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HAWAIIAN SKIRT , an Arabidopsis F-Box Gene, Involves in Specifying Organ

Boundaries and Growth 

Figures

Col-0 hws-1 hws-2   Pro 35S ::HWS 

A

B

Figure 1 Seedlings of Col-0,hws-1

,hws-2 

andPro 

35S ::HWS 

 (A) Stage 1.10, 10 rosette leaves > 1 mm in length, plants (note leaf number from both hws mutants washigher than WT and Pro 35S ::HWS ). (B) Stage 6, first flower open, plants at flowering stage (notePro 35S ::HWS plant exhibited smallest seedling without primary inflorescence). Bar = 1 cm. 

Col-0

hws-1 

Pro 35S ::HWS 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20buds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24buds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 2 3 4 5 6buds

hws-2 

buds

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Figure 2 Buds, flowers and developing siliques from different inflorescences from Stage 6.50,midflowering, plants of Col-0, the hws mutants and Pro 35S ::HWS . The numbers represent the positions along the entire inflorescences. Note position 1 is the first stage atwhich petals can be seen. Bar = 2.5 mm 

Pro 35S ::HWS Col-0 hws-1 hws-2 

Ep

Co

En

A

B

Figure 3 Comparative phenotypic analysis of roots.

(A) The 8 day old seedlings of Col-0, the hws mutants and Pro 35S ::HWS  growing vertically on MSmedium with 0.8% Bacto agar. Bar= 1 cm. (B)Transverse light micrographs from primary roots of 3 dayold seedlings. (Ep), epidermis; (Co), cortex; (En), endodermis. Bar= 20 μm.

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24 h

Col-0 hws-1 hws-2  Pro 35S ::HWS 

Figure 4 Comparative gravitropic response of roots.The 3 days old seedlings were grown horizontally for 24 h on ½MS medium with 1.5% (W/V) bacto agar.Note the hws-2 mutant produced more root hairs (arrows). Bar= 5 mm.

A

sepal

Stigmaticpapillae

Woundingarea

Pollen

EB C D

 

Figure 5 Analysis of expression from Pro HWS ::GUS plants.Cleared whole-mount GUS stained tissue samples after different incubation periods.(A) After 40 min staining (Note the GUS expression in roots, junction between root and hypocotyls(arrow), and junctions of cotyledonary leaves (circle)). (B) Floral bud. (C) A flower from position 1. (D) Aflower from position 4. (E) A flower from position 8 (Note the GUS expression in sepals, stamen, pollen,stigmatic papillae).Bar= 1 mm.

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Pro HWS ::GFP  Emerging lateralroot and vascular tissue 

Pro HWS ::GFP  Root cap

B CIII

II I III

II I

Col-0Root

A III

II I

 

Figure 6 Confocal images of roots from eight days old Col-0 (A) and Pro HWS ::GFP plants (B andC).(A and B) Root cap. (C) Emerging lateral root. The materials was observed and photographs were takenat the same time under different light sources including normal light image in bright field (I) and imagefrom sample excited at 488 nm with argon ion laser (II), and another image (III) is the overlapped imagesof I and II. Bar= 50 μm (A), 17.8 μm (B), 77.49 μm (C). 

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 Pro HWS ::GFP  Col-0

A

B

C

D

Stigmaticpapillae

pollen

Stigmaticpapillae

pollen

Seed coat

Figure 7 Light images using UV fluorescence light source from Pro HWS ::GFP (A, B) and Col-0 (C,D).(A) Pollen and stigmatic surface  from Pro HWS ::GFP  flower at position 4. (B) Developing seeds isolatedfrom Pro HWS ::GFP young siliques (C) Stigmatic surface  from Col-0 flower at position 4. (D) developingseeds isolated from Col-0 young silique. Bar= 50 μm (A, and C), 100 μm (B and D).

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 RT RL CL BD FL ST GS YS genomic

Col-0

RT RL CL BD FL ST GS YS genomic

hws-1

HWS 

UBQ  

HWS 

UBQ  

1 Kb

1 Kb

ATG STOP

ORF (1236bp)

590Rev

5’UTR

419bp

532bp

12bp

intron

590 5’UTR

Figure 8 RT-PCR analysis of expression of  the HWS gene in hws-1 and Col-0 using HWS genespecific primer 5905'UTR and 590Rev. The positive control for UBQ giving a fragment of 1.251 Kb withubiquitin specific primers was amplified at comparable levels from all tissues. (RT), Roots; (RL), Rosetteleaves; (CL), Cauline leaves; (BD), Floral buds; (FL), Open flowers; (ST), Inflorescence stems; (GS),Green siliques; (YS), Yellow siliques.