YABBYs and the transcriptional corepressors LEUNIG and LEUNIG_HOMOLOG maintain leaf polarity and meristem activity in Arabidopsis

Abstract

In Arabidopsis thaliana, FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) encode YABBY domain proteins that regulate abaxial patterning, growth of lateral organs, and inflorescence phyllotaxy. In this study, we show that YABs physically interact with components of a transcriptional repressor complex that include LEUNIG (LUG), LEUNIG_HOMOLOG (LUH), the LUG-associated coregulator SEUSS, and related SEUSS-LIKE proteins. Consistent with the formation of a LUG-YAB complex, we find that lug mutants enhance the polarity and growth defects of fil yab3 mutant leaves and that this enhancement is due to a loss of LUG activity from the abaxial domain. We performed a more extensive genetic analysis, which included the characterization of yab triple and quadruple mutants, lug luh/+ (heterozygous only for luh) mutants, and plants expressing artificial microRNAs targeting LUG or LUH. These analyses showed that the LUG-YAB complex also promotes adaxial cell identity in leaves as well as embryonic shoot apical meristem (SAM) initiation and postembryonic SAM maintenance. Based on the likely formation of the LUG-YAB complex in the abaxial domain of cotyledons and leaves, we propose that this complex has numerous non-cell-autonomous functions during plant development.

Figure 1.

BRET Assays Detect Protein Interactions between LUG, YABs, and SLKs in Planta. Y:B luminescence ratios from onion slices transiently coexpressing RLUC-LUG and the indicated YAB fusion protein (A), RLUC-LUG or YAB5-RLUC, and the indicated SLK fusion protein (B) and FIL-RLUC or YAB5-RLUC, and the indicated YAB fusion protein (C). The superscript letters R and Y indicate the positions of the RLUC and YFP tags, respectively. Results are means ± se from at least three replicates, with the exception of LUG/YAB5 and LUG/SLK2 combinations, which were only repeated twice. Statistical difference from the RLUC-fusion/YFP control was calculated using a Student's t test, with P < 0.01 indicated by one asterisk and P < 0.001 indicated by two asterisks.

Figure 2.

Enhancement of fil yab3 Polarity Defects in lug and seu Mutant Backgrounds. (A) to (D) Sixteen-day-old wild-type (A), fil-8 yab3-2 (B), fil-8 yab3-2 lug-1 (C), and fil-8 yab3-2 seu-2 (D) plants. (E) and (F) Scanning electron micrographs of fil-8 yab3-2 (E), fil-8 yab3-2 lug-1 (F), and wild-type seedlings showing the first two leaves at an early stage of development. (G) and (H) Expression of CYCB1_pro:GUS reporter in emerging leaves of fil-5 yab3-1 (G) and fil-5 yab3-1 lug-1 (H) mutants. (I) to (K) Scanning electron micrographs showing the abaxial epidermis of fully expanded wild-type (I), fil-8 yab3-2 (J), and fil-8 yab3-2 lug-1 (K) leaves. (L) to (N) Transverse sections through fully expanded wild-type (L), fil-8 yab3-2 (M), and fil-8 yab3-2 lug-1 (N) leaves. Arrowheads mark adaxialized spongy mesophyll cells. (O) to (S) Distribution of YAB3:GUS ([O] to [Q]) and PHB RNA ([R] and [S]) in transverse sections of yab3-2 (O), fil-8 yab3-2 (P), fil-8 yab3-2 lug-1 ([Q] and [S]), and wild-type (R) apices. Asterisks mark the position of the meristem, and arrowhead indicates expanded domain of PHB expression. (T) lug-444 and wild-type (inset) flowers. (U) and (V) Representative flowers from 35S_pro:amiR-LUG plants displaying an intermediate (U) and strong (V) lug mutant phenotype. (W) and (X) Representative 16-d-old fil-8 yab3-2 35S_pro:amiR-LUG (W) and fil-8 yab3-2 FIL_pro:amiR-LUG (X) lines. c, cotyledons; pm, adaxial palisade mesophyll cells; sm, abaxial spongy mesophyll cells; ad, adaxial side of primordia; ab, abaxial side of the primordia. Bars = 2 mm in (A) to (D), (W), and (X), 1 mm in (E), (F), and (T) to (V), and the inset in (T), 200 μm in (O) to (S), and 100 μm in (G) to (N).

Figure 3.

Leaf Polarity and Meristem Defects of lug luh/+ Plants. (A) to (D) Histochemical localization LUG_pro:GUS ([A] and [C]) and LUH _pro:GUS ([B] and [D]) in 8-d-old plants viewed in transverse sections through the apex ([A] and [B]) or as whole mounts ([C] and [D]). Arrowheads indicate stipules. (E) and (F) Twenty-eight-day-old lug-444 (E) and lug-444 luh-4/+ (F) plants. lug luh/+ leaves are narrow and aberrant phyllotaxy is often observed (asterisks). (G) lug-444 luh-4/+ plants with small needle-like and trumpet-shaped leaves (arrowheads). (H) Transverse section through a mature lug-444 luh-4/+ trumpet-like leaf. (I) Midrib vasculature of leaf shown in (H). Position of phloem and xylem are indicated. (J) Scanning electron micrograph of the epidermis of a lug-444 luh-4/+ needle-like leaf. (K) and (L) Transverse section through a lug-444 luh-4/+ needle (K) and vasculature showing laterally displaced phloem (L). (M) Histochemical localization of YAB3:GUS in yab3-2 lug-1 luh-4/+ plant revealing ectopic YAB3 expression in the adaxial domain of a needle-like leaf. p, phloem; x, xylem. Bars = 5 mm in (A) and (B), 2 mm in (C), 200 μm in (F), 100 μm in (D), (F), and (G), and 20 μm in (E) and (H).

Figure 4.

Vegetative Phenotypes of yab Triple and Quadruple Mutants. (A) Structure of the YAB2 and YAB5 genes. Boxes depict the 5′ and 3′ untranslated regions (white), Zn-finger domain (red), and YABBY domain (green). The position of the point mutations (arrows) is measured from the start ATG of the genomic sequence. (B) and (C) Polycotyledonous fil-8 yab3-2 yab5-1 (B) and fil-8 yab2-1 yab3-2 yab5-1 (C) seedlings. (D) fil-8 yab3-2 yab5-1 mutants with narrow or radial leaves. (E) Transverse section through a narrow fil-8 yab3-2 yab5-1 leaf. Normal palisade and spongy mesophyll tissue is present between arrowheads. (F) Vasculature of leaf shown in (E) showing phloem surrounding central xylem tissue. (G) Transverse section through a radial fil-8 yab3-2 yab5-1 leaf and associated vasculature (inset). (H) and (I) Distribution of GUS activity (from YAB3:GUS) (H) and PHB RNA (I) in transverse sections of fil-8 yab3-2 yab5-1 apices. Asterisks mark the position of the meristem. Bars = 2 mm in (B) to (D), 100 μm in (E) and (G), 50 μm in (H) and (I), and 20 μm in (F) and the inset in (G).

Figure 5.

SAM Defects of yab and lug luh/+ Mutants. (A) to (C) Longitudinal sections through apices of 10-d-old short-day-grown yab2-1 yab3-2 yab5-1 (A), fil-8 yab3-2 (B), and fil-8 yab2-1 yab3-2 yab5-1 (C) mutant plants. Arrows indicate anticlinal L1 and L2 cell layers. (D) Section through a quadruple yab mutant showing the establishment of a secondary shoot SAM (arrowhead) in the axil of a cotyledon (c). Inset: close-up of secondary shoot meristem with clear L1 and L2 anticlinal cell layers (arrows). (E) fil-8 yab2-1 yab3-2 yab5-1 quadruple mutant showing a fasciated primary shoot (arrowhead) and two secondary shoots (arrows) arising from the axils of cotyledons (c). (F) Formation of ectopic meristems from the adaxial surface of a yab quadruple mutant leaf (asterisks). (G) and (H) Section through apices of 14-d-old short-day-grown lug (G) and lug-444 luh-4/+ (H) plants. (I) and (J) RNA in situ hybridization using STM as a probe on wild-type (I) and lug-444 luh-4/+ (J) apical sections. (K) and (L) Inflorescences of lug-444 (K) and lug-444 luh-4/+ (L). (M) and (N) Sections through of lug-444 (M) and lug-444 luh-4/+ (N) inflorescences. Bars = 1 mm in (E) and (F), 200 μm in (D), 100 μm for (M) and (N), and 50 μm in (A) to (C), (G) to (J), and the inset in (D).

Figure 6.

Genetic Interactions between Corepressor and YAB Mutants Reveal a Role in Embryonic SAM Development. (A) Six-day-old fil-8 lug-1 luh-3/+ plants lack a SAM. Inset: scanning electron micrograph of shoot apex. (B) to (D) Longitudinal sections through apices of 4-d-old wild-type (B), lug-1 luh-3/+ (C), and fil-8 lug-1 luh-3/+ (D) seedlings. Layers in which cells are dividing anticlinally are indicated with arrows. Note the absence of a layered SAM structure in fil-8 lug-1 luh-3/+ plants. (E) A 30-d-old fil-8 yab3-2 lug-1 luh-3/+ plant with extremely narrow and needle-like leaves. (F) SAM of a 16-d-old luh-4 AS1_pro>>amiR-LUG plant is inactive, but signs of organ formation are apparent (arrows). Inset: view of entire seedling. (G) Section through a terminated luh-4 AS1_pro>>amiR-LUG apex revealing the absence of cytoplasmically dense cells. Inset: view of a wild-type apex with cytoplasmically dense cells. Bars = 1 mm in (A) to (E) and the inset in (F), 500 μm in (F), and 50 μm in (B) to (D) and (G).

Figure 7.

Phenotypes of seu slk2/+ and seu slk2 Mutants. (A) RT-PCR expression analysis of SEU, SLK1-3, and the tubulin gene TUB7 in roots, vegetative shoot, and inflorescence tissue. SLK1 cDNA is distinguished from SLK3 cDNA following digestions with HpaI (see Methods). (B) Phylogeny of the Arabidopsis SEU/SLK family of coregulators. The tree was constructed by the neighbor-joining method and bootstrap values are indicated. (C) to (E) Flowers of lug-444 (C), seu-4 (D), and seu-4 slk2-1/+ (E) mutants with several outer whorl organs removed to reveal the carpel. Inset is a wild-type flower. Arrowheads indicate stigmatic horns. (F) Twenty-eight-day-old seu-4 slk2-1 double mutants lack an active SAM. View of the shoot apex reveals fusion between cotyledons (inset). (G) Longitudinal section through a 14-d-old seu-4 slk2-1 shoot apex revealing a complete absence of cytologically dense cells typical of an active SAM. (H) and (I) DIC microscopy of slk2 (H) and seu slk2 (I) embryos at the heart stage of development. (J) and (K) CLV3_pro:YFP-ER marker expression in slk2 (J) and seu slk2 (K) embryos at the heart stage of development. Note the complete absence of YFP florescence in seu slk2 double mutant embryos. (L) lug-444 35S_pro:amiR-LUH plants lack a SAM. (M) Section of a lug-444 35S_pro:amiR-LUH plants revealing the absence of cytoplasmically dense cells. Bars = 1 mm in (F) and (L), 0.5 mm in (C) to (E) and the inset in (F), 200 μm in (G), 50 μm in (M), and 20 μm in (H) to (K).

Figure 8.

Proposed Composition of the LUG-YAB Complex and Its Role in Signaling during Vegetative Development. (A) Based on protein–protein interactions in yeast and in planta, it is likely that the LUG-YAB complex includes two YAB proteins (forming either homodimers or heterodimers) in close association with LUG or LUH and SEU or one of the three SLKs. According to our model (see text for details), the LUG-YAB complex is recruited to specific cis-regulatory elements (black line) by SEU-interacting transcription factors (TF). (B) Given the expression patterns of LUG, LUH, and FIL, it is likely that the LUG-YAB complex forms in the abaxial domain of developing cotyledons and leaves (shaded). Once formed, this complex activates two signaling pathways. The first promotes adaxial cell identity (arrowheads) and the other regulates SAM initiation and maintenance (arrows) possibly by promoting boundary formation (dotted line).