Transcriptional, posttranscriptional, and posttranslational regulation of SHOOT MERISTEMLESS gene expression in Arabidopsis determines gene function in the shoot apex

Abstract

The activity of SHOOT MERISTEMLESS (STM) is required for the functioning of the shoot apical meristem (SAM). STM is expressed in the SAM but is down-regulated at the site of leaf initiation. STM is also required for the formation of compound leaves. However, how the activity of STM is regulated at the transcriptional, posttranscriptional, and posttranslational levels is poorly understood. We previously found two conserved noncoding sequences in the promoters of STM-like genes across angiosperms, the K-box and the RB-box. Here, we characterize the function of the RB-box in Arabidopsis (Arabidopsis thaliana). The RB-box, along with the K-box, regulates the expression of STM in leaf sinuses, which are areas on the leaf blade with meristematic potential. The RB-box also contributes to restrict STM expression to the SAM. We identified FAR1-RELATED SEQUENCES-RELATED FACTOR1 (FRF1) as a binding factor to the RB-box region. FRF1 is an uncharacterized member of a subfamily of four truncated proteins related to the FAR1-RELATED SEQUENCES factors. Internal deletion analysis of the STM promoter identified a region required to repress the expression of STM in hypocotyls. Expression of STM in leaf primordia under the control of the JAGGED promoter produced plants with partially undifferentiated leaves. We further found that the ELK domain has a role in the posttranslational regulation of STM by affecting the nuclear localization of STM.

Figure 1.

RB-box presence in STM genes. A, The RB-box is present in all STM gene promoters analyzed. The multiple sequence alignment was made with MUSCLE and displayed with Boxshade. The line over the alignment indicates the location of the large RB-box. The box demarcates the highly conserved core RB-box. B, Relative location of the core RB-box in the STM gene promoters of the species analyzed. The numbers at top indicate positions relative to the translation initiation codon. Brassicaceae species are characterized by having a relatively distant location from the RB-box with respect to the transcriptional start site.

Figure 2.

Expression of GUS in Arabidopsis STM promoter constructs harboring combinations of RB-box and K-box deletions. A, Schemes of the STM promoter fragments used. Numbers indicate positions relative to the translation initiation codon ATG. Solid lines indicate fragments of the promoters used, dashed boxes indicate the promoter regions deleted for every construct, and vertical dashed lines connect the boxes between constructs for clarification. B to Q, Whole-mount and transverse sections of 8-d-old T3 seedlings harboring the constructs depicted in A. B and C, Staining of plants with the construct ProSTM:GUS with the native fragment of 3,379 bp. Seedling (B) and transverse section (C) through the shoot apex are shown. D and E, Staining of plants with the construct ProSTM-ΔLK:GUS harboring the STM promoter without the long K-box. F and G, Seedling (F) and cross section (G) of plants with the construct ProSTM-ΔCRB:GUS having the STM promoter without the core RB-box. H and I, GUS expression pattern in ProSTM-ΔCRB-CK:GUS plants lacking both the core RB-box and the core K-box. J and K, Staining of plants with the construct ProSTM-ΔLRB:GUS without the large RB-box. Seedling (J) and cross section through the hypocotyl (K) are shown. L to O, GUS expression pattern in ProSTM-ΔLRB-CK:GUS plants lacking both the large RB-box and the core K-box. Seedling (L) and transverse sections through the shoot apex (M), the hypocotyl (N), and a young leaf with GUS accumulation in sinuses (O) are shown. P and Q, Staining of plants with the construct ProSTM-ΔLRB-LK:GUS harboring deletions of the large RB-box and the large K-box. Seedling (P) and transverse section (Q) through the shoot apex are shown. Dashed lines outline leaf primordia. Bars = 0.5 mm in B, D, F, H, J, L, and P; 50 µm in C, E, G, I, K, and N; and 100 µm in M and Q.

Figure 3.

Characterization of FRF1 and related factors. A, FRF1 is capable of binding the RB-box through EMSA. C+ indicates the positive control from the manufacturer’s kit. Arrows mark the shifted bands, and arrowheads mark the free probe of 246 bp for the RB-box and 60 bp for C+. B, FRF factors are related to FRS factors. The protein domain structures of FRF and FRS factors are shown. The FAR1 DNA-binding domain is indicated by pale gray boxes, the Mutator-like element transposase domain by dark gray boxes, and the zinc finger of the SWIM type by black boxes. Numbers indicate the size of each factor. C, Phylogenetic analysis of FRF factors in Arabidopsis, tomato, M. truncatula, grape, and rice. A neighbor-joining tree with 1,000 bootstrap replications using the conserved FAR1 DNA-binding domain of the FRF factors is shown. Branch lengths are indicated. The tree was generated using PAUP* 4.0 (Swofford, 2003; paup.csit.fsu.edu).

Figure 4.

GUS expression pattern in constructs with internal deletions in the Arabidopsis STM promoter. A, Schemes of the STM promoter with the internal deletion fragments used. Numbers indicate positions relative to the translation initiation codon ATG. The positions of the RB-box and the K-box are also indicated. Solid lines indicate the fragment of the promoter used, dashed boxes indicate the internal deletion on each construct, and vertical dashed lines connect the boxes between constructs for clarification. B to R, Whole-mount and transverse sections of 8-d-old T3 seedlings harboring the constructs indicated in A. B and C, Staining of plants with the construct ProSTM-ΔF1:GUS. Seedling (B) and transverse section through the shoot apex (C) are shown. D and E, Staining of plants with the construct ProSTM-ΔF2:GUS. Seedling (D) and cross section through the shoot apex (E) are shown. F and G, Seedling (F) and cross section through the hypocotyl (G) of stained plants with the construct ProSTM-ΔF3:GUS. H and I, GUS expression pattern in ProSTM-ΔF4:GUS plants. Whole-mount stained seedling (H) and cross section through the shoot apical region (I) are shown. J and K, Staining of plants with the construct ProSTM-ΔF5:GUS. Seedling (J) and cross section through the shoot apex (K) are shown. L and M, GUS expression pattern in ProSTM-ΔF6:GUS plants. Seedling (L) and transverse section through the shoot apex (M) are shown. N and O, Staining of plants with the construct ProSTM-ΔF7:GUS. Seedling (N) and cross section through the shoot apical region (O) are shown. P to R, Staining of plants with the construct ProSTM-ΔF8:GUS. Seedling (P) and cross sections through the shoot apex (Q) and through the hypocotyl (R) are shown. Leaf primordia are outlined with dashed lines. Bars = 0.5 mm in B, D, F, H, J, L, N, and P, 25 µm in C, E, and O, 50 µm in G, K, M, and R, and 100 µm in I and Q.

Figure 5.

CNSs in STM-like gene promoters of Brassicaceae species upstream of the RB-box. A, mVISTA alignment of the B. rapa STM promoter compared with the corresponding genes of T. halophila, C. rubella, Arabidopsis, A. lyrata, and C. hirsuta. Three regions, Fa, Fb, and Fc, are identified. B, Sequence alignment in the Fa region. C, Alignment of the sequences for the Fb region. D, Sequence alignment in the Fc region. Sequence alignments were made with MUSCLE and displayed with Boxshade.

Figure 6.

Expression of STM under the control of the JAG promoter. A and B, Consecutive transverse sections through the shoot apex of transgenic Arabidopsis ProJAG:GUS plants expressing the uidA gene under the control of the JAG promoter. A, GUS is expressed in P₀ leaf primordia (arrowhead). B, GUS is found in P₀ (arrowhead) and in the developing leaves (arrow). C to E, Expression of STM directed by the JAG promoter. C, Rosette of a wild-type plant. D, Representative rosette of a ProJAG:STM plant with a strong phenotype. Plants are very small with partially undifferentiated leaves. E, Rosette of a ProJAG:STM plant with a mild phenotype. The plants are small and have deeply lobed leaves. F to H, Confocal analysis of plants expressing GFP-STM under the control of the STM or JAG promoter. F, Confocal merged image of ProSTM:GFP-STM plants. GFP signal is found at the base of the shoot apex (arrowhead). G, Confocal merged image of the shoot apex of a ProJAG:GFP-STM plant with a strong phenotype showing intense GFP expression in stipule-like structures. H, Confocal merged image of a ProJAG:GFP-STM plant with highly lobed leaves and GFP expression in the base of developing leaves. Bars = 50 µm in A and B, 0.5 cm in C, 0.25 cm in D and E, 25 µm in F, and 100 µm in G and H.

Figure 7.

Altering the ELK domain affects leaf development and the cellular localization of STM. A, Diagram depicting the conserved domains of STM KNOX1, KNOX2, GSE, ELK, and HD. Numbers at top indicate relative positions from the first amino acid. B to D, Effect of the S272A mutation in the ELK domain on leaf development. Compared with ProSTM:GFP-STM plants that have a phenotype similar to the wild type (Uchida et al., 2007), ProSTM:GFP-STM-S272A plants produce smaller and lobed rosette leaves (B) and highly lobed cauline leaves (C) while overall plant architecture remains unaffected (D). Representative plants and isolated leaves are 5 weeks old. E and F, The mutation S272A alters the cellular distribution of STM. In agroinfiltration experiments in N. benthamiana leaves, STM (GFP-STM) is distributed throughout the cytoplasm (E); however, in the S272A mutation (GFP-STM-S272A), STM is located in the cell nucleus (F). The arrow indicates a cell nucleus. STM distribution was visualized with confocal microscopy. Bars = 1 cm in B, 5 mm in C, 2 cm in D, and 20 µm in E and F.