Kazuki Motomura

Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.

Gametogenesis, which is essential to the sexual reproductive system, has drastically changed during plant evolution. Bryophytes, lycophytes and ferns develop reproductive organs called gametangia—antheridia and archegonia for sperm and egg production, respectively. However, the molecular mechanism of early gametangium development remains unclear. Here we identified a ‘non-canonical’ type of BZR/BES transcription factor, MpBZR3, as a regulator of gametangium development in a model bryophyte, Marchantia polymorpha. Interestingly, overexpression of MpBZR3 induced ectopic gametangia. Genetic analysis revealed that MpBZR3 promotes the early phase of antheridium development in male plants. By contrast, MpBZR3 is required for the late phase of archegonium development in female plants. We demonstrate that MpBZR3 is necessary for the successful development of both antheridia and archegonia but functions in a different manner between the two sexes. Together, the functional specialization of this ‘non-canonical’ type of BZR/BES member may have contributed to the evolution of reproductive systems.

The pollen tube plays a pivotal role in double fertilization by delivering sperm cells (SCs) to the ovule. In Arabidopsis thaliana , a pair of SCs tightly connects with the vegetative nucleus (VN) to form the male germ unit (MGU), which is located in the apical region during pollen-tube growth, keeping the VN ahead of the SCs. MGU transport relies on independent motility of VN and SC pairs. However, the complexity of this dual motive force has hindered our understanding of MGU behavior, including its positioning and nuclear order. We used Arabidopsis mutants or transgenic plants that produced semi-motile MGUs with aberrant VNs or SCs to analyze the positioning of VN or SCs after stochastically disconnecting the MGU. In pollen tubes with an immotile SC pair, the VN was ∼70 μm away from the tip, whereas in pollen tubes with an immotile VN, the SC pair was ∼100 μm away from the tip, implying that the VN and SCs have independent home positions. The position of MGU moved forward owing to the loss of the microtubule-destabilizing kinesin KINESIN-13A . Conversely, microtubule depolymerization by oryzalin treatment or introducing mutations in TUBULIN BETA 4 ( TUB4 ) deregulated the position of the MGU and shifted its position backward. In addition, tub4 plants exhibited reduced fertility. These data indicate a significant role of microtubules in stable MGU positioning to ensure reproductive success.

花粉管は被子植物の生殖プロセスに必須であることから，重要な研究対象である．特に花粉管の発芽や伸長，方向制御を担う分子の同定が進んでおり，これまでに重要な分子群が次々に明らかになってきている．近年，筆者らはこのような分子をコードする遺伝子群がいつ発現を開始して，花粉管機能がどのように獲得，維持されるかについて新たな仮説を提案した．そこで本総説では，この分野の近年の研究成果をレビューし，特にシロイヌナズナを用いた研究を中心に，時間的な遺伝子発現のタイミングに焦点を当てて考察する．さらにそれに基づき，植物の花粉管の遺伝子発現研究における今後の展望についても述べる．

The vegetative cell nucleus proceeds ahead of a pair of sperm cells located beneath the pollen tube tip during germination. The tip-localized vegetative nucleus had been considered to play a pivotal role in the control of directional pollen tube growth and double fertilization. However, we recently reported the female-targeting behavior of pollen tubes from mutant plants, of which the vegetative nucleus and sperm nuclei were artificially immotile. We showed that the apical region of the mutant pollen tubes became physiologically enucleated after the first callose plug formation, indicating the autonomously growing nature of pollen tubes without the vegetative nucleus and sperm cells. Thus, in this study, we further analyzed another Arabidopsis thaliana mutant producing physiologically enucleated pollen tubes and discussed the mechanism by which a pollen tube can grow without de novo transcription from the vegetative nucleus. We propose several possible molecular mechanisms for persistent pollen tube growth, such as the contribution of transcripts before and immediately after germination and the use of persistent transcripts, which may be important for a competitive race among pollen tubes.

The pollen tube is a male reproductive tissue in angiosperms that transports sperm cells to the ovule where double fertilization takes place. It was believed that the nuclei, always present at the apical region of the pollen tube, were responsible for the pollen-tube growth and directional control during its long journey to the ovule. However, we recently showed that pollen tubes could reach the ovule even when de novo transcripts are not supplied to a tip during pollen-tube elongation. This review focuses on recent topics about the directional control of pollen tubes including our research and discusses the importance of these studies for plant reproduction.

During the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in Arabidopsis thaliana whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.