OsJAG gene mainly promotes the specialization and development of stamen primordia. However, DL gene specificity promotes the specialization and development of pistil primordia.In short, two important regulatory genes of floral organ development, OsJAG and DL, sparately or jointly regulated the development four floral organ, but its have different roles and chronological order. Among them, the OsJAG gene individually re-gulated the development of palea and lodicules primordial. OsJAG+DL co-regulated the development of lemma OsJAG and DL interacted decisions the development of stamen, but the development of pistil mainly determined by the DL gene.