Regulation of SHOOT MERISTEMLESS genes via an upstream-conserved noncoding sequence coordinates leaf development

Abstract

The indeterminate shoot apical meristem of plants is characterized by the expression of the Class 1 KNOTTED1-LIKE HOMEOBOX (KNOX1) genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch in fate from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS (STM) in Arabidopsis (hereafter called STM genes) in the initiating primordia. In simple leafed plants, this down-regulation persists during leaf formation. In compound leafed plants, however, KNOX1 gene expression is reestablished later in the developing primordia, creating an indeterminate environment for leaflet formation. Despite this knowledge, most aspects of how STM gene expression is regulated remain largely unknown. Here, we identify two evolutionarily conserved noncoding sequences within the 5' upstream region of STM genes in both simple and compound leafed species across monocots and dicots. We show that one of these elements is involved in the regulation of the persistent repression and/or the reestablishment of STM expression in the developing leaves but is not involved in the initial down-regulation in the initiating primordia. We also show evidence that this regulation is developmentally significant for leaf formation in the pathway involving ASYMMETRIC LEAVES1/2 (AS1/2) gene expression; these genes are known to function in leaf development. Together, these findings reveal a regulatory point of leaf development mediated through a conserved, noncoding sequence in STM genes.

Fig. 1.

Two CNSs in the 5′ upstream regions of STM orthologous genes. (A) The positions of RB-boxes (white boxes) and K-boxes (black boxes) in the 5′ upstream region from the translation start codon are illustrated. s and c indicate simple and compound leafed species, respectively. Halved white boxes for some RB-boxes show that sequences only downstream of the core RB box are available because the 5′ degenerate primer for the core RB-box was used to obtain upstream sequences. (B and C) The alignment of sequences of the RB-box and K-box are illustrated, respectively.

Fig. 2.

GUS expression from the NTH15 upstream region in tobacco and the determination of the transcription initiation sites. (A–C) GUS expression in transgenic tobacco seedlings harboring NTH15p–GUS, ΔK-GUS, and ΔRB-GUS transgenes were detected, respectively. Arrows indicate ectopic expression of GUS proteins in leaves. (D and E) GUS expression in transverse sections of leaves of transgenic tobacco harboring NTH15p–GUS and ΔK-GUS transgenes are shown, respectively. (F and G) Agarose gel electrophoresis of nested PCR products from the RLM-RACE procedure for endogenous NTH15 gene and GUS reporter transgenes, respectively. Molecular size markers (base pairs) are shown on the left. Arrows on the right mark the major PCR products. (H) Sequence around the core K-box of the NTH15 gene is illustrated, and arrows above the sequence indicate the identified transcription start sites. Horizontal arrow below the sequence indicates the core K-box. (Scale bars: A–C, 2 mm; D and E, 0.4 mm.)

Fig. 3.

GUS expression from the STM upstream region in Arabidopsis and the determination of the transcription initiation sites. (A and B) GUS expression in transgenic Arabidopsis seedlings harboring STMp–GUS and ΔK-GUS transgenes were detected, respectively. Arrows indicate ectopic expression of GUS proteins in leaf petioles. (C and D) Agarose gel electrophoresis of nested PCR products from the RLM-RACE procedure for endogenous STM gene and GUS reporter transgenes, respectively. Molecular size markers (base pairs) are indicated on the left. Thick arrows and thin arrows on the right mark the major and minor PCR products, respectively. (E) The identified transcription start sites are illustrated. (Scale bars: 1.5 mm.)

Fig. 4.

Paraffin sections of GUS-stained Arabidopsis SAM tissues. GUS expression in longitudinal (A and B) and transverse (C and D) sections of SAM of transgenic Arabidopsis seedlings harboring STMp–GUS (A and C) and ΔK-GUS (B and D) transgenes. Arrowheads indicate down-regulation of GUS expression in young leaf primordia. Asterisks indicate the reestablishment of GUS expression in the developing leaf primordia. (Scale bars: 50 μm.)

Fig. 5.

Transgenic Arabidopsis plants expressing the STM protein from the STM upstream region with or without the K-box. Vegetative rosettes (A–D) and cleared rosette leaves (E–H) of wild-type (Col) (A and E), STMp–STM (B and F), and ΔK-STM (C and G) and as1-1 (D and H) Arabidopsis plants are shown. (Scale bars: A–D, 5 mm; E–H, 2 mm.)

Fig. 6.

Involvement of STM regulation by K-box in the AS1 pathway during leaf development. (A and B) Products by RT-PCR for AS1, AS2, and ACT2 mRNA in tissues containing the SAM and leaf primordia of Arabidopsis plants (A) and KNAT1 and ACT2 mRNA in leaves of Arabidopsis plants (B) are shown. (C and D) GUS expression in transgenic Arabidopsis seedlings harboring KNAT1p–GUS with or without ΔK-STM, respectively. Arrow and arrowheads indicate GUS expression in the leaf midvein and leaf hydathodes, respectively. (Scale bars: 2 mm.)