Mysteries of plant reproduction revealed by real-time imagery

Imaging of a Ladies' Arabette, with its pollen seeds highlighted by green fluorescence. | Valuchova, Mikulkova et al.

A new technique based on microscopy has been used by researchers to observe stages of reproduction of flowering plants never described before. The results obtained reveal crucial information which will help us to understand the different processes involved.

In flowering plants, reproduction takes place in two organs: the anther, which is the male reproductive system and the place where pollen is formed, as well as in the ovary, the female reproductive organ containing the waiting eggs. to be fertilized by contact with pollen.

Also called germ cells, pollen grains and eggs multiply during meiosis and mitosis, which are cell division processes. After fertilization, the resulting cell will develop into a plant.

Although dissection of flowering plants under the microscope and the study of abnormalities caused by gene mutations have so far been the most widely used techniques to study the different stages of germ cell division and reproduction, they do not provide any information on the exact moment when this occurs. Questions that a team of researchers wanted to answer using real-time imagery.

High resolution three-dimensional images

"Real-time imaging has been instrumental in researching root growth and development, but imaging cellular processes within the flower is technically much more difficult," said the co-author of the Sona Valuchova study, from Masaryk University, in the Czech Republic. "It is necessary to develop methods of imaging organs or plants in their entirety".

To get to the end, Valuchova and her colleagues used light-sheet fluorescence microscopy (LSFM), where plant samples trapped in agar were passed through a thin plane of laser light. A detector then produced data to obtain high-resolution three-dimensional images, thus making it possible, (thanks to the detailed structures of the flower imaged) to follow the fate of an individual germ cell from the inside.

Maximum intensity projection of a cut flower from the bud, at the height indicated above. The second line of images shows anthers in more detail. Credits: Sona Valuchova et al.

Having shown that LSFM could provide high-resolution images of flowers, the team's next goal was to establish that it could detect specific events in reproduction. To achieve this, they used flowers that had been engineered to have fluorescent labels on key molecules involved in meiosis and mitosis. They were able to capture the entire process of meiosis in male germ cells by detecting changes in the amount and location of a molecule called ASY1 every hour for four days.

The team went on to show that live imaging could be successfully used to study plant hormone levels during different stages of flower development and to watch the movement of chromosomes across the cell during cell division.

Imaging of a 0.5 mm flower bud expressing the protein ASY1 (here in green), seen from eight different angles. Credits: Sona Valuchova et al.

Detailed observation of germ cells during cell division

The last objective of their research was to observe the production of female germ cells during meiosis. This process has often been overlooked in the past, as these are rarer than their male counterparts. In addition, they have a strong resemblance to other cells, making it difficult to trace them during division.

The team had to set up another method to be able to follow meiosis of the eggs. They carefully dissected the bud, revealing the eggs. These were then passed every ten minutes (for 24 hours) under the laser to obtain a 3D film. The researchers were thus able to visualize the two stages of meiosis in female germ cells, as well as determine their duration.



"This work demonstrates the power of light-layer microscopy to provide new information on plant reproduction, which could not previously be studied by other types of microscopy," said Karel Riha, researcher and director of the Masaryk University Institute of Technology. "Our success in developing a real-time imaging protocol for female meiosis represents major technical progress in plant cell biology."

Bibliography: Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy Sona Valuchova DOI: 10.7554/eLife.52546