Map-based Cloning And Functional Characterization Of BLS1, A Gene Required For Lemma And Palea Development In Rice(Orzya Sativa L.)

Grass species, like rice (Oryza sativa L), have highly specialized flowers that are different fromthose of eudicots. A typical eudicot flower comprises of sepals, petals, stamens, and carpel from outer toinner, whereas rice flower consists of stamens and carpel like eudicots, but the surroundingstructures, lodicules, lemma and palea, are unique to grasses. At present, the origin and molecularmechanism of lemma and palea development are poorly understood. In this study, we characterized tworice mutants, bls1-1and bls1-2(beak like spikelet1) with deformed lemmas and paleas, were obtainedby screening a T-DNA insertion population and spontaneous mutant population, respectively.Map-based cloning and functional analysis of the bls1mutant revealed the genetic and molecularmechanism that caused the mutant phenotype of bls1. These results enhanced our understanding of themolecular mechanisms of lemma and palea development in rice spikelet. Results from our experimentare summarized as follows:1. bls1lemma and palea exhibited slender phenotype, especially at the top where it displayed abeak like structure. However, the morphology of other floral organs, including rudimentary glumes,sterile lemmas, lodicules, stamens and pistils was normal. This suggested that the alterations in themutant were due to shape changes, rather than homeotic transformation of the lemma and palea. In ahistological analysis, the bls1cell sizes of four types of the lemma and palea, including silicified cell,fibrous sclerenchyma, spongy parenchymatous cell and nonsilicified cell were obviously smaller thanthose of WT. With the statistic analysis of the innermost nonsilicified cell in lemma and palea, there wasno difference in the cell number between bls1and WT, while the average cell area reduced significantlyin bls1. Scanning electron microscope analysis showed that the floral-organ primordia initiation andfloral organ patterning of bls1mutant were normal. By Sp8with formation of ovule and pollen, thelemma and palea of WT started to develop laterally, whereas the developmental process of bls1seemedto be delayed and the lemma and palea were prevented from growing horizontally.2. Real-time PCR analysis revealed that the expression of several floral organ identity genes relatedlemma and palea, including AP1-like, SEP-like and AGL6-like gene were apparently not affected in bls1mutant. In addition, in bls1, palea-related genes REP1and DP1or lemma-related gene DL displayedsimilar expression level as in WT. No significant alterations in these genes expression suggested thateither BLS1functions as the downstream of genes measured or BLS1represents a new pathway relatedto lemma and palea development.3. Genetic analysis revealed that bls1phenotype is controlled by a single recessive nuclear locus.Through mapping the F2population produced by bls1-1crossing Dular, the bls1locus was mappedwithin a region of about110kb on the long arm of chromosome2. A chromosome segment ofapproximately87kb was deleted within the restricted region in bls1-1. Then we used another mutantbls1-2, which is an allele mutant with bsl1-1to cross with Peiai64, and refined the bls1locus to a65kbregion. There was also an approximately50kb deletion within the restricted region in bls1-2. Sevenputative genes were including, among them, mere LOC_Os02g56610expressed in the young inflorescence of WT. Complement test and RNA interference experiment confirmed that the beak likespikelet of bls1mutant was caused by the deletion of gene LOC_Os02g56610, which was subsequentlydesignated as BLS1.4. BLS1encodes a protein containing287amino acids with a conserved DUF640domain withunknown function. A GenBank database search suggested that DUF640domain may be specific to landplants. Histochemical staining of the transgenic plants showed that GUS activity was strong in theyoung inflorescence, specifically the young lemmas and paleas of spikelets, corresponding to the resultsrevealed by Real-time PCR analysis. Both the results of transiently expression in rice leaf protoplastsand transgenic rice revealed that BLS1localizes in the nucleus.