The Lukowitz Lab Plant Developmental Biology at UGA

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Formation of the root is normally initiated at the boundary between the suspensor and the proembryo. In this process, the uppermost suspensor cell, called hypophysis, divides to produce a lens-shaped daughter cell, which forms the center of the developing root meristem. Root formation is dependent on auxin, a pervasive plant hormone. A reporter gene of transcriptional responses to auxin becomes expressed in the hypophysis just before it divides, suggesting that auxin accumulation in the hypophysis is important.

Mutations in HAN abolish all anatomical hallmarks of root formation. In normal development (right), the hypophysis divides asymmetrically to form a small the lens shaped daughter (l), the center of the root meristem, and slender, elongated cells occupy the center of the lower tier (upper/lower tier boundary marked by arrows). The hypophysis of han mutants (h) never divides and the cells of the lower tier appear swollen and irregularly shaped.

A GATA-type transcription factor regulates the position of the embryonic root.

The GATA-type transcription factor HANABA TARANU (HAN) was first discovered because it regulates the number of petals and other floral organs. Hajime Sakai named the gene “fewer floral leaves” in Japanese, as an homage to the Drosophila mutant FUSHI TARAZU or “fewer segments”. Strikingly, han mutant embryos also show none of the anatomical hallmarks of root formation: their basal pole is occupied by large and irregularly shaped cells, and the hypophysis never divides to produce the lens-shaped cell.

An analysis of gene expression domains reveals a coordinated apical shift in han mutant embryos. See text for details.

Despite this suggestive phenotype, a loss of HAN does not cause a loss of the embryonic root. The root is merely initiated at a different position, in the center of han embryos, rather than at the boundary between proembryo and suspensor. An analysis with cell fate markers further reveals that the expression domains of genes mediating auxin transport and root formation are coordinately shifted in han mutants. This change can be traced back to the octant stage, when the proembryo is a small shpere of eight cells, when the auxin transporter PIN7 accumulates at the apical base of the uppermost suspensor cell of wild tye embryos. In contrast, han embryos accumulate PIN7 in the apical membrane of the lower tier cells. Slightly later, the auxin transporter PIN1 becomes expressed throughout the proembryo of wild type embryos. PIN1 expression is restricted to the upper tier of han mutants, PIN7 is. The coordinated expression of PIN1 and PIN7 is thought to mediate auxin accumulation in the uppermost suspensor cell, the hypophys, of wild type embryos. Consistent with this view, a reporter of auxin-dependent gene transcription, DR5, becomes strongly expressed in the hypophysis. DR5 expression shifts to all cells of the lower tier of han mutants.

These findings demonstrate that HAN is a key regulator of auxin flux and perception in the early embryo. Normally the anatomical boundary between the proembryo and the suspensor also marks an inductive boundary across which root formation is organized. This boundary is marked by the mutually exclusive expression domains of PIN1 and PIN 7 and, presumably, results in the accumulation of auxin in the hypophysis. We propose that this instructive boundary is shifted in han mutants and now corresponds to the anatomical boundary between upper and lower tier.

Where do we go from here?

Present work on this project focuses on the following questions:

Can the effect of han mutations on embryo patterning be fully explained by the observed changes in auxin transport and perception? If so, we would predict that the PIN1 and/or PIN7 genes may be targets of HAN-dependent regulation.

What are the primary targets of HAN-dependent gene regulation, and how, mechanistically, are they regulated by HAN?

What is the contribution of two closely related HAN-like genes to embryo patterning? Could these genes provide partially overlapping function?