Activation of CRABS CLAW in the Nectaries and Carpels of Arabidopsis

Abstract

CRABS CLAW (CRC), a member of the YABBY gene family, is required for nectary and carpel development. To further understand CRC regulation in Arabidopsis thaliana, we performed phylogenetic footprinting analyses of 5' upstream regions of CRC orthologs from three Brassicaceae species, including Arabidopsis. Phylogenetic footprinting efficiently identified functionally important regulatory regions (modules), indicating that CRC expression is regulated by a combination of positive and negative regulatory elements in the modules. Within the conserved modules, we identified putative binding sites of LEAFY and MADS box proteins, and functional in vivo analyses revealed their importance for CRC expression. Both expression and genetic studies demonstrate that potential binding sites for MADS box proteins within the conserved regions are functionally significant for the transcriptional regulation of CRC in nectaries. We propose that in wild-type flowers, a combination of floral homeotic gene activities, specifically the B class genes APETALA3 and PISTILLATA and the C class gene AGAMOUS act redundantly with each other and in combination with SEPALLATA genes to activate CRC in the nectaries and carpels. In the absence of B and C class gene activities, other genes such as SHATTERPROOF1/2 can substitute if they are ectopically expressed, as in an A class mutant background (apetala2). These MADS box proteins may provide general floral factors that must work in conjunction with specific factors in the activation of CRC in the nectaries and carpels.

Figure 1.

Sequence Comparison of 5′ Upstream Regions of CRC from Arabidopsis, Lepidium, and Brassica. VISTA analysis (75% identity and 50 base sliding window) identified five conserved regions in three species (see supplemental data online for complete alignment). Regions with >75% identity shared by three species are shaded in pink. The top comparison is between Arabidopsis and Lepidium and so on. Five conserved domains were found between Arabidopsis and Lepidium, denoted regions A, B, C, D, and E, and used for the functional analyses. A larger fragment than the conserved region of region A that extends to just before the start codon was used as the transcriptional enhancer instead of a CaMV-derived TATA box. Sequence comparison with Brassica showed that the same regions are conserved but some of the conserved domains do not overlap with the domains conserved between Arabidopsis and Lepidium. CArG boxes (line with diamond) and putative LEAFY binding sites (line with oval) are shown along aligned sequences; the top row represents Arabidopsis, the second row represents Lepidium, and the third row represents Brassica. The CArG boxes and LFY binding sites functionally analyzed are labeled (EM1, EM2, EL1, CL2, and CL3). The translation start site is identified by a vertical line, and the exons of CRC are denoted by blue boxes. Numbers below denote base pairs of Arabidopsis sequence.

Figure 2.

GUS Expression Patterns Regulated by Conserved Regions of CRC Promoter in Arabidopsis Expressed in Wild-Type Landsberg erecta and crc-1. All whole-mount images ([A] to [H]) are Landsberg erecta (Ler). C:A≫GUS ([A] to [C]); E:A≫GUS ([D] to [F] and [I] to [O]); E:D:C:B:A≫GUS ([G], [P], and [Q]); pCRC 3.8kb≫GUS ([H] and [R]). Region C drives GUS expression in developing carpels and the margins of sepals. Expression in carpels is very high and lasts until late stages of flower development (after stage 15) (C). Though at early stages (stage 10) GUS is expressed throughout the carpels (A), it is restricted to valves at stage 12 (B). Region E drives GUS expression in carpels and nectaries. In carpels, GUS expression is throughout at the early stages and is restricted to the upper regions of the carpels later ([E] and [F]). Expression in nectaries starts before stage 10 (D) and lasts until after stage 17 (F). GUS expression regulated by the fusion of all the conserved regions (G) is very similar to the regulation by the 3.8-kb-long 5′ upstream region of CRC (H). Expression in carpels is absent at stage 12 (insets in [G] and [H]) in contrast with the late expression observed with regions E and C ([C] and [F]). (I) In a cross section of a crc-1 inflorescence, E:A≫GUS is expressed in developing carpels and ovules. By stage 13, ovule expression disappears but expression in the septum is detected (arrow). (J) In a longitudinal section of a stage 9 flower, E:A≫GUS is detected in the nectary anlagen (arrow). (K) Longitudinal section of a stage 12 crc-1 flower in which E:A≫GUS is expressed in cells that would form a nectary in the wild type (arrow). (L) Longitudinal section of a crc-1 inflorescence meristem; E:A≫GUS expression in carpels starts in stage 6, but it is very weak. Stage 7 flowers exhibit very strong expression in carpels. Numbers refer to floral stages (Smyth et al., 1990). (M) Longitudinal section of a Ler inflorescence meristem; E:A≫GUS expression in the carpel is very similar to (L). (N) Cross section of a Ler inflorescence; the E:A≫GUS expression pattern in wild-type flowers is similar to that of crc-1 flowers. (O) Longitudinal section of a stage 15 to 16 flower. Nectaries are demarcated with arrows. (P) Cross section of a crc-1 inflorescence with E:D:C:B:A≫GUS. Expression in septum and ovules disappears in stage 11 to 12 flowers (arrows), whereas septum expression persists with E:A≫GUS. (Q) Stage 10 crc-1 flower; E:D:C:B:A≫GUS expression in nectary anlagen is clearly visible (arrows). (R) Stage 9 crc-1 flower with pCRC 3.8kb≫GUS showing expression in the nectary anlagen (arrow).

Figure 3.

Complementation of the crc Mutant Phenotype. As compared with wild-type fruits (A), the composite construct EDCBA≫CRC fully complements the carpel defects of crc mutants (D). EA≫CRC partially complements crc carpel defects, with carpel fusion restored, but carpel (fruit) growth only partially restored (C). By contrast, CA≫CRC fails to complement crc (B), with the transgenic lines resembling crc mutants (E). Stamens are identified as medial (m) and lateral (l). Nectary glands are found at the abaxial bases of all stamens in wild-type flowers (A). Both the EA≫CRC and EDCBA≫CRC transgenes are able to rescue lateral nectaries, although medial nectaries are rarely present ([C] and [D]). By contrast, the CA≫CRC transgene is unable to rescue nectary development (B) (compare with crc-1 mutants in [E]).

Figure 4.

Nectary Phenotypes in Genotypes Lacking Combinations of MADS Box Gene Activity. (A) to (D) Flower phenotypes. (A) The wild type. (B) sep1-1 sep2-1 sep3-2. (C) ap2-2 pi-1 ag-1 shp1-1 shp2-1. (D) ap2-2 sep1-1 sep2-1 sep3-2. (E) to (J) Nectary phenotypes. (E) The wild type. Stomata and cuticular patterning characteristic of nectary morphology are visible in the inset. (F) shp1-1 shp2-1. (G) ap2-2 sep1-1 sep2-1 sep3-2. (H) sep1-1 sep2-1 sep3-2. (I) ap2-2 pi-1 ag-1 shp1-1 shp2-1. No cuticular patterning characteristic of nectaries is visible on the pedicel (inset). (J) ap2-2 pi-1 ag-1. Nectaries are present in wild-type, shp1 shp2, and ap2 pi ag flowers (arrows) but are lacking in the other genotypes. The slender outgrowths in ap2 sep1 sep2 sep3 and ap2 pi ag shp1 shp2 flowers are stipules that develop at the base of the floral organs because of their leaf-like character.

Figure 5.

Model of CRC Regulation in Arabidopsis. Based on previous genetic analyses (Baum et al., 2001), B (AP3 and PI) and C (AG) genes are regulating nectary development in combination. Because sep1/2/3 does not have any nectaries, SEPs are likely interacting with B and/or C proteins directly to activate CRC, whose protein interaction was shown by Honma and Goto (2001). In wild-type flowers, SHP1 and 2 are expressed in developing carpels not in the third whorl; however, in ap2 mutants, they are activated in all the whorls (Savidge et al., 1995; Flanagan et al., 1996; Pinyopich et al., 2003). It is likely that SHP1/2 might be redundantly interacting with SEP genes (Favaro et al., 2003) to activate CRC (shown as dashed line). To restrict the CRC expression at the base of stamens, there should be other unidentified floral factors (?) that fine tune the gene regulation. LFY may activate the CRC expression within flowers by activating BC genes directly and SEPs directly or indirectly (Schmid et al., 2003).