SUMMARYMutant analyses in different eudicotyledonous flowering plants demonstrated that SEPALLATA-like MADSbox genes are required for the specification of sepals, petals, stamens and carpels, and for floral determinacy, thus defining class E floral organ identity genes. SEP-like genes encode MADS-domain transcription factors and constitute an angiosperm-specific gene clade whose members show remarkably different degrees of redundancy and sub-functionalization within eudicots. To better understand the evolutionary dynamics of SEP-like genes throughout the angiosperms we have knocked down SEP-like genes of rice (Oryza sativa), a distant relative of eudicots within the flowering plants. Plants affected in both OsMADS7 and OsMADS8 show severe phenotypes including late flowering, homeotic changes of lodicules, stamens and carpels into palea/ lemma-like organs, and a loss of floral determinacy. Simultaneous knockdown of the four rice SEP-like genes OsMADS1, OsMADS5, OsMADS7 and OsMADS8, leads to homeotic transformation of all floral organs except the lemma into leaf-like organs. This mimics the phenotype observed with the sep1 sep2 sep3 sep4 quadruple mutant of Arabidopsis. Detailed analyses of the spatial and temporal mRNA expression and protein interaction patterns corresponding to the different rice SEP-like genes show strong similarities, but also gene-specific differences. These findings reveal conservation of SEP-like genes in specifying floral determinacy and organ identities since the separation of eudicots and monocots about 150 million years ago. However, they indicate also monocot-specific neo-and sub-functionalization events and hence underscore the evolutionary dynamics of SEP-like genes. Moreover, our findings corroborate the view that the lodicules of grasses are homologous to eudicot petals.

Summary• In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleo-AP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana.• The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively.• Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3.• Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most destructive diseases and can cause severe yield losses in many regions of the world. Because of the large size and complexity of wheat genome, it is difficult to study the molecular mechanism of interaction between wheat and PST. Brachypodium distachyon has become a model system for temperate grasses’ functional genomics research. The phenotypic evaluation showed that the response of Brachypodium distachyon to PST was nonhost resistance (NHR), which allowed us to present this plant-pathogen system as a model to explore the immune response and the molecular mechanism underlying wheat and PST. Here we reported the generation of about 7,000 T-DNA insertion lines based on a highly efficient Agrobacterium-mediated transformation system. Hundreds of mutants either more susceptible or more resistant to PST than that of the wild type Bd21 were obtained. The three putative target genes, Bradi5g17540, BdMYB102 and Bradi5g11590, of three T-DNA insertion mutants could be involved in NHR of Brachypodium distachyon to wheat stripe rust. The systemic pathologic study of this T-DNA mutants would broaden our knowledge of NHR, and assist in breeding wheat cultivars with durable resistance.

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B. distachyon. The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B. distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T 0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.

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