LEUNIG, a putative transcriptional corepressor that regulates AGAMOUS expression during flower development

Abstract

Regulation of homeotic gene expression is critical for proper developmental patterns in both animals and plants. LEUNIG is a key regulator of the Arabidopsis floral homeotic gene AGAMOUS. Mutations in LEUNIG cause ectopic AGAMOUS mRNA expression in the outer two whorls of a flower, leading to homeotic transformations of floral organ identity as well as loss of floral organs. We isolated the LEUNIG gene by using a map-based approach and showed that LEUNIG encodes a glutamine-rich protein with seven WD repeats and is similar in motif structure to a class of functionally related transcriptional corepressors including Tup1 from yeast and Groucho from Drosophila. The nuclear localization of LEUNIG-GFP is consistent with a role of LEUNIG as a transcriptional regulator. The detection of LEUNIG mRNA in all floral whorls at the time of their inception suggests that the restricted activity of LEUNIG in the outer two floral whorls must depend on interactions with other spatially restricted factors or on posttranslational regulation. Our finding suggests that both animals and plants use similar repressor proteins to regulate critical developmental processes.

Figure 1

Micrographs of lug flowers. (A) lug-16, a weak allele. The pair of arrows indicate the split stigma characteristic of lug mutant flowers. (B) lug-12, a strong allele. The flower has no petal and exhibits split stigma (arrows). The smaller size sepals are caused by partial homeotic transformation into carpelloid sepals. (C) The flower of a lug-16 mutant rescued by cosmid 31-G. The flower and its stigma are similar to wild type.

Figure 2

Molecular cloning of LUG. (A) A physical map of LUG on chromosome 4. Open bars represent yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones. The relative position of several PCR-based markers is placed above the chromosome. Cosmids 31–1 and 31-G both complemented the lug-16 mutant. (B) The protein sequence of LUG from L-er. The position of five lug mutations is indicated above the amino acid sequence. The two glutamine-rich regions are in bold. The seven WD repeats are underlined and are identified by using http://pfam.wustl.edu/hmmsearch.shtml. The specific base pair changes in each lug allele are described below based on the sequences of cosmid L23H3 (AL050398). lug-3 causes a C to T change at 25309; lug-12 causes a C to T change at 25198; lug-16 causes a G to A change at 24693; lug-1 mutation causes a G to A change at 26267; lug-14 mutation inserts an A after 29080. (C) Structural similarity between LUG, LUH (AC 003974), the S. cerevisiae Tup1 (19), and the Drosophila Gro (20). Numbers above and in parentheses correspond to amino acids. The percentage between the amino acid numbers indicates the level of identity between LUG and LUH. The LUFS domain in LUG and LUH is indicated by a dotted region. A second highly conserved region between LUG and LUH immediately precedes the WD repeats and is marked by wavy lines. The level of similarity is identified by using http://www.bioinformatix.com/sas/. (D) Sequence alignment in the LUFS domain among LUG, LUH (AC003974), Flo8, and a human single-strand DNA-binding protein (AF077048). Numbers correspond to amino acids. Flo8 has extra 21 aa indicated by (X)21. Amino acids conserved in all four genes are in bold and are indicated by *. Amino acids conserved in three of the four genes are in bold and are indicated by :.

Figure 3

LUG mRNA expression. (A) LUG mRNA is expressed in all tissues including roots (lane 1), shoots (lane 2), stems (lane 3), cauline leaves (lane 4), and inflorescences (lane 5). A decreased level of LUG mRNA is detected in lug-1 (lane 6) and lug-12 (lane 7) mutant inflorescences. The mRNA level is corrected with the actin loading control, and the relative level is derived by comparing all signals to the stem (lane 3), which equals to 1.0. (B) LUG mRNA expression in inflorescences of wild-type (lane 1), ap1–1 (lane 2), ap2–1 (lane 3), ap2–2 (lane 4), pi-1 (lane 5), ag-2 (lane 6), lfy-6 (lane 7), and lug-16 (lane 8). With the exception of ag-2, LUG mRNA level in most mutants appears unchanged. In lug-16, LUG transcript is reduced in size and increased in abundance (arrowheads).

Figure 4

In situ expression pattern of LUG mRNA. (A) LUG expression in 14-day-old seedlings. A low level of LUG mRNA is present in the shoot apical meristem and in the first few young leaves. LUG mRNA level increases dramatically in more developed leaves (arrow). (B) LUG sense probe to 14-day-old seedlings. (C) LUG mRNA is detected in the secondary inflorescence meristem and in the stage 1 and 3 floral meristems (numbers indicate the stage of respective floral meristems; ref. 33). LUG mRNA is detected in vascular tissues (arrowhead). LUG mRNA level is also more abundant in the adaxial side of the cauline leaf (arrow). (D) LUG mRNA level is low in the inflorescence meristem but increases in young flowers (stages 3 and 5, respectively). (E) LUG is strongly expressed in the sepal primordia and the central dome of the stage 4 and 5 flowers. (F) At stage 7, LUG mRNA is not detected in sepals, but is present in the carpel (ca) and stamen (st) primordia. LUG mRNA is persistently detected in petals (pe) as shown in this stage 11 flower. (G) In stage 10 carpels, LUG mRNA is strongly expressed in the placenta/ovule primordia (arrows) and weakly expressed in the carpel valves. (H) A cross section of a stage 9 flower. A high level of LUG mRNA is detected in placenta (p) and locules (lo) of the anther. (I) LUG mRNA is detected in developing ovules at stage 12.

Figure 5

Nuclear localization of GFP-LUG. (A and B) Dark-field images. (C and D) Corresponding phase-contrast microscopic images. (A and C) An onion epidermal cell transiently expressing the GFP-LUG chimeric protein. (B and D) An onion epidermal cell transiently expressing the GFP protein (vector pAVA 393; ref. 15). Arrows mark the location of nucleus.