Nonomura Lab
Research
Figure 2. Rice MEL2 forms RNA granules in the cytoplasm of gametocytes immediately prior to meiosis
(A) A cross section of a rice anther just before meiosis (left). In the pollen mother cell of the anther locus, MEL2-GFP (green) forms a strong granular signal in addition to a weak signal that spreads throughout the cytoplasm. The right shows an enlarged image of the cytoplasm.
(B) Transverse section of rice anther at the prophase of meiosis I (left). Once the meiotic phase begins, no MEL2-GFP signal can be detected. This indicates that MEL2 functions only in gametocytes immediately prior to meiosis and controls the timing of the transition to meiosis. The bar indicates 5 µm.
Figure 1. Rice AGO4a functions in DNA methylation in meiotic cells
(A) AGO4a localizes to meiotic cell nuclei (arrowheads) and frequently binds to meiosis-specific 24nt phasiRNAs followed by 24nt TE-siRNAs derived from transposable elements (TEs). (B) Overview of meiotic cell isolation.
(C) Of the three DNA methylating domains CG, CHG, and CHH (H=A, T, C), CHH methylation in microspores immediately after meiosis was significantly reduced in the ago4a mutant.
(Presentation that won the Best Paper Award at the 96th Annual Meeting of the Genetics Society of Japan)
1. Research on molecular mechanisms that promote reproductive development and meiosis in plants
Plants, which are unable to move to a different location even if their habitat environment deteriorates, have evolved unique systems responsible to their environments and reproductive systems. For example, the double fertilization of angiosperms has been well studied as a reproductive mode unique to plants. We are interested in the entire process of plant reproduction.
In particular, we are focusing on the molecular mechanisms that promote meiosis. Meiosis plays an important role in creating diverse haplotypes through homologous chromosome pairing and recombination mechanisms and transmitting them to the next generation. It also contributes to the maintenance of species through monitoring the number and structure of chromosomes during the chromosome pairing process.
Despite their importance, there were few meiosis-related plant genes with known functions when we started the research in 2000. We have selected a number of sterile lines from rice mutant populations, identified five novel genes that promote germ cell development, including meiosis, and analyzed those functions.
An example is the rice germline-specific Argonaute (AGO) protein MEL1, which binds to its target RNA via a small RNA 20 to 30 bases long as a guide molecule. MEL1 specifically expressed in the cytoplasm of germ cells leading to meiosis, suggesting its function primarily in mRNA degradation, translation inhibition, and defense against foreign viruses. The Nonomura lab aims to elucidate the biological functions of ME1 and other AGOs functioning in the germline by identifying target genes (Fig. 1).
Meiotic recombination is a fundamental principle of breeding that creates new genetic combinations in conventional breeding through crossbreeding. In the latter case, the genetic linkage between good and bad traits cannot be resolved even after repeated crossbreeding, which often leads to a “chain trapping” problem. If we can artificially induce recombination in such areas, we can dramatically improve the efficiency of breeding. In the future, we plan to work on the development of new breeding techniques that combine mutagen and genome editing technologies.
2. Research on mechanisms of meiotic initiation and transition in plants
In 2011, we identified the rice MEL2 gene. The mel2 mutants show abnormal timing of meiotic transition and eventually all meiotic cells die during meiosis. we found that MEL2 forms cytoplasmic RNA granules in the spore mother cells just before meiosis (Figure 2). RNA granules generally regulate the degradation, stabilization and translational repression of target mRNAs, and MEL2 granules may regulate the timing of meiotic transition through similar mechanisms. In the future, we intend to explore MEL2 functions by identifying MEL2-targeted mRNAs and MEL2-interacting proteins.
One of the genes that genetically interacts with MEL2 is OsGSL5, encoding a synthase of callose, a cell wall component. Just prior to meiosis, meiotic cells in plants are wrapped with a thick callose wall, which has long been recognized by plant cytological researchers as an indicator of meiosis initiation. The osgsl5 mutant lost the callose wall and showed meiotic abnormalities in addition to previously reported pollen formation defects. In other words, our study revealed for the first time that callose wall synthesis plays an important role downstream of MEL2-dependent regulation of meiotic timing transition.
It is known that meiotic progression in plants is influenced by the environment. In rice, low temperatures during the meiosis-progressing flowers at a booting stage often cause sterility. Cytoplasmic MEL2 granules change dynamically in size and number responsible to the extracellular ambient temperature, a kind of environmental abiotic stress. Callose is also incorporated into the defense response to the pathogen attack, a biotic stress. In other words, it is highly possible that the meiotic control system including MEL2, or its upstream pathway, is environmentally responsive. We plan to analyze the MEL2-dependent regulatory mechanism of meiotic transition in the future.
3. Research on meiosis and land plant evolution
Land plants have two distinct bodies (reproductive generations): gametophyte and sporophyte. Gametes produced in the gametophyte are fertilized, and the fertilized egg repeats somatic cell division to form a sporophyte. This reproduction mode is called “alteration of generation” (Figure 3). On the other hand, the life cycle of Charophyte algae, an ancestral species of land plants, consists only of gametophyte. This is because fertilized eggs go directly into meiosis and quickly revert to the gametophytic generation (Figure. 3). The increase in sporophytic body size that accompanied the land plants evolution suggests that the sporophyte acquisition contributed significantly to their adaptation to the harsh terrestrial environment.
How did land plants manage to acquire sporophytes? In 1908, British botanist Dr. Frederick Orpen Bower proposed an important hypothesis concerning the relationship between meiosis and the evolution of land plants. He proposed that “meiotic retardation” occurred in the life cycle of pre-land plants, probably leading to insertion of multiple mitoses , and consequently sporophyte formation, between the fertilized egg and subsequent meiosis. Although another hypothesis has been proposed, the meiotic insertion (meiosis retardation) theory is now considered more plausible.
What was the molecular mechanism behind the “meiotic retardation” in plants prior to their emergence on land? We believe that genes such as rice MEL2 were the key. In fact, MEL2-like proteins are widely conserved in Streptophytes, a phylogenetic lineage including land plants, but not in Chlorophytes, another lineage (Figure 4).
In the future, we would like to research not only on angiosperms but also on Charophytes and Conjugatophytes belonging to the lineage connecting to land plants, and investigate the relationship between meiosis initiation and transition regulation of MEL2 and related genes.
Figure 3. Life cycles of Chara algae and land plants
(A) The life cycle of a charophyte. After fertilization of the gametes (sperm and egg cell), the zygote (2n) immediately undergoes meiosis to form the gametophyte (n).
(B) The life cycle of land plants. After fertilization of the gametes (sperm cells and egg cells), the zygote undergoes repeated somatic cell division to form a multicellular sporophyte (2n). Part of the sporophyte becomes the reproductive organ, and the gametocytes undergo meiosis to form gametes (sperm cells and egg cells) in the gametophyte (pollen and embryo sac). The life cycle in which gametophytes and sporophytes alternate is called "alternation of generations." The prevailing theory behind the acquisition of sporophytes by land plants is that a delay in meiosis created room for somatic cell division to occur between the zygote and meiosis.
Figure 4. MEL2-like proteins are conserved in many land plants and in charophytes that are related to land plants.