Expression Profiling of the Whole Arabidopsis Shaggy-Like Kinase Multigene Family by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Abstract

The recent publication of the complete sequence of the Arabidopsis genome allowed us to identify and characterize the last two members of the SHAGGY-like kinase (AtSK) gene family. As a result, the study of the overall spatio-temporal organization of the whole AtSK family in Arabidopsis has become an achievable and necessary aim to understand the role of each SHAGGY-like kinase during plant development. An analysis of the transcript level of the 10 members of the family has been performed using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction. Transcript levels in several organs, under different growth conditions, were analyzed. To calibrate the results obtained, a number of other genes, such as those coding for the two MAP3K⑀s and the two MAP4K␣s, as well as the stress response marker RD29A; the small subunit of the Rubisco photosynthetic enzyme Ats1A; the MEDEA chromatin remodeling factor; and the SCARECROW, ASYMMETRIC LEAVES 1, and SUPERMAN transcription factors all involved in key steps of plant development were used. The analysis of our data revealed that eight of the 10 genes of the AtSK family displayed a pseudo-constitutive expression pattern at the organ level. Conversely, AtSK13 responded to osmotic changes and saline treatment, whereas AtSK31 was flower specific and responded to osmotic changes and darkness.The SHAGGY/GSK3-like kinases are non-receptor Ser-Thr (S/T) kinases playing numerous roles (for review, see Kim and Kimmel, 2000). In animals, they are involved in the determination of cell destiny, resulting in the spatial organization of the body plan. In Drosophila melanogaster, a pool of several isoenzymes called SHAGGY and encoded by a single gene, is involved both in the definition of boundaries between the embryonic segments of the larvae (Siegfried et al., 1992), and in the development of the central and peripheral nervous system (Heitzler and Simpson, 1991). In the sea urchin embryo, the SHAGGY-like enzyme is involved in the definition of the animal/vegetal axis (Emily-Fenouil et al., 1998). In Xenopus laevis embryo, a deficiency for the activity of this kinase results in a defect of the dorso-ventral plan formation, leading to the formation of two heads (He et al., 1995). Finally, in mammals, two enzymes named GSK3␣ and GSK3␤ (for glycogensynthase kinase), encoded by two genes, are involved in the regulation of glycogen metabolism (Oreñ a et al., 2000), in the stability of the cytoskeleton (Zumbrunn et al., 2001), and in numerous other processes related to oncogenesis (Webster et al., 2001).In higher plants, the SHAGGY-like genes are present as small gene families. They have been characterized from a number of plant species (Pay et al., 1993; Tichtinsky et al., 1998; Jonak et al., 2000). Before the completion of the sequencing of the whole Arabidopsis genome, eight genes were known to belong to the SHAGGY-like gene family (AtSK; Jonak et al., 1995; Dornelas et al., 1998 Dornelas et al., , 1999 Tichtinsky et al., 1998). Recently, two additional ge...

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