Morphogenesis, Gene Mapping And Functional Analysis Of Floral Organs Variation In Naked Seed Rice

Genetic and molecular mechanism underlying the regulation of floral development has become one of important subjects for plant developmental biology. The mutants of floral development were important for the studies on genes expression and function. In monocot plants, most of genes involved in floral development were isolated by screening the cDNA or DNA library with the probes homologous to sequences of dicot plants, and their functions were identified by the altered morphology of floral organs in transgenic plant. Understanding of the regulation on floral development was limited greatly, since some changed traits in transgenic plant were not consistent with the function of genes. Naked seed rice (nsr) was characterized by overdeveloped palea/lemma, the transformation of lodicules to glume-like structures, the variable number of palea/lemma, lodicules, stamens and pistils, it could be, hence, served as typical material for the researches on the molecular regulation of floral development in rice, as well as in grass plants.The investigation of spikelets in nsr showed that the length of florets ranged from 0.98cm to 3.55cm with an average of 1.82cm, and the florets were significantly longer than that of wild-type rice. Compared to those in wild-type spikelet, the palea/lemma appeared thinner and displayed fewer and smaller epidermal cells and shorter trichomes with higher density in nsr. Furthermore, the epicuticula presented regular hexagonal cells, while those in nsr were thin and long oblong cells. The variable number of floral organs was observed in nsr spikelets. 75.6% of the investigated spikelets had one palea and on lemma and others contained extra palea or lemma. In nsr, the number of anthers varied from one to ten, and most of the spikelets were three anthers (accounted for 33.7% of the investigated spikelets ). The number of stigmas ranged from two to eleven and a majority of spikelets (67.6%) had two stigmas. The number of ovaries varied from zero to five and most of spikelets contained one ovary. Moreover, a majority of spikelets (91:3%) produced two pairs of lodicules, while a minority of ones (8.5%) contained only one pair of lodicules.The observation of SEM (Scanning Electronic Microscope) found that the primorida of palea/lemma, lodicule, stamens and carpel were successively initiated by floral primordium in wild-type rice. In nsr spikelet, lemma and palea were observed before the formation of the lodicule primordium. Then the irregular differentiation of floral primordium was exhibited in nsr spikelet, in which lodiculeprimordium was formed inner to lemma or two lodicules primordia were also observed inner to both lemma and palea. The floral primordium was elongated in the direction of lemma and palea and then was divided. Consequently, two separate floral primordia or syncarpy were formed in nsr spikelet. After the formation of lodicules primordia, the floral primordium could aslo generate primordia of stamens and carpels irregularly, and the number of stamen varied from two to four. Hence, the position and shape of stamen and carpel primordia for nsr were different from those of wild-type rice.Ft hybrids were derived from the cross between nsr and japonica rice variety "Zhenongda 104", which showed normal phenotype as wild type plants. The F2 population segregated in a ratio of 3 wild-type plants : 1 mutant plants, indicating that nsr was a monogenic recessive trait. The nsr locus was primarily mapped to chromosome 3 between microsatellite markers, RM3548 and RM2326. The genetic distance from nsr locus to RM3548 and RM2326 were 2.26cM and 1.7cM, respectively. Subsequent fine mapping showed that nsr locus was flanked by RM3417 (0.26cM) and RM7576 (0.2lcM). The physical distance between RM3417 and RM7576 is about 400kb. Annotation of this region identified an open reading frame (ORF) encoding a MADS-box protein, OsMADSl. Jeon et al. (2000) reported that OsMADSl was located at the region between RG100 and RZ313. The nsr locus found in this experiment was also located at this region. Thereby, the sequence analysis on coding regions of the OsMADSl gene of wild-type rice and nsr showed that five nucleotides of A, G, A, A and T at positions 58, 80, 287, 527 and 666, in coding region were changed to G, A, G, G and C in nsr, respectively. Consequently, the deduced amino acid of Thr20, Gly27, Lys96 and Asn176 were replaced with Ala, Asp, Arg and Ser, respectively, in OsMADSl protein of nsr, while His222 was not changed. So, nsr contained both missense and nonsense mutant in the. coding region of OsMADSl.To elucidate the function of OsMADSl, the expressing vector of 3SS::OsMADSl and RNA interference (RNAi) of OsMADSl were constructed and transformed into Nippobare rice embryogenic calli by Agrobaterium-mcdiattd transformation. The results showed that 17.9% of outer glumes in flowers of 35S::OsMADSl transgenic lines were transformed to palea/lemma-like structures, indicating a distinct role of OsMADSl in specifying the palea and lemma. The decreased number of stamens was observed in 35S::OsMADSl transgenic lines. The flowerof OsMADSl RNAi...