Conservation and diversification of SCARECROW in maize

Abstract

The SCARECROW (SCR) gene in Arabidopsis is required for asymmetric cell divisions responsible for ground tissue formation in the root and shoot. Previously, we reported that Zea mays SCARECROW (ZmSCR) is the likely maize ortholog of SCR. Here we describe conserved and divergent aspects of ZmSCR. Its ability to complement the Arabidopsis scr mutant phenotype suggests conservation of function, yet its expression pattern during embryogenesis and in the shoot system indicates divergence. ZmSCR expression was detected early during embryogenesis and localized to the endodermal lineage in the root, showing a gradual regionalization of expression. Expression of ZmSCR appeared to be analogous to that of SCR during leaf formation. However, its absence from the maize shoot meristem and its early expression pattern during embryogenesis suggest a diversification of ZmSCR in the patterning processes in maize. To further investigate the evolutionary relationship of SCR and ZmSCR, we performed a phylogenetic analysis using Arabidopsis, rice and maize SCARECROW-LIKE genes (SCLs). We found SCL23 to be the most closely related to SCR in both eudicots and monocots, suggesting that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots.

Figure 1

Complementation of scr with ZmSCR. (A–C) Transverse sections of primary roots (wild type plants: A, D, G, J; scr: B, E, H, K; scr with pSCR::ZmSCR: C, F, I, L). (D–F) Casparian strip staining of transverse sections of primary roots. The white arrowheads point to Casparian strip staining in the endodermis, which indicates the restoration of endodermal characteristics in the inner ground tissue layer. (G–I) Gravitropic response of inflorescence stems. (J–L) Longitudinal sections through inflorescence stems. The black arrowheads indicate the amyloplasts that sediment in response to gravity. The direction of the gravity vector is shown by the arrows. Note the restoration of two ground tissue layers in scr with the introduction of pSCR::ZmSCR. co, cortex; en, endodermis; ep, epidermis; m, mutant cell layer. Bars in (A) to (L) = 50 μm.

Figure 2

Analysis of ZmSCR expression during embryogenesis. Longitudinal sections through embryos at sequential stages of maize embryogenesis. (A) ZmSCR mRNA was localized in the apical region of the embryo (6 days after pollination [DAP]). (B) ZmSCR mRNA was detected in the apical region of the embryo proper, not in the suspensor (8 DAP). (C) Expression of ZmSCR was observed on the anterior side of the embryo that would give rise to the prospective SAM and RAM (10 DAP). (D) ZmSCR expression was restricted to the root meristem and leaf primordia (14 DAP). (E) ZmSCR expression was detected in the endodermal cell layer of the root, leaf primordia, and in young leaves, but not in the SAM (18 DAP). The stages are adapted from Randolph (1936). (F) Embryo probed with a sense probe (12 DAP). co, coleoptile; lp, leaf primordia; sc, scutellum; su, suspensor; yl, young leaf.

Figure 3

Expression of ZmSCR in the maize shoot system. (A and B) ZmSCR mRNA was expressed in leaf primordia and young leaves, but not in the SAM. (C and D) ZmSCR expression was observed in the parallel veins of young leaves. (E) Close-up of the box in D is shown. ZmSCR expression was detected in 2–3 cells of the parallel veins. (F) ZmSCR mRNA was detected in places where vascular bundles would develop, showing characteristic morphology with regular spacing (transverse section of the young leaves). co, coleoptile; lp, leaf primordia; SAM, shoot apical meristem Bars in (A) and (B) = 50 μm.

Figure 4

Phylogenetic relationship of Arabidopsis, rice and maize GRAS genes. The Neighbor-joining tree was constructed using the culled alignment of 33 Arabidopsis, 52 rice and 50 maize GRAS proteins. The tree was rooted using a human STAT (HsSRC) as an outgroup as previously reported (Bolle, 2004). The pair of SCR and SCL23 is marked with a black box. Bootstrap values of 2000 replicates are shown for each branch.