Functional And Expressional Analysis Of Three Pivotal Genes For Floral Organ Development And A SUPERMAN-like Gene In Rice (Oryza Sativa L.)

Research of the genetic basis of plant reproductive development (especially flower development) is very significant in theory and practice. With three rice floral organ development mutants, we analyzed the expression and function of three key floral development genes SNG (Supper-Number Glume), OsMADS6 and OsJAG. In addition, the expression and function of a rice gene ZOS2-01, which is homologous to SUPMAN, a key gene for floral organ development in Arabidopsis, was also analyzed. The main results are as follows:1. Research of SNG: The vegetative growth and inflorescence development of sng mutants are all normal, but its spikelets are obviously deformed and swollened. At the early stage of lemma/palea development, the developmental directions of floral organ primordia are changed. Lodicules, stamens and pistils are completely degenerated, replaced by multiple whorls of extra symmetric glumes. An apparent petiole grows upward at the centre of each whorl of the extra glumes, forming a typical“flower in flower”phenotype. These mutant phenotypes suggest that SNG is a key gene for rice floral development and meristematic determination. Fine mapping had previously suggested that two phosphatase genes, named here as SNG1 and SNG2, could be the candidates of SNG. Q-PCR and GUS expression analyses showed that SNG1 is expressed in all the tissues examined, whereas SNG2 is expressed strongly in leaves and spikelets but weakly in other tissues. Genetic complementation test indicated that SNG2 could rescure the sng mutant phenotypes, confirming that SNG2 should be the SNG gene. Subcellular localization analysis indicated that both SNG1 and SNG2 are located in the nucleus. Q-PCR analysis of floral organ identity genes showed that class A genes were obviously up-regulated in the sng mutant, while classes B and C genes, class E genes OsMADS7/8 and AGL6-like genes were obviously down-regulated. The result of in situ hybridization also showed that OsMADS7/8 are expressed mainly in lemma-like primordium, while OsMADS6/17/3 are expressed very weakly in lemma-like primordium. So, we deduced that class B, class C and AGL6-like genes may be directly or indirectly regulated by SNG.2. Research of OsJAG: The vegetative growth of osjag mutant was normal, but all floral organs developed abnormally in the Osjag mutant. Although all floral organ primordia can be inititated at their correct positions, their development directions are changed. The most obviously feature is that all stamens are transformed into carpels. By fine mapping, a single C2H2 zinc finger gene was identified as the candidate gene of OsJAG, which was confirmed by genetic complementation experiment. Q-PCR and in situ hybridization analyses of floral organ identity genes indicated that class C genes OsMADS3/58, DL gene and OsMADS6 were obviously up-regulated in the Osjag mutant, and the expression domain also expanded from whorl 4 to whorl 3; while the expression of OsMADS17 was obviously reduced. This suggests that the expression of OsMADS3, DL and OsMADS6 may be negatively regulated by OsJAG.3. Research of OsMADS6: The vegetative growth and inflorescence development of osmads6 are normal, but its spikelets are obviously deformed and swollened. Although all floral organ primordia can be initiated at their correct positions, their development directions are changed. Palea, lodicules, stamens and carpel are all homologously transformed into lemma or lemma-like organs and the floral meristem determination is lossed. These results suggest that OsMADS6 is a key gene for rice floral development and meristematic determinination. OsMADS17 is also an AGL6-like gene, but its RNAi transgenic plants did not show visible abnormal phenotypes. By transfering OsMADS17 RNAi vector into Osmads6 mutants, the vegetative growth of the transgenic plants (double mutants) was severely blocked and could not differentiate into floral organs. Q-PCR analysis showed that in the double mutant, the expression of class A genes was significantly down-regulated, while that of OsMADS1 was significantly up-regulated. The results suggest that the double mutant phenotypes might be caused by the down-regulation of class A genes and the up-regulation of OMADS1 gene.4. Research of ZOS2-01: According to the functional domain of Arabidopsis SUPERMAN (SUP) gene, a SUP-like gene ZOS2-01 was isolated from rice. Q-RT-PCR showed that ZOS2-01 is expressed almost in all tissues, with the strongest expression in young panicles, implying that ZOS2-01 might be probably involved in the regulation of vegetative growth and floral organ development in rice. However, decrease and increase of ZOS2-01 expression mediated by RNA interference and over-expression did not exhibit obvious influence on rice growth and development. These results suggest that although rice ZOS2-01 and Arabidopsis SUP have the same functional domain, they might have diverged in expression and function, or there might be several SUP-like genes with redundant functions in rice.