Mutation of the gene for AHA1, an H+-ATPase isoform, provides new insights into the stomatal regulation of Arabidopsis in response to blue light.

The most frequently used method to identify mutations induced by a commonly used mutagen, EMS (ethyl methane sulfonate), in Arabidopsis thaliana has been map-based cloning. The first step of this method is crossing a mutant with a plant of another accession as it requires polymorphisms between accessions for linkage analysis. Therefore, to perform the method routinely, it is greatly preferred to use accession combinations between which enough polymorphisms are already known. Further, it requires laborious examination of a large number of F₂ recombinants using many markers to detect each polymorphism. After linkage analysis narrows down the chromosomal region containing the causal mutation, sequencing candidate genes one by one within the region is necessary until the mutation is finally identified. Overall, this method is generally time-consuming and labor intensive, and it becomes harder when multiple loci are involved in phenotypes. A few recent reports showed that causal mutations induced by EMS could be identified by deep-sequencing technologies with less labor compared with the conventional method when mutants were generated in the Arabidopsis reference Columbia background whose genome organization is well known. Here we report that we succeeded in rapid identification of EMS-induced causal mutations in a non-reference accession background, whose whole genome sequence is not publicly available, using one round of whole genome sequencing. Moreover, in our case, we could monitor the causal locus and the transgenic reporter locus simultaneously, implying that this methodology could theoretically be applicable to analyzing even complex traits. We describe the pipeline of this methodology and discuss its characteristics.

Significance: FLOWERING LOCUS T (FT) acts as a mobile floral activator that is synthesized in leaf and transported to shoot apex. A Rice FT-like protein, Heading date 3a (Hd3a), requires interaction with 14-3-3 proteins and transcription factor FD to induce flowering. We confirm that Hd3a and its interactors, as well as their transcriptional target, coexist in the shoot apex at the appropriate time during floral transition. RNA-sequencing analysis of shoot apices from wild-type and RNA-interference plants for FT-like genes showed that 58% of classified transposable elements are transcribed, and >200 are down-regulated in response to FT-like. Our results indicate a link between reproductive development and transposon behavior in the shoot apical meristem, supporting and extending recent evidence for such a link during gametophyte development.

In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore), and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.