A PRELIMINARY STUDY OF ROOT-TO-SHOOT REGENERATION BY ECTOPIC EXPRESSION OF WUS IN ARABIDOPSIS THALIANA ROOTS

Abstract

In plants, new organs and tissues generate from the meristems. The two main meristems located in root apices and shoot apices, namely the shoot apical meristem (SAM) and the root apical meristem (RAM), orchestrate the balance between cell differentiation and cell division with related regulators. For example, the homeodomain protein WUSCHEL (WUS) and its counterpart WOX5 are responsible to maintain the stem cell potency in the SAM and RAM, respectively. WUS is first expressed in the 16-cell embryo within the region that will develop into embryonic shoot. Ectopic expression of WUS has been shown to induce somatic embryogenesis, indicating that WUS can promote the embryonic identity. Intriguingly, when expressed in the root, WUS induces shoot stem cell identity and leaf development (without additional cues), floral development (together with LEAFY), or embryogenesis (in response to increased auxin), suggesting that WUS establishes stem cells with intrinsic identity. To elucidate the mechanism underlying stem cell formation and regeneration in plants, we developed a tissue/cell-specific GAL4-GR (GVG)-UAS inducible system to ectopically express WUS in the Arabidopsis root. UAS::WUS lines have been generated and crossed with tissue/cell-specific drive lines including pSCR::GVG, pWOX5::GVG, pPIN2::GVG, and pADF5::GVG. Thus, upon DEX application, WUS expression can be ectopically induced in specific root tissues/cells. Our induction experiments showed that, with inducible expression of WUS in pADF5::GVG-UAS::WUS and pADF5::GVG-UAS::WUS-mCherry lines, seedlings induced by DEX for 6 days exhibited a new cluster of stem cells in the root cap region. This formation of the new cluster of stem cells also abolished the root cap cell identity. Moreover, extended induction of WUS expression in the SCR-expressing root endodermis induced leaf formation from the position of lateral roots or at the basal end of lateral roots, suggesting the involvement of a lateral root development program. In order to test whether ectopic expression of WUS in endodermis is sufficient to induce regeneration, we made an artificial J shape of pSCR::GVG-UAS::WUS roots. After 4days of induction with J-shape roots, more leaf primordia formed at the curve of the J shape roots. The lateral root primordia development was also examined with or without induction of WUS in endodermis. Our results indicated that with induction of WUS in endodermis the lateral root primordia development became different since stage III due to the extra cell divisions in the WUS-inducible lines. In addition, the epidermis, cortex, and endodermis specific markers were introduced in the pSCR::GVG-UAS::WUS line. The ectopic expression of WUS in endodermis led to extra cell divisions in endodermis, cortex, and epidermis at somewhat extent. Out data also indicated some cells in the cortex lost their identity due to ectopic expression of WUS in endodermis. In the future studies, fluorescence activated cell sorting and microarray assay will be used to unearth the changes in epidermis, cortex, and endodermis cell layers and reveal the molecular framework for leaf regeneration in Arabidopsis roots.