PMC

Loss of YAB1 Expression in Organ Primordia Stimulates Altered Distribution of Meristem-Specific Markers.
(A) to (C) Aerial view of inflorescences expressing the PCLV3≫ER-GFP marker. The green fluorescent signal is superimposed on the light image viewed by a stereoscope. Wild-type (A), fil-8 (B), and fil-8 yab3-2 (C) apices are shown. White two-headed arrows mark outward expansion of PCLV3≫ER-GFP expression in the mutants compared with the wild type, and arrows mark abaxial expression in fil-8 yab3-2 filaments.
(D) to (F) Longitudinal sections through inflorescences expressing the PWUS≫ER-GFP marker. In the wild type (D), the PWUS≫ER-GFP signal marks a limited domain above the rib meristem. In the fil-8 mutant apex (E), PWUS≫ER-GFP expression has considerably expanded in both IM and FM. In fil-8 yab3-2 mutant apices (F), it has expanded in the IM, while filamentous flowers fail to initiate PWUS≫ER-GFP expression. White two-headed arrows mark expansion of marker expression in the mutants compared with the wild type.
Bars = 100 μm in (A) to (E) and 50 μm in (F).

Effects of a Domain-Specific Reduction in Floral YAB1 Activity Generated by an Artificial miRNA.
(A) to (C) Scanning electron micrographs of phyllotactic patterns in apices lacking YAB1 activity in bracts. Disrupted phyllotactic patterns and abnormal flower initiation are seen either in fil-8 yab3-2 (A) or PANT≫amiR-YAB1 inflorescences ([B] and [C]).
(D) PANT≫amiR-YAB1 flower with reduced numbers and altered positions of organ primordia.
(E) PAP1≫amiR-YAB1 inflorescences have normal flower initiation phyllotaxis.
(F) and (G) Phyllotactic patterns in PAP1≫amiR-YAB1 flowers. Disrupted numbers and positions of floral organs are evident (cf. Figure 1K).
(H) Mature PAP3≫amiR-YAB1 flower. The radial petals are formed at the correct position, whereas the adjacent medial stamens are closer to each other than in wild-type or PAP3≫miR165 flowers (cf. stamen proximity marked by the arrows in [H] and [I]).
(I) Mature PAP3≫miR165 flower. The radial petals and stamens are formed in the correct positions.
Asterisks indicate the sepal. Numbers represent phyllotactic order. b, bract; f, filament; pe, petal; st, stamen. Bars = 50 μm in (A) to (F) and 100 μm in (G) to (I).

Autonomous Distribution of Functional GFP-Tagged YAB1 Proteins.
(A) to (F) Complementation of fil-8 by GFP-tagged YAB1. Inflorescences ([A], [B], [D], and [E]) and flowers ([C] and [F]) are shown from wild-type ([A] to [C]) and fil-8 mutant ([D] to [F]) plants with ([B], [C], [E], and [F]) or without ([A] and [D]) PFIL≫YAB3-GFP. The numbers mark the order of flower initiation and outline the phyllotactic pattern. Note the partial complementation in older flowers (arrow in [F]).
(G) to (J) GFP fluorescence activated by the FIL promoter. Sections through inflorescences ([G] and [H]) and flowers ([I] and [J]) showing the distribution of fluorescence emitted from the ER-localized GFP ([G] and [I]) or from GFP-tagged YAB1 ([H] and [J]) reporter lines driven by the same FIL promoter line. The top rows of images are transverse sections, and the bottom rows are longitudinal sections, each at different magnifications. All apices were counterstained by propidium iodide (PI), excluding the longitudinal sections in (I) and (J). The asterisk indicates the sepal primordia. cb, cryptic bract.
Bars = 1 mm in (A) to (F) and 20 μm in (G) to (J).

The LAS Gene Mediates YAB1-Derived Signals.
(A) to (F) Effect of the las-11 mutant on flowers with altered levels of YAB1 activity.
(A) PAP1:YAB3 flower with one sepal removed revealing additional petals, additional stamens, and disrupted gynoecium development.
(B) las-11 PAP1:YAB3 flower with a nearly normal number of petals and stamens (one sepal removed).
(C) PAP3:YAB3 flower with additional stamens and disrupted gynoecium development (one sepal removed).
(D) las-11 PAP3:YAB3 flower with a near wild-type appearance (one sepal removed).
(E) fil-8 inflorescence.
(F) las-11 fil-8 inflorescence, comprising primarily filamentous flowers.
(G) to (R) Distribution of PLAS≫LAS-GFP fluorescence. Transverse ([G] and [I]) and longitudinal ([H] and [J]) sections through LAS≫LAS-GFP inflorescences ([G] and [H]) and flowers ([I] and [J]). Transverse section of a fil-8 PLAS≫LAS-GFP inflorescence (K) and longitudinal section of a flower (L) with expression between the floral organs and the FM (b, bract). Transverse section of a fil-8 yab3-2 PLAS≫LAS-GFP inflorescence (M) and longitudinal section of a flower (N) with almost no expression between the sepal and the FM. Transverse section of a PAP1:YAB3 PLAS≫LAS-GFP inflorescence (O) and longitudinal section of a flower (P) with significant expansion of the LAS≫LAS-GFP expression domain marked by arrows. Longitudinal section through stage 4 wild-type (Q) and PAP3:YAB3 (R) flowers, where expansion of PLAS≫LAS-GFP expression is marked with arrows.
(S) A model of IM expression territories. A representation of the expression domains of genes examined in this study and the direction of influence (arrows) established or speculated based on the results. PLAS≫LAS-GFP signal is present, while the others have been painted in as representative domains. Primordial YAB1 activity (pink) nonautonomously communicates with the floral (FM) and inflorescence (IM) meristems and regulates the expression of CLV3 (purple) and WUS (yellow). LAS, acting at the organ-meristem boundary (bou), mediates this signaling process. cb, cryptic bract.
Asterisks indicate the sepal. Numbers represent the order of flower initiation. Bars = 1 mm in (A) to (F) and 50 μm in (G) to (R).

Autonomous YAB1 Expression Initiates Morphogenic Cues.
(A) to (C) Scanning electron micrographs of near-anthesis flowers. Wild-type flowers (A) have a stereotypic phyllotaxis (three sepals and two petals were removed), while PAP3≫DTA flowers ([B]; one sepal removed) have arrested petal and stamen development. PAP3≫KAN1 flowers (C) lack petals and have radialized stamens, as also illustrated in the inset (here, two sepals and the gynoecium have been removed).
(D) to (H) Flowers with graded effects of ectopic YAB1 expression. Flowers of a weak PAP3≫FIL-GFP line (D) have normal petals and an excess of stamens and carpels. An intermediate (E) and a strong ([F] and [G]) line of PAP3:YAB3 have abnormal, filamentous organs that occupy the 2nd and 3rd whorls in addition to naked placental mounds bearing malformed ovules at the center of the flowers. Sepals but no other organs were removed. The effects of PAP3:YAB3 on carpel development are likely sequential, as ectopic YAB1 expression throughout carpel primordia, as in PCRC≫GFP-FIL, caused minor defects only ([H], right) by sharp contrast with the miniature, abnormal carpels of CRC≫KAN1 plants (left).
(I) to (K) GFP fluorescence in floral apices. Distribution of the NLS-GFPx2 (I) or GFP-FIL (J) fluorescent reporters transactivated by the PAP3:LhG4 driver line in stage 4 to 5 flowers. Top images are transverse sections, and bottom images are longitudinal. (K) shows the specific distribution in petal primordia of YAB3-GFP expressed by the PRBE:LhG4 driver.
(L) and (M) Petal-specific responses. PRBE≫YAB1-GFP flowers (L) have small radial petals (arrows and inset), while other organs are normal. Similarly, PRBE≫DTA flowers (M) lack petals.
Asterisks indicate sepals. FM, floral meristem; pe - petals; st, stamens; ca, carpels; f, filaments; n, nectaries; ov, ovules. Bars = 100 μm in (A) to (C), (E), and (G), 1 mm in (D), (H), (L), and (M), and 20 μm in (I) to (K).

Loss of YAB1, Which Is Expressed in Organ Primordia, Results in Aberrant Phyllotaxis.
(A) and (B) Recognizable domains within floral Arabidopsis apices. (A) shows the side view, and (B) shows the top view. Yellow color marks the SHOOTMERISTEMLESS expression domain, and green marks cryptic bract primordia. The youngest primordium is marked as p1 and later ones as p2, p3, etc. Incipient primordia are labeled i1, i2, etc. The red color marks the organ primordia boundary, the purple color marks flower domains differentiating into sepals and pedicels, and blue color marks the flower B class domain.
(C) Wild-type inflorescence shoot. Flowers and siliques are produced in spiral phyllotaxis with progressive, even, internodal growth between them.
(D) and (E) Disrupted phyllotactic patterns in the fil-8 (D) and fil-8 yab3-2 (E) mutant shoots showing clustered (arrows) or dispersed flowers positioned at variable distances and angles relative to each other.
(F) to (H) Phyllotaxis of IMs. Scanning electron micrographs of wild-type (F), fil-8 mutant (G), and fil-8 yab3 double mutant (H) IMs producing flowers and bract-like structures. Numbers represent phyllotactic order of developing primordia: 0 marks oldest incipient primordia, and 1 marks first morphologically detectible primordia (p1). In fil-8 and fil-8 yab3 mutants ([G] and [H]), relative positions of adjacent primordia, marked by two-headed arrows, are different from relative positions of same primordia pairs in wild-type apex (F).
(I) and (J) FIL mRNA distribution. Transverse (I) or longitudinal (J) sections of apices from wild-type flowering plants. Expression is limited to the organ domain, either cryptic bracts (cb) at IM periphery or sepal primordia in stage 3 flowers.
(K) to (M) Phyllotaxis of FMs. Aerial-view, scanning electron micrographs of wild-type (K), fil-8 mutant (L), and fil-8 yab3 double mutant (M) flowers. (K) shows the stereotypic organization of a stage 3 to 4 wild-type flower, and disrupted phyllotactic patterns of organ initiation can be seen in the mutant flowers ([L] and [M]). Asterisks mark sepal primordia and f marks filamentous organs.
Bars = 1 mm in (C) to (E), 100 μm in (A), (B), and (F) to (J), and 50 μm (K) to (M).