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First published online May 16, 2007
doi: 10.1242/10.1242/dev.005017

Development 134, 2073-2081 (2007)
Published by The Company of Biologists 2007
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Integration of cytokinin and gibberellin signalling by Arabidopsis transcription factors GIS, ZFP8 and GIS2 in the regulation of epidermal cell fate

Yinbo Gan1, Chang Liu2, Hao Yu2 and Pierre Broun1,*

1 CNAP, Department of Biology (7), University of York, York YO10 5YW, UK.
2 Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore, 117543, Singapore.


Figure 1
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  		Fig. 1. Induction of trichome initiation by cytokinins in wild-type Arabidopsis plants and GIS clade mutants. (A) Trichome initiation on sepals (top panel) and first (bottom panel) and third (middle panel) cauline leaves of wild-type, gis, zfp8, gis2 and gis gis2 ZFP8-RNAi line 2 (termed gis ZFP8-R gis2) plants that were treated with increasing concentrations of 6-benzylaminopurine. Values represent averages and standard error for 20 plants. (B) Expression of ZFP8 and GIS2 in loss-of-function mutants and RNAi lines; left panel: RT-PCR analysis of GIS2 expression in gis2 and gis ZFP8-R gis2 mutants; right panel: real-time PCR analysis of ZFP8 expression in wild-type (1), zfp8 (2), gis ZFP8-R1 (3), gis ZFP8-R2 (4), gis2 ZFP8-R1 (5), gis2 ZFP8-R2 (6), gis ZFP8-R1 gis2 (7) and gis ZFP8-R2 gis2 (8). Values are ratios to wild type of normalized transcript levels. Sepal trichome values represent the total number of trichomes for 12 flowers. WT, wild type.

 

Figure 2
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  		Fig. 2. Inflorescence trichome initiation in response to gibberellins in wild-type Arabidopsis plants and GIS clade mutants. (A) Trichome initiation on inflorescence organs of GA-deficient ga1-3 mutants treated with increasing concentrations of GA3. (*) Sepal trichome values represent the total number of trichomes for 12 flowers. (B) Differential response of GIS clade loss-of-function mutants to GA applications at increasing concentrations. Values represent averages and standard errors for 20 plants. WT, wild type.

 

Figure 3
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  		Fig. 3. GIS, ZFP8 and GIS2 have equivalent activities. (A,B) Trichome initiation on sepals of wild-type (A) and gis2 Arabidopsis flowers (B). (C) Trichome production on zfp8 (left) and wild-type cauline leaves (right). (D) Production of ectopic trichomes on carpels of wild-type 35S:GIS, 35S:ZFP8 and 35S:GIS2 plants. (E) Effects of ZFP8 and GIS2 overexpression on trichome initiation in wild type and in the gis mutant background (second branches). (F) Trichome branching and density on the main stems of gis (left), pGIS:GIS gis, pGIS:ZFP8 gis or pGIS:GIS2 gis mutants compared with wild-type stems (right). WT, wild type.

 

Figure 4
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  		Fig. 4. Expression of ZFP8, GIS2 and GL1 in response to GA and cytokinin treatments. Left: expression of GIS, ZFP8, GIS2, GL1 and primary response regulator ARR5 in inflorescence organs of Arabidopsis plants 2 hours after 6-benzylaminopurine applications (100 µM) to wild-type plants. Middle: expression of GIS, GL1 and ARR5 in response to 6-BA applications (100 µM) in the spy-3 mutant background. Right: induction by GA of ZFP8, GIS2 and GL1 expression in the inflorescence of ga1-3 mutants 4 hours after treatment. Black bars: mock treatments; grey bars: hormone treatments. Transcript levels were measured by real-time PCR and the values represent ratios of normalized levels to controls.

 

Figure 5
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  		Fig. 5. Genetic interactions between GIS2, SPY and trichome initiation regulators in Arabidopsis. (A,B) Trichome initiation on flowers of spy-3 (A) and 35S:GIS2 spy-3 mutants (B). (C) Trichome initiation on stems of gl1, gl3, 35S:GIS2 gl1 and 35S:GIS2 gl3 plants. (D) Effect of R overexpression on trichome initiation on gis2 flowers (left) and third branches (right). (E) GL1 expression in inflorescence organs of two 35S:GIS2 overexpressor lines (left) and in the gis2 mutant (right). Transcript levels were measured by real-time PCR and the values represent ratios of normalized levels to controls. U.I., upper developing inflorescence; W.I., whole developing inflorescence; WT, wild type.

 

Figure 6
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  		Fig. 6. Distribution of ZFP8 and GIS2 transcripts in different Arabidopsis plant tissues. (A-D) In situ hybridization of GIS2 (A,C) and ZFP8 (B,D) probes to developing inflorescence organs of wild-type plants. GIS2 is strongly expressed in inflorescence meristems, floral meristems and the epidermis (arrows, A). ZFP8 is most highly expressed in developing cauline leaves (B). (C,D) Sections that were hybridized using sense RNA. (E) Real-time PCR analysis of ZFP8 (top) and GIS2 (bottom) expression in various plant tissues. Values are normalized to the expression of the UBQ10 gene. 1stB, first branch; 2ndB, second branch; CL, cauline leaf; DI, developing Inflorescence; Fl, flower; RL, rosette leaf; Sil, silique; St, main stem (base).

 

Figure 7
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  		Fig. 7. Model of GIS, ZFP8 and GIS2 action in Arabidopsis inflorescence organs in response to GA and cytokinins. Broken lines: the interaction between GIS and SPY was previously examined. The antagonistic role of SPY in the regulation by ZFP8 and GIS2 of GA signalling is only suggested, as is the possibility that the regulation of trichome initiation by cytokinins is not solely dependent on GL1.

 




© The Company of Biologists Ltd 2007