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First published online 13 May 2004
doi: 10.1242/dev.01144

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A broad competence to respond to SHORT ROOT revealed by tissue-specific ectopic expression

Giovanni Sena^1,*, Jee W. Jung¹ and Philip N. Benfey^2,

¹ NYU, Department of Biology, The Silver Center, room 1009; 100 Washington Square East, New York, NY 10003, USA
² Duke University, Department of Biology, Box 91000, Durham, NC 27708, USA

View larger version (127K): [in a new window]   Fig. 1. SHR::GFP does not move from phloem companion cells (CC), where the native SHR promoter is not active. Longitudinal (A-C) and transverse (D,E,G) confocal images of GFP fluorescence of pSUC2::GFP (A), pSUC2::GFPER (GFPER contains ER targeting and retention signals) (B,D), pSUC2::SHR::GFP (C,E) and pSHR::SHR::GFP (G) transgenic roots. (F) In situ hybridization with GFP antisense probe on transverse section from pSHR::SHR::GFP transgenic root. The SUC2 promoter is active in the phloem CC (B and arrows in D). Non-targeted GFP can move from the CC to the epidermis (A), while the fusion protein SHR::GFP does not leave the CC (C and arrows in E). The native SHR promoter is not active in the phloem CC (arrows in F), yet the SHR::GFP fusion protein can be found throughout the stele and in the endodermis when transcribed under the same promoter (G). Arrowheads in D and E indicate protoxylem. ep, epidermis; co, cortex; en, endodermis; st, stele. Scale bars: 50 µm in A-C; 25 µm in D-G.

View larger version (215K): [in a new window]   Fig. 2. SHR::GFP moves only into the first of the supernumerary endodermis-like layers. Longitudinal confocal image of a pSCR::SHR, pSHR::SHR::GFP transgenic root. The pSCR::SHR background produces supernumerary layers as described previously (Nakajima et al., 2001) and the fusion protein SHR::GFP is detected in the stele and in the first layer in contact with it (arrowheads). ep, epidermis; sn, supernumerary layers; st, stele. Scale bar: 25 µm.

View larger version (79K): [in a new window]   Fig. 3. SHR::GFP does not move from the epidermis even when supernumerary layers are present. Longitudinal confocal images (A-D) and transverse section (E) of pGL2::YFPER (inset in A), pWER::YFPER (inset in C), pGL2::SHR::GFP (A,B) and pWER::SHR::GFP (C-E) transgenic roots. pWER::SHR::GFP induces a pattern perturbation resulting in supernumerary layers (C,E), while pGL2::SHR::GFP does not alter the pattern (A). SHR::GFP does not appear to move from the epidermis in either pGL2::SHR::GFP (B) or in pWER::SHR::GFP (D) transgenic roots. Arrow in the inset of C indicates ep/LRC initial. ep, epidermis; co, cortex; en, endodermis; sn, supernumerary layers. Scale bars: 50 µm in A,C,E; 25 µm in B,D.

View larger version (127K): [in a new window]   Fig. 4. SCR is required for pWER::SHR::GFP-mediated ectopic divisions and movement of SHR::GFP from the epidermis occurs in a scr mutant background. Longitudinal confocal images of pWER::SHR::GFP transgenic roots in a scr-4 background (A, red channel only) and (B) a pSHR::SHR::GFP transgenic root in scr-1 background (inset in B), and in situ hybridizations with GFP antisense probe on transverse sections from pWER::SHR::GFP transgenic roots in wild-type (C) and scr-4 (D) backgrounds. The resulting radial pattern (A) is identical to that of scr (Di Laurenzio et al., 1996). The fusion protein SHR::GFP is found in the epidermis as well as in the mutant ground tissue layer (B), whereas no RNA expression of SHR::GFP is detectable in the mutant layer of the scr-4 root (D), indicating movement of the fusion protein from the epidermis to the mutant layer. SHR::GFP is detected both in the nuclei and in the cytoplasm of the mutant layer, when moving either from the epidermis [as in pWER::SHR::GFP in scr-4 (B)] or from the stele [as in pSHR::SHR::GFP in scr-1 (inset in B)]. mut, mutant layer of scr; LRC, lateral root cap; ep, epidermis; sn, supernumerary layers; st, stele. Arrowheads in the inset indicate the mutant layer. Scale bars: 50 µm in A; 25 µm in B-D.

View larger version (124K): [in a new window]   Fig. 5. SCR is induced only in initials by pWER::SHR::GFP. Longitudinal (A-D) and transverse (E,F) confocal images of the same pSCR::YFPER, pWER::SHR::GFP transgenic root. In the GFP+YFP mode (see Materials and methods), signals from both GFP and YFPER are rendered in green (A,C,E); in the YFP mode, the signal from YFPER alone is rendered in yellow (B,D,F). The SCR promoter is active in its normal expression pattern (endodermis, en/co initial and daughter and QC) and in cells in a position that corresponds to the location of the ep/LRC initials (arrowheads) (B,D,F). The SCR promoter is not active in the outermost tissue, even though SHR::GFP is visible in its nuclei (arrow in A,B). QC, quiescent center; st, stele; en, endodermis. Scale bars: 50 µm in A,B; 25 µm in C-F.

View larger version (106K): [in a new window]   Fig. 6. Competence to respond to SHR::GFP-mediated cell fate changes. Endodermis-specific markers. Casparian strip histochemical staining (A,C,E) and JIM13 immunostaining (B,D,F) of transverse sections of wild-type (A,B), pGL2::SHR::GFP (C,D) and pWER::SHR::GFP (E,F) transgenic roots. C,D and E,F are consecutive sections (the star and the asterisk indicate the same cell in C,D and E,F, respectively). In both transgenic lines, Casparian strip is detected in the normal location of the endodermis (arrowheads in A,C,E), as well as in some cells of the outermost tissue (arrows in C,E). Lignin in the xylem is also stained. JIM13 is also detected in the normal location of the endodermis (B,D,F) and in some cells of the outermost tissue (D,F). Some cells in the stele are also stained by JIM13. ep, epidermis; co, cortex; en, endodermis; st, stele. Scale bars: 25 µm.

View larger version (76K): [in a new window]   Fig. 7. Epidermal cell fate is not lost when SHR is expressed in the epidermis. Longitudinal confocal images of the same pGL2::YFPER, pWER::SHR::GFP transgenic root (A-C). In the GFP+YFP mode (see Materials and methods) signals from both GFP and YFPER are rendered in green (A,B) while in the YFP mode the signal from YFPER alone is rendered in yellow (C). The epidermis-specific GL2 promoter is active in cells above the initials containing SHR::GFP (arrowheads), while younger cells contain SHR::GFP but do not show any YFPER (arrows). st, stele. Scale bars: 50 µm in A; 25 µm in B,C.

View larger version (32K): [in a new window]   Fig. 8. Summary of the SHR::GFP localization experiments along the radial axis. pSHR::SHR::GFP in wild type (A) (Nakajima et al., 2001); pSHR::SHR::GFP in pSCR::SHR (B); pSHR::SHR::GFP in scr-1 (C) (Nakajima et al., 2001); pGL2::SHR::GFP in wild type (D); pWER::SHR::GFP in wild type (E); pWER::SHR::GFP in scr-4 (F). Green circle represents nuclear GFP; (strong or weak) green cell represents (strong or weak accumulation of) cytoplasmic GFP. st, stele. Refer to the text for discussion.

View larger version (58K): [in a new window]   Fig. 9. Relationship between SHR movement and competence to respond to SHR in regulating root radial patterning. Movement of SHR protein (asterisks) from the stele is limited to the adjacent layer (red arrows), while the competence to respond to SHR extends beyond the zone of movement. Cells that are competent to respond to SHR-mediated cell specification are rendered in green (cortex competence has yet to be directly tested); initial cells competent to respond to SHR-mediated cell periclinal divisions are rendered in yellow. The fact that competence to respond to SHR exists outside of the zone of SHR movement highlights the crucial role of regulated SHR movement in root radial patterning. in, initial; ep, epidermis; co, cortex; en, endodermis; st, stele; LRC, lateral root cap.

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