Plants have versatile morphologies and create complex organs including flowers, leaves, roots and stems. The basis of morphology formation  is to establish proper orientation known as the body axis. In most plants, fertilized eggs divide asymmetrically to establish the vertical axis (Figure). However, since fertilized eggs of flowering plants develop deeply within flowers or seeds, how the fertilized egg becomes polarized and how it divides asymmetrically remains to be answered.

Using Arabidopsis thaliana, we successfully observed the internal structure of fertilized plant eggs in real time. We discovered the spatiotemporal dynamics of cytoskeleton and organelles in fertilized eggs, which contribute to the polarity establishment. For example, before fertilization, microtubules are oriented along the vertical axis, but this alignment collapses upon fertilization. The microtubules then form a ring-like structure that moves along the elongating zygotes. On the other hand, actin fibers are oriented along the vertical axis and are responsible for the apical movement of the nucleus.

Live-imaging of microtubules in zygote
Kimata et al. (2016), Movie S1

The vacuole, which makes up the majority of plant cells’ volume, shrinks rapidly after fertilization. The vacuole then forms filamentous tubular structures around the nucleus and gradually moves toward the basal end. In sgr2 mutant plants, where the flexibility of the vacuole is reduced, the tubular structure is not formed and the vacuole cannot move toward the basal end. As a result, a large vacuole is left at the apical end of the fertilized egg and prevents the nucleus from reaching the apical end, resulting in a near symmetrical division. This indicates that  the flexible vacuole plays a role in asymmetric division by changing its shape and moving toward the basal end to support the movement of the nucleus to the opposite side.

When a fertilized egg divides asymmetrically, it produces a smaller apical cell (precursor of embryo proper) with few vacuoles and a larger basal cell (precursor of suspensor) with large vacuoles. In the sgr2 mutant plants, both the apical and basal cells are similar in size and inherit large vacuoles. As embryogenesis proceeds, the giant vacuoles remain in the embryo proper, impair embryonic patterning and ultimately result in an abnormal number of cotyledons. It is known that actively dividing cells contain few vacuoles in plants, so the abnormal embryonic morphology in sgr2 mutant plants is thought to be a result of the inheritance of excess vacuoles in the apical cell.

Live-imaging of vacuoles in wild type zygote
Kimata et al. (2019), Movie S1

Live-imaging of vacuoles in sgr2 zygote
Kimata et al. (2019), Movie S2

The discovery that vacuoles actively contribute to cell polarization and embryonic morphology has overturned the conventional impression of vacuoles as mere passive “water bags”. Therefore, we aim to overcome the limitations of conventional research methods and elucidate the developmental mechanisms at the intracellular level without preconceptions by performing precise live imaging of various intracellular structures and determining their roles using inhibitors or mutants.

Kimata et al. (2016) Proc Nat Acad Sci 113 (49), pp14157-14162, DOI:10.1073/pnas.1613979113
Kurihara et al. (2017) JoVE (127), 55975, DOI: 10.3791/55975
Kimata et al. (2019) Proc Nat Acad Sci 116 (6), pp2338-2343, DOI: 10.1073/pnas.1814160116
Kimata et al., (2020) Quant Plant Biol 1, e3, DOI: 10.1017/qpb.2020.4