SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa

In this article, we report that carpel specification in the Oryza sativa (rice) flower is regulated by the floral homeotic gene DROOPING LEAF (DL) that is distinct from the well-known ABC genes. Severe loss-of-function mutations of DL cause complete homeotic transformation of carpels into stamens. Molecular cloning reveals that DL is a member of the YABBY gene family and is closely related to the CRABS CLAW (CRC) gene of Arabidopsis thaliana. DL is expressed in the presumptive region (carpel anlagen), where carpel primordia would initiate, and in carpel primordia. These results suggest that carpel specification is regulated by DL in rice flower development. Whereas CRC plays only a partial role in carpel identity, DL may have been recruited to have the more essential function of specifying carpels during the evolution of rice. We also show that DL interacts antagonistically with class B genes and controls floral meristem determinacy. In addition, severe and weak dl alleles fail to form a midrib in the leaf. The phenotypic analysis of dl mutants, together with analyses of the spatial expression patterns and ectopic expression of DL, demonstrate that DL regulates midrib formation by promoting cell proliferation in the central region of the rice leaf.     

Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants

To develop an efficient gene tagging system in rice, a plasmid was constructed carrying a non-autonomous maize Ds element in the untranslated leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid containing the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-resistant calli, regenerated plants and their progeny showed that the introduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hygromycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alterations are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 by target duplications is observed at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated stably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stably integrated into three different chromosomes, one of which also contained the functional hph gene restored by Ds excision. The results indicate that a dispersed distribution of Ds throughout genomes not bearing the active Ac transposase gene can be achieved by simultaneous transfection with Ds and the Ac transposase gene.     

TEAseq-based identification of 35,696 Dissociation insertional mutations facilitates functional genomic studies in maize

In plants, transposable element (TE)-triggered mutants are important resources for functional genomic studies. However, conventional approaches for genome-wide identification of TE insertion sites are costly and laborious. This study developed a novel, rapid, and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing (TEAseq). Using TEAseq, we systemically profiled the Dissociation (Ds) insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background. The Ac-containing individuals were excluded for getting rid of the potential somatic insertions. We characterized 35,696 germinal Ds insertions tagging 10,323 genes, representing approximately 23.3% of the total genes in the maize genome. The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci, and insertions occurred preferentially in gene body regions. Furthermore, we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development. We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations. Overall, our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.     

Functional characterization of transposon-tagged abiotic stress-responsive rice genes and their molecular polymorphisms among various stress-tolerant genotypes

Dissociation (Ds) insertional mutagenesis has been regarded as an efficient tool to generate insertion mutants for functional genomics and molecular breeding. However, little is known about the application of the tool on exploring biological functions of abiotic stress-related genes and their molecular breeding experience. In this study, a total of 833 Ds insertion lines have been obtained, which showed significantly higher tolerance or sensitivity to high salinity, drought or cold stress, by screening around 20,000 Ds lines. Analysis of Ds flanking sequence tags revealed that 165 rice genes were tagged by Ds insertion. Gene Ontology (GO) and gene set enrichment analysis showed that over-represented Ds-tagged genes might function in the response to exogenous stimuli. These Ds-tagged genes showed expression divergence among five high salinity and five drought tolerant rice lines under either high salinity or drought stress. Higher percentages of Ds-tagged genes were down- or up-regulated by these abiotic stresses. These Ds-tagged genes were also frequently reduced or suppressed by various phytohormones including abscisic acid and jasmonate. On the other hand, we have also detected single nucleotide polymorphisms (SNPs) and 1–10 base pairs of insertion and deletions (indels) of these Ds-tagged genes among ten high salinity/drought tolerant rice lines by comparing with the reference genome Nipponbare. Our data showed that SNPs were detected among 102 out of 165 genes and indels were identified in 39 out of 165 genes. All the data provided additional information to further explore the biological functions of these genes or to carry out molecular breeding.