The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion

The GSK3/Shaggy family of serine/threonine protein kinases is involved in a series of biological processes in animals, plants and yeast [Charrier et al. (2002) Plant Physiol 130:577-590; Jope and Johnson (2004) Trends Biochem Sci 29:95-102; Li and Nam (2002) Science 295:1299-1301; Piao et al. (2001) Plant J 27:305-314]. In Arabidopsis thaliana, out of the 10 members of the GSK3/Shaggy-like gene family (AtSKs), a biological function has been assigned to only 1 member (AtSK2-1) by mutation. In the present work, a study was undertaken to elucidate the function of AtSK3-2. We have generated mutated versions of the A. thaliana Shaggy-like kinase 3-2 (AtSK3-2), in which Lys(167) and Arg(178), respectively homologus to Lys(85) and Arg(96) of the mammal GSK3 beta, were modified into Ala by site-directed mutagenesis. In vitro kinase activity assays of the mutated recombinant protein AtSK3-2-R178A showed that the "primed activity" of the mutated kinase was reduced by 90% while the "non-primed" activity was only 20% reduced compared to the wild-type protein kinase. However, the mutant protein AtSK3-2-K167A showed no activity. Arabidopsis transgenic lines over-expressing AtSK3-2-R178A displayed smaller floral organs, namely pedicels, sepals and petals. Conversely, over-expression of both the wild-type AtSK3-2 protein and the AtSK3-2-K167A mutated version, displayed no altered morphogenesis. Scanning electron microscopic analyses of the AtSK3-2-R178A transgenic plants clearly showed a reduced cell size in flower organs, in which quantitative RT-PCR expression analyses of cell wall expansion enzymes showed reduced transcript levels of three xyloglucan endotransglycosylases (XET), namely XTH22 (TCH4), XTH23 (XTR6) and XTH30 (XTR4). Our data show that AtSK3-2 plays an important role in the control of cell elongation in flower development.