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Functional genetics of rice PISTILLATA genes reveals new roles and target genes in flowering time, female fertility, and parthenocarpy.

Floral organ identity is controlled largely by the combinatorial action of MADS domain homeotic transcription factors. Lodicules are specialized plant organs in cereals and grasses that are involved in floret opening and facilitate pollination and fertility in rice (Oryza sativa L.). To understand the mechanisms underlying the specification of the rice lodicule, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2, and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and revealed new roles in flowering time and floral organ number and fate. Doubly perturbed osmads2 osmads4kd florets had severe abnormalities, were female infertile, yet could initiate parthenocarpy. Ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities. We also utilized genome-wide binding analyses and transcriptome profiling to identify putative target genes contributing to OsMADS2 functions. In osmads2d8/d8 null mutant, we observed deregulated genes in a plethora of processes including lodicule and stamen development, floral organ number, and cell wall development. Some examples are cell division regulators (Cyclin D6, Cyclin-P4-1-like), an aquaporin (PIP1A), a peptide transporter, a vascular developmental regulator (HOX1), and a cell wall modulator (GH9B16). The deregulation of these genes may be associated with the disrupted cell division, tissue differentiation, and physiology of the malformed lodicules in osmads2 and osmads2 osmads4kd florets. Altogether, we reveal novel roles for the rice PI paralogs in flowering time, panicle exsertion, and embryo sac differentiation, identify gene targets for lodicule development, and provide mechanistic insights on the functional diversification of rice PI paralogs.

Oryza↗

Identification of class B and class C floral organ identity genes from rice plants.

The functions of two rice MADS-box genes were studied by the loss-of-function approach. The first gene, OsMADS4, shows a significant homology to members in the PISTILLATA (PI) family, which is required to specify petal and stamen identity. The second gene, OsMADS3, is highly homologous to the members in the AGAMOUS (AG) family that is essential for the normal development of the internal two whorls, the stamen and carpel, of the flower. These two rice MADS box cDNA clones were connected to the maize ubiquitin promoter in an antisense orientation and the fusion molecules were introduced to rice plants by the Agrobacterium-mediated transformation method. Transgenic plants expressing antisense OsMADS4 displayed alterations of the second and third whorls. The second-whorl lodicules, which are equivalent to the petals of dicot plants in grasses, were altered into palea/lemma-like organs, and the third whorl stamens were changed to carpel-like organs. Loss-of-function analysis of OsMADS3 showed alterations in the third and fourth whorls. In the third whorl, the filaments of the transgenic plants were changed into thick and fleshy bodies, similar to lodicules. Rather than making a carpel, the fourth whorl produced several abnormal flowers. These phenotypes are similar to those of the agamous and plena mutants in Arabidopsis and Antirrhinum, respectively. These results suggest that OsMADS4 belongs to the class B gene family and OsMADS3 belongs to the class C gene family of floral organ identity determination.

Cloning, Molecular↗

Pistillody is caused by alterations to the class-B MADS-box gene expression pattern in alloplasmic wheats.

Class-B floral homeotic genes are involved in specifying petal and stamen identity during flower development in plant species. Homeotic transformation of stamens into pistil-like structures (called pistillody) has been observed in alloplasmic lines of bread wheat ( Triticum aestivum L.) having the cytoplasm of a wild relative species, Aegilops crassa Boiss. To obtain information about the molecular mechanism underlying pistillody induction, we isolated two PISTILLATA ( PI)-type class-B MADS-box genes, WPI1 ( wheat PI STILLATA # 1) and WPI2, from wheat. Phylogenetic reconstruction indicated that WPI1 is orthologous to OsMADS4 and that WPI2 is probably an ortholog of OsMADS2. Both OsMADS4 and OsMADS2 genes were suggested to be PI orthologs in rice, and the function of OsMADS4 as a class-B gene was proven by the transgenic study. An in situ hybridization study demonstrated that the WPI1 gene is expressed in primordia of lodicules and stamens in developing florets in wheat. In the alloplasmic wheat line exhibiting pistillody, the WPI1 transcripts were not detected in the primordia of pistil-like stamens, whereas WPI1 was expressed in the lodicules. The wheat APETALA3 ( AP3)-type class-B MADS-box gene WAP3 ( wheat AP3) /TaMADS#82 showed an expression pattern similar to that of WPI1. These results suggest that pistillody in alloplasmic wheats is caused by alterations to the expression pattern of class-B MADS-box genes.

Amino Acid Sequence↗