The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development

Abstract

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

Keywords: ALOG domain; flower development; plant transcription factors.

Fig. 1.

Expression pattern of LSHs (A–L) and analysis of the lsh1 lsh3 lsh4 (M–R) triple mutant. Expression profile of LSH1 (A, B, G, and H), LSH3 (C, D, I, and J), and LSH4 (E, F, K, and L) in reproductive tissues analyzed by in situ hybridization in WT (A–F) and lfy-2 (G–L) backgrounds. (A, C, E, G, I, and K) are longitudinal and (B, D, F, H, J, and L) are transversal sections. (Scale bar, 50 μm.) Black arrowheads in (A and C) indicate signal in the cryptic bract region, white arrowheads in (I and K) indicate bract primordia. See SI Appendix, Fig. S13 for negative controls. (M and N) Stereo microscope pictures of WT (M) and lsh1 lsh3 lsh4 triple mutant (N) inflorescences. The white arrowheads in (N) indicate bracts. (Scale bar, 3 mm.) (O–R) SEM pictures of WT and lsh1 lsh3 lsh4 plants. (O), floral bud, the white arrowhead indicates the bract. (Scale bar, 100 µm.) lsh1 lsh3 lsh4 triple mutant bract (P and Q). The white arrowhead indicates the stigmatic papillae. The red arrowhead indicates the sepal-like cells. (Scale bars: P = 100 µm; Q = 50 µm.) WT sepal (R, Scale bar, 50 µm).

Fig. 2.

LSHs regulate PUCHI expression. (A) Genome browser view showing ampDAP-seq binding of LSH proteins to the PUCHI promoter. Regions I to III analyzed by ChIP in (B) are presented below. Y axis indicates count per millions of reads mapped. (B) ChIP assays analyzed by real-time PCR show that LSH4-GFP bound region I and III of PUCHI promoter but not region II. Error bars represent the propagated error value using three technical replicates. ChIP results of one representative experiment out of two independent biological replicates is shown. (C–E) Analysis of PUCHI expression profile by in situ hybridization. WT or mutant backgrounds are indicated below pictures. (Scale bar, 50 µm.) Asterisks indicate the bracts that subtend the primordia and the arrow points to the PUCHI expression domain, adaxial to the flower primordia in WT and abaxial in the lsh1 lsh3 lsh4 mutant. (F) Model showing the interactions involved in bract repression. Arrows indicate regulations (with solid arrows when direct) and the interaction between BOP and LSH proteins is shown.

Fig. 3.

Determination of ALOG binding specificity by ampDAP-seq. (A) Logo obtained for LSH3 in ampDAP-seq, using the 600 peaks with the strongest signal. (B) Receiver operating characteristic (ROC) curve for LSH3 using all peaks except those used to build the logo. The value of the Area Under the Curve (AUC) is indicated. (C) EMSA with ALOG highest-score sequence DNA probe (WT ALOG motif) and LSH3_S45-S190. Based on the analysis of three independent EMSAs, we found an apparent Kd of 28 nM for LSH3-DBD/DNA. (D) EMSA with LSH1_M1-L166, LSH3_S27-S199 and indicated DNA probes. The WT ALOG probe (Top Left) was mutated at positions indicated on the LSH3 logos (Bottom Left). Scores between brackets were obtained by scanning each DNA probe sequence with the LSH3 PWM (the best binding sites correspond to the less negative score values). EMSA with described DNA probes (Right). Uncropped gels are provided in SI Appendix, Fig. S14.

Fig. 4.

Structure of LSH3 DBD in complex with DNA. (A) Alignment of studied ALOG proteins. Blue dotted lines indicate the limits of the ALOG domain. The zinc ribbon is highlighted in orange, with the key histidine and cysteine residues indicated by blue triangles. Green stars denote residues in direct contact with DNA bases. NLS = Nuclear Localization Signal. Numbers are relative to AtLSH3. At = Arabidopsis thaliana, Sl = Solanum lycopersicum, Mp = Marchantia polymorpha, Os = Oryza Sativa. (B) ALOG-DBD/DNA complex. Throughout this figure, LSH3 DBD is shown in blue except helix 3 colored in cyan. The DNA duplex is depicted in gray with bases with the highest information content in orange. The Zn²⁺ ion is shown in yellow. A 180° rotation along the y-axis was applied to obtain the bottom picture. (C) Protein-DNA interactions. (D) Ribbon diagram of LSH3 DBD bound to its cognate DNA, with a focus on helix 3. Interactions are indicated by black dashed lines. For clarity, helix 1 was removed. (E) Close-up view of LSH3 C-terminal tail in contact with DNA. (F) EMSA with ALOG highest-score sequence DNA probe (WT ALOG motif) and indicated LSH3_S27-S190 versions. (G) Close-up view of the zinc ribbon. Side chains of the residues coordinating the zinc ion are represented. (H) EMSA with WT ALOG motif DNA probe and indicated _LSH3S27-S190 and LSH1_M1-L166 versions. Uncropped gels are provided in SI Appendix, Fig. S14.