Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen.

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.

The Arabidopsis CUL4-DDB1 complex interacts with MSI1 and is required to maintain MEDEA parental imprinting.

Protein ubiquitylation regulates a broad variety of biological processes in all eukaryotes. Recent work identified a novel class of cullin-containing ubiquitin ligases (E3s) composed of CUL4, DDB1, and one WD40 protein, believed to act as a substrate receptor. Strikingly, CUL4-based E3 ligases (CRL4s) have important functions at the chromatin level, including responses to DNA damage in metazoans and plants and, in fission yeast, in heterochromatin silencing. Among putative CRL4 receptors we identified MULTICOPY SUPPRESSOR OF IRA1 (MSI1), which belongs to an evolutionary conserved protein family. MSI1-like proteins contribute to different protein complexes, including the epigenetic regulatory Polycomb repressive complex 2 (PRC2). Here, we provide evidence that Arabidopsis MSI1 physically interacts with DDB1A and is part of a multimeric protein complex including CUL4. CUL4 and DDB1 loss-of-function lead to embryo lethality. Interestingly, as in fis class mutants, cul4 mutants exhibit autonomous endosperm initiation and loss of parental imprinting of MEDEA, a target gene of the Arabidopsis PRC2 complex. In addition, after pollination both MEDEA transcript and protein accumulate in a cul4 mutant background. Overall, our work provides the first evidence of a physical and functional link between a CRL4 E3 ligase and a PRC2 complex, thus indicating a novel role of ubiquitylation in the repression of gene expression.

Disruption of the pollen-expressed FERONIA homologs ANXUR1 and ANXUR2 triggers pollen tube discharge.

The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.

Determining the plasmotypic structure of rye populations by SCAR markers.

In rye (Secale cereale L.), 2 types of cytoplasmic male sterility are known: Pampa type (CMS-P) and Vavilovii type (CMS-V). As an alternative method to the conventional plasmotype-genotype interaction test, for identification of the cytoplasm type, the use of sequence-characterised amplified region (SCAR) markers was validated in this study. In over 2600 individual rye plants, representing 26 populations originating from Poland (18 cultivars), Iran (5 populations of primitive rye), and South America (3 populations), the cytoplasm type was determined by using a set of 3 SCAR markers. For about 10% of these individuals, the plasmotype-genotype interaction test was performed in parallel. The results of both tests were fully consistent. In the majority of the Polish populations, CMS-V was present, and only 4 populations contained CMS-P. Primitive Iranian populations contained predominantly normal cytoplasm, and only occasionally CMS-P was identified in them. South American populations displayed a mixture of normal cytoplasm, CMS-P and CMS-V. This work validates the use of SCAR markers as a reliable and quick method to determine the plasmotypic diversity of rye populations on a large scale.

Rye SCAR markers for male fertility restoration in the P cytoplasm are also applicable to marker-assisted selection in the C cytoplasm.

The study aimed at testing the usefulness of recently developed SCAR markers on rye (Secale cereale L.) chromosome 4R in hybrid breeding based on the C source of male sterility-inducing cytoplasm. Of 10 markers studied, 4 revealed polymorphisms between 2 inbred lines (544cms-C and Ot0-20) crossed to develop F2 and BC1 mapping populations. Analyses performed on 94 F2 and 93 BC1 plants allowed to extend a formerly constructed genetic map of chromosome arm 4RL. Three SCAR markers (SCP14M55, SCP15M55 and SCP16M58) were mapped in the vicinity of gene Rfc1, which restores male fertility in the C cytoplasm. The 3 tested SCAR markers proved to be effective in marker-assisted selection (MAS) for male fertility/sterility.